BIOCATALYST, METHOD TO PRODUCE A BIOCATALYST, METABOLIC PROCESS, PROCESS TO METABOLIZE ORGANIC CARBO

专利摘要:
biocatalyst, method for producing a biocatalyst, metabolic process, process for metabolizing organic carbon, process for the biological reduction of phosphate, process for treating water and process for bioconverting the substrate. these are micro-organism-containing biocatalysts revealed to have a large population of the microorganisms irreversibly retained within the biocatalysts. Biocatalysts have a surprisingly stable population of microorganisms and have an essential absence of waste generation from the metabolic activity of the microorganisms. composed of highly hydrophilic polymer and have an open, internal porous structure that promotes phenotypic community changes.
公开号:BR112014031262B1
申请号:R112014031262-1
申请日:2013-06-14
公开日:2021-09-08
发明作者:Fatemeh Razavi-Shirazi；Ameen(nmn) Razavi；Farhad DORRI-NOWKOORANI；Mohammad Ali Dorri
申请人:Microvi Biotech Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[001] Priority is claimed in Provisional Patent Applications No.: U.S. 61/689,921, filed June 15, 2012; U.S. 61/689,922, filed June 15, 2012; U.S. 61/689,923, filed June 15, 2015; U.S. 61/689,924, filed June 15, 2015; U.S. 61/689,925, filed June 15, 2015; U.S. 61/689,929, filed June 15, 2015; U.S. 61/689,930, filed June 15, 2015; U.S. 61/689,932, filed June 15, 2015; U.S. 61/689,933, filed June 15, 2015; U.S. 61/689,935, filed June 15, 2015; U.S. 61/689,939, filed June 15, 2015; U.S. 61/689,940, filed June 15, 2015; U.S. 61/689,943, filed June 15, 2015; U.S. 61/689,945, filed June 15, 2015; U.S. 61/689,953, filed June 15, 2015; U.S. 61/849,725, filed February 1, 2013; U.S. 61/850,631, filed February 20, 2013; U.S. 61/851,467, filed March 8, 2013; and U.S. 61/852,451, filed March 15, 2013, each of which is hereby incorporated in its entirety by way of reference. A right is hereby reserved to have patentability determinations made based on the applicable sections of Public Law 112-29. STATEMENT RELATED TO FEDERAL SPONSORED RESEARCH OR DEVELOPMENT
[002] The use of biocatalysts for the continuous degradation of 1,4-dioxane at ultra-low concentrations in water was first reduced to practice with Government support under Contract 1R43ES022123-01, awarded by the National Institutes of Health. The Government has certain rights to it. FIELD OF THE INVENTION
[003] This invention relates to innovative biocatalysts and their use. BACKGROUND OF THE INVENTION
[004] Metabolic processes have long been proposed for anabolic and catabolic bioconversions. Microorganisms of several types have been proposed for such bioconversions and include bacteria and archaea, both of which are prokaryotes; fungi; and algae. Metabolic processes are used by nature and some have been adapted for use by man for millennia for anabolic and catabolic bioconversions in the range of yogurt culture and sugar fermentation to produce alcohol for water treatment to remove contaminants. Metabolic processes offer the potential for high-efficiency, low-energy bioconversions in relatively inexpensive processing equipment and thus can and often are viable alternatives to chemical synthesis and degradation methods. Often, anabolic processes can use raw materials that are preferred from a renewable or environmental standpoint, but are not desirable for chemical synthesis, eg, the conversion of carbon dioxide into biofuels and other bioproducts. Catabolic bioconversions can degrade substrates and have long been used to treat wastewater. There are considerable interests regarding the improvement of metabolic processes for industrial use and expansion of the range of alternatives for the metabolic process in relation to chemical syntheses and degradations.
[005] Several types of process techniques have been proposed for anabolic and catabolic bioconversions. Such processes include the use of suspended microorganisms, that is, planktonic processes. Furthermore, process techniques in which microorganisms are located on or within a solid support have been disclosed.
[006] Employees are faced with a number of challenges related to improving metabolic processes and providing metabolic processes that are viable enough economically to be of commercial interest. Some problems can be inherited with the raw material itself, including the presence of toxins, phages and fortuitous competing microorganisms. Other problems may arise from the microorganism to be used for bioconversion, such as low metabolic conversion rate, low population growth rate, self-mutation, significant substrate consumption to support population growth, the need for inducers, cometabolites, promoters and performance enhancing additives and the lack of a microorganism that has the targeted metabolic conversion. And yet, additional problems can arise from the process used for bioconversion, such as costs with bioproducts from recovering an aqueous fermentation broth. Especially, with sustained microorganisms, problems can arise from the instability of biofilms, including their physical degradation; overgrowth of the microorganism population causing suffocation; the detachment of microorganisms from the support; and the susceptibility to competing microorganisms. Additionally, metabolic processes are characterized by generating solid debris from dead cells or lysates and debris must be accommodated in the process to remove such solids. In some cases, the waste has value as food supplements, such as distillation grains from ethanol manufacture, but in other metabolic processes such as for urban wastewater treatment, costs may need to be incurred to dispose of the waste from an environmentally acceptable way. Genetic engineering, which has been proposed to overcome one or more of these problems, can be problematic in itself.
[007] Microorganisms, including but not limited to bacteria, archaea, fungi and algae, can become attached or adhered to a surface. Studies were conducted that refer to the effect of a change in planktonic growth on the growth of microorganisms on surfaces, including the formation of biofilms on surfaces. Several employees are investigating the prevention or degradation of biofilms in an animal or human body to enhance the effectiveness of antibiotic treatments to heal the animal or human body.
[008] Tuson, et al., in "Bacteria-surface Interactions", Soft Matter, volume 1, article 608 (2013) mentionable as DOI: 10.1039/c3sm27705d, provides a review of work in the field of bacteria and surface interactions. The authors describe the processes involved in attaching a microorganism to a surface and state that attachment to surfaces generates phenotypic changes in cells and that the surface can provide benefits to attached cells. The authors state that organic matter can concentrate on horizontal surfaces in order to stimulate the growth of surface-associated bacteria, and increasing the substrate surface area provides more area over which nutrients can be absorbed to allow for cells grow in nutrient concentrations that would normally be too low to support growth. The authors further state that in addition to surface fixation that facilitates nutrient uptake, some bacteria obtain the necessary metabolites and cofactors directly from the surfaces to which they adhere.
[009] Some of the observations reported in such an article include that nucleation of cell growth in communities on surfaces protects cells from predation and other environmental threats and facilitates genotype conservation. The authors state that where microorganisms form biofilms, resistance to antibiotic treatment has been observed. Such resistance was attributed to one or more of the barrier function of the biofilm matrix; the presence of inactive persistent cells and highly resistant colony variants and upregulation of several biofilm-specific antibiotic resistance genes. A group of officials postulates that some non-biofilm-associated adhesion cells have antibiotic resistance due to primary mechanisms of reducing net negative charge in bacterial cells and enhancing membrane stability. Tuson, et al., point to a conclusion drawn in one paper that attaching bacteria to surfaces alters their metabolic state and reduces susceptibility to antibodies, which is a common recourse for bacteria during the stationary phase of cell growth.
[010] Regarding cell activities that refer to the association of bacteria with surfaces, the authors argue that surface sensitivity is a precursor to agglomeration which is an important adaptive behavior in which the contact between cells and surfaces programs morphological changes that facilitate cooperative behavior, rapid community growth, and community migration. Cells in bacterial communities such as clusters or biofilms interact with each other in a number of different ways. Bacteria can communicate through the use of small molecule chemical messengers in a process called quorum detection.
[011] "The dense grouping of cells in bacterial communities facilitates and increases the concentration of small molecules that transfer information between cells and trigger physiological changes. The format of chemical gradients very close to surfaces intensifies the exchange of chemical information in biofilms and communities attached to surfaces.
[012] "Cho, et al., in "Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control", PLOS Biology, volume 5, article 11, November 2007, pages 2614 to 2623, refers to their findings that E. coli in microchambers communicate to provide colony growth to escape the confines of the microchambers without a "stamp-like" blockage of the exit and to provide channels to facilitate nutrient transport into the colony.
[013] Tuson, et al., further describes the steps for the formation of an attachment of a cell to a surface. Initial fixation is reversible and involves hydrodynamic and electrostatic interactions and the second step of fixation is irreversible and involves van der Waals interactions between the hydrophobic region of the outer cell wall and the surface. Irreversible fixation is facilitated by the production of extracellular polymeric substance.
[014] "Thermodynamics plays a major role in regulating the attachment of bacteria to surfaces. Cells attach preferentially to hydrophilic materials (ie materials with a high surface energy) when the surface energy of the bacteria is greater than the surface energy of the liquid in which they are suspended. The surface energy of bacteria is typically less than the surface energy of liquids in which the cells are suspended and such incompatibility causes the cells to fixate preferentially , to hydrophobic materials (ie, materials with low surface energies) Bacteria can attach to a wide variety of different materials, including glass, aluminum, stainless steel, various organic polymers, and necessary materials such as Teflon™.
[015] "Tuson, et al., report that surface sensitivity triggers a variety of cellular changes. Many of these changes are morphological and facilitate attachment to surfaces. It is stated: "Interestingly, the physical properties of surfaces can influence cell morphology and community structure." ... "Cells adhere uniformly to hydrophobic surfaces, form microcolonies, and grow to become tightly packed multilayer biofilms. Fewer cells attach to hydrophilic surfaces and changes in cell division lead to the formation of chains of cells that are >100 µm in length. Such strands become loosely entangled to form relatively unstructured and densely packed biofilms.
[016] "Tuson, et al., in their concluding remarks, state: "The findings of the interaction of bacteria and surfaces are remarkably incomplete. Such a topic seems ideally suited for collaborations between microbiologist and materials scientists, chemists and engineers because it is bound to benefit from multidisciplinary approaches that are formulated to penetrate several areas, including: (1) identifying the properties of surfaces that are detected by bacteria; (2) elucidate the molecular mechanisms bacteria used to send surfaces and their biochemical responses; and (3) determine how to modulate surface properties to elicit a desired cellular response, including changes in morphology, changes in bioenergetics, or cell death."
[017] There are several proposals for the use of a solid carrier or support for microorganisms to effect a plethora of anabolic and catabolic bioconversions; however, despite the potential process advantages provided by the use of a solid, commercial success has been limited to relatively few applications. The proposals were made so that the microorganisms were supported on the surface of a carrier or in pores of a carrier and for the microorganisms to be located inside the carrier. See, for example, Zhou, et al., "Recent Patents on Immobilized Microorganisms Technology and Its Engineering Application in Wastewater Treatment, Recent Patents on Engineering, 2008, 2, 28 to 35.
[018] As a general rule, solid debris is generated as a result of biological ability, for example, from the instability of the biofilm formed on the carrier and from the death and deterioration of the cell mass. For example, Sato, et al., in U.S. Patent 6,610,205, discloses processes for nitrifying and denitrifying organic wastewater using a thermoplastic microbe carrier. Patent holders claim that a single carrier can affect bioconversions that require both aerobic and anaerobic conditions. The carrier, once formed, is connected to activated sludge that contains microorganisms. The patent holders claim that the nitrifying bacteria are "thickly cultured" on the surface of the carrier and the denitrifying bacteria are "adsorbed onto the carrier and thus firmly immobilized thereon". Its Figure 1 represents an apparatus that uses the carrier and includes the settling tank 9 to remove the sludge. Consequently, such processes appear to require a means of removing debris from the support or carrier.
[019] Several employees formed an aqueous mixture of microorganisms and polymer as a solution, dispersion or emulsion. Some employees submitted the mixture to the spray dryer and others proposed cross-linking to obtain a solid structure that contains microorganisms within the solid structure. The following discussion is provided as an illustration of proposals for forming the solid structures of an aqueous medium that also contains microorganisms.
[020] Hino, et al., in Patent No. US 4,148,689 discloses the use of microorganisms in a hydrophilic complex gel by dispersing microorganisms in a certain homogeneous solution and then gelling the mixture and treating it chemically or by drying to obtain a xerogel. Xerogel is said to have the desired strength and is composed of gelled water soluble polymer such as polymers, polyvinyl alcohol, polyethylene glycol and polyethylene imine and silica. The xerogels used in the examples appear to provide bioconversion, but in fewer activities than suspended cell fermentations. Most examples appear to demonstrate bioactivity over a short duration, eg less than 30 hours. Such examples that appear to report activity over longer durations also indicate deactivation over time. In fact, patent holders predict that an advantage of their xerogel is that the polymer can be recovered and recycled upon deactivation. See column 9, lines 66 et seq.
[021] Fukui, et al., in U.S. Patent 4,195,129, discloses mixing microbial cells with photo-curable resin and irradiating the mixture to provide a cured product that contains immobilized cells. The product, according to the examples, does not have the bioactivity of a free cell suspension. Patent holders do not provide any data regarding the performance of cells immobilized for a long period.
[022] Yamada, et al., in U.S. Patent 4,546,081 discloses a process for continuous fermentation with yeast to produce alcohol. The yeast is immobilized in a thin film which is then placed inside a vessel for fermentation. Patent holders cite several different techniques for producing the film containing the yeast. Although a process in which a mixture of yeast and polyvinyl alcohol is gelled by radiation and formed into the desired shape, no performance difference between the films prepared by the various techniques is specifically mentioned in the patent.
[023] Ishimura, et al., in U.S. Patent 4,727,030, has an objective to obtain a molded porous article that contains microbial cells. A process for immobilizing enzymes or cells is disclosed in which the enzymes or cells are mixed with polyvinyl alcohol and activated carbon and then the mixture is partially dried, then molded and further dehydrated under specified conditions. The porous gel is said to have little expansion upon hydration.
[024] In the 1990s a process was developed in Japan which is called the Pegasus Process, see eg Stowa Pagasus/Pegazur/Bio-tube Process Sheets, June 13, 2006, which uses pellets of organic gels composed of a mixture of polyethylene glycol and activated nitrification slurry. See also U.S. Patent 4,791,061 which is in the same family of patents as KR9312103 referenced herein. The pellets are said to have a diameter of 3 millimeters and a polyethylene glycol fraction of 15 percent and a microorganism fraction of 2 percent with a biofilm thickness of about 60 micrometers. The patent discloses preparing the pellets from a mixture containing an activated slurry and prepolymer and pouring the mixture into a water solution of polyvalent metal ion and persulfate to form particles with immobilized microorganisms. It is found that the process reduces the loss of activity of microorganisms in the formation of pellets.
[025] Chen, et al., in U.S. Patent 5,290,693 the immobilization of microorganisms or enzymes in polyvinyl alcohol microspheres. They form a mixture of polyvinyl alcohol and microorganisms and then conduct a two-stage freezing and hardening step using boric acid and then phosphoric acid or phosphate. Patent holders claim that their process provides strong microspheres that are not detrimental to immobilized microorganisms or enzymes. The examples are instructive. Example 1, for example, is concerned with producing and using microspheres for the denitrification of water containing 100 ppm of potassium nitrate. They state in column 5, lines 7 to 11: "On the seventh day, the denitrification rate of the immobilized microorganisms reached 0.65 mg NC31-N/g of gel/h (sic), which remained unchanged until the 30th day . The biochemical vitality of microorganisms remained stable."
[026] The solution used to produce the microspheres contained about 25 g/l of denitrification slurry microorganisms. Such an example appears to indicate that 7 days of growth of the microorganism population were necessary to reach the activity and that after 30 days, the stable activity was lost. The comparative control reported in such an example, which used boric acid only to gel and harden the PVA, provided a denitrification rate of 0.55 mg N031-N/g gel/h and became unstable after 15 days. Examples 2 and 3 report data for continuous operations spanning 10 and 20 days, respectively. Example 4 refers to the production of ethanol using Sacchramyces cerevisa (about 15 g/l in the mixture with polyvinyl alcohol) and only 8 hours of use were reported and the microspheres containing the immobilized microorganisms are slightly inferior in ethanol production than non-sustained yeast.
[027] Nagadomi, et al., in "Treatment of Aquarium Water by Denitrifying Photosynthetic Bacteria Using Immobilized Polyvinyl Alcohol Beads", Journal of Bioscience and Bioengineering, 87, 2, 189 to 193 (1999), confirms the observations of Chen, et al. al. Boric acid has been found to be harmful to microorganisms. It was also observed that the population growth of microorganisms immobilized on alginate microspheres and on polyvinyl alcohol microspheres. The data reported by the authors did not extend much beyond 15 days of operation.
[028] Willuwait, et al., in U.S. Patent 7,384,777 B2, immobilizes the bacteria in polymeric matrices. Matrices are used for the controlled release of microorganisms. As explained in column 3, lines 63 to 67: "Through the cleaning process, the microorganisms multiply until the retention capacity of the capsules/spheres or until the gel has been reached and the wall bursts, that is, until the microorganisms are released."
[029] It is not surprising, therefore, that the large volume of activities aimed at improving metabolic processes have focused on changing the genotype of the microorganism, for example, through genetic engineering. Genotypic alterations are often provided at significant cost and require substantial time to achieve the targeted performance of a microorganism. Typically, most genetically modified microorganisms do not have the robustness, for example, they are slow growing and are competitively disadvantaged compared to invasive microorganisms and are subject to plasmid loss during the ascending scale to sufficient quantities to fill the scale bioreactors commercial and during the bioconversion process itself. Additionally, genetically modified microorganisms may need to be carefully contained so that they do not escape into the environment, and waste disposal from metabolic processes using genetically modified microorganisms may be treated as hazardous waste. BRIEF DESCRIPTION OF THE INVENTION
[030] Biocatalysts containing the microorganism of this invention have a large population of microorganisms irreversibly retained within the biocatalysts and biocatalysts have a surprisingly stable population of microorganisms and, therefore, stable bioactivity and an essential absence of waste generation from the metabolic activity of microorganisms and biocatalysts can exhibit such phenomenon over extended periods of time. Such phenomena are contrary to conventional expectations that microorganisms either escape from physically restricted regions or that the physically restricted region becomes obstructed or overpopulated leading to a loss of metabolic activity and the total death of the microorganism population.
[031] The microorganisms in the biocatalysts of this invention exhibit phenotypic changes that, in combination with a cavity-containing internal structure of the biocatalyst, provide highly advantageous biocatalysts, including, but not limited to, a metabolic shift in the growth of microorganisms and their population to bioconversion activity (anabolic or catabolic); enhanced tolerance to toxins; heightened ability to enter a substantial state of stasis, even over extended periods of time; and enhanced ability to effectively bioconvert the substrate. Such phenotypic changes add significantly to the fact that microorganisms that perform bioconversion are retained within the biocatalyst to provide beneficial metabolic processes, especially metabolic processes in which the biocatalysts of this invention provide desirable bioconversion activity over periods of time extended, preferably at least about 3, preferably at least about 6, and often in excess of 12 or 24 months and sometimes up to 5 years or more.
[032] Although it is not desired to stick to theory, it is believed that the ability of the biocatalysts of this invention to have the stable population of microorganisms within it is due to phenotypic changes to microorganisms that occur during the manufacture of biocatalysts. It is believed that such phenotype changes are due to the confluence of three primary factors. First is the use of a high concentration of microorganisms to produce the biocatalyst, for example, at least about 60, preferably at least about 100 grams of cells per liter, so that communication can take place between the cells at the time of formation of the biocatalyst. All references to cell mass in this document are in relation to wet cell mass. The high concentration of cells is also preferable due to the fact that due to the occurrence of the phenotypic change, there is little if there is net growth in the population of microorganisms in the biocatalyst. In some cases, the net growth in the microorganism population can be up to three or four times until the steady-state population occurs. However, in most cases, the population of steady-state microorganisms is about 50, often about 30 percent of the concentration of cells initially used in preparing the biocatalyst.
[033] The second main factor is that the biocatalyst, when formed, contains microorganisms in a plurality of larger interconnected cavities between about 5 and 100 microns in the smallest dimension. Preferably, on a volumetric basis, at least about 20, and preferably at least about 50 percent of the interior structure of the biocatalyst (excluding microorganisms) is composed of larger cavities in this range. Although the larger main cavities may be present, preferably less than about 25 percent of the interior of the solid structure is composed of such larger main cavities. Preferably, the interconnected cavities in the biocatalyst are quiescent. The high preponderance of interconnected larger cavities that have a smaller dimension between about 5 and 100 microns is believed to enhance the ability of microorganisms located in the cavities to communicate, so that microorganisms, as a community, are subjected to phenotypic change .
[034] The third major factor is that the polymeric material component of the biocatalyst is hydrated and hydrophilic. Microorganisms located within the biocatalyst as it is produced, especially those in the larger and smaller cavities, are believed to detect the hydrophilic capacity of the surface and such detection of the environment also contributes to the change in phenotype. The polymeric material is highly hydrated, however, it contains sufficient hydrophobic capacity so that the polymeric material in the biocatalyst is not dissolved or dispersed in water under anticipated conditions of use. The hydrophilic capacity and hydrophobic capacity of the polymer occur such that the microorganisms become substantially irreversibly trapped within the biocatalyst. Due to the fact that the retention of microorganisms in the biocatalyst is due to detection by the microorganisms and their response, such irreversible retention can be described as metabolic retention. Biocatalysts can be characterized by having a Hydration Expansion Volume (HEV) of at least about 1,000, preferably, at least about 5,000, and more often at least about 10,000. The Hydration Expansion Volume is indicative of the hydrophilic capacity of the polymeric material and the higher the HEV, the greater the hydrophilic capacity of the polymeric material.
[035] Consequently, in its broadest aspects, the biocatalyst composition of this invention comprises: a solid structure of hydrated hydrophilic polymer that defines an interior structure that has a plurality of interconnected larger cavities that have a smaller dimension between about 5 and 100 microns and a HEV of at least about 1,000, preferably at least about 5,000, and a population of microorganisms substantially irreversibly retained in the interior structure, said microorganisms being at a concentration of at least about 60 grams per liter based on the volume defined by the exterior of the solid structure when fully hydrated, where microorganisms maintain their population substantially stable.
[036] Preferably, the hydrophilic polymer also forms a skin on the outside of the biocatalyst composition. Although it has been found that microorganisms become substantially irreversibly trapped within the biocatalyst, in general the case is that the microorganisms are not, significantly, if they are, in direct contact with the polymer, although they may be in contact through fibrils, for example extracellular polymeric substance or polymer filaments. The high hydrophilic capacity of the polymer is believed to reduce the ability of microorganisms to adhere to the external surfaces of the biocatalyst under conditions of physical stress, such as fluid flow over the exterior of the biocatalyst.
[037] Another broad aspect of this invention relates to methods for producing biocatalyst compositions comprising: a. forming a liquid dispersion, preferably an aqueous dispersion, of solubilized precursor to the hydrophilic polymer and microorganisms to said biocatalyst, wherein the concentration of microorganisms in the liquid dispersion is at least about 60, preferably at least about 100 grams per liter; B. subjecting said dispersion to solidification conditions to form a solid structure of the hydrophilic polymer, wherein the solid structure has an interior structure that has a plurality of interconnected larger cavities containing said microorganisms, said larger cavities having a smaller dimension. between about 5 and 100 microns and wherein the solid structure has a HEV of at least about 1,000, preferably at least about 5,000, wherein said solidification conditions do not unduly adversely affect the population of said microorganisms; and c. maintain the solid structure containing the microorganisms under conditions that do not adversely affect the population of said microorganisms within the solid structure for a time sufficient to enable the microorganisms to undergo a phenotypic change to maintain their population substantially stable and become substantially irreversibly retained within the solid structure.
[038] The solidification conditions may, in some cases, include the presence of a crosslinking agent and the precursor is a solubilized prepolymer. Alternatively, solidification conditions can comprise a reduction in the temperature of the liquid dispersion so that the polymer becomes solidified to form the solid structure. Often, the liquid dispersion not enclosed within the solid structure formed in step (b) is separated during or before step (c).
[039] Other broader aspects of this invention relate to metabolic processes in which the biocatalysts of this invention are subjected to metabolic conditions that include the presence of substrate to bioconvert said substrate into bioproduct. Metabolic processes can be anabolic or catabolic. In preferred processes, microorganisms show a metabolic shift compared to planktonic metabolism under substantially the same metabolic conditions. BRIEF DESCRIPTION OF THE FIGURES
[040] Figure 1 is an SEM image of a portion of a cross-section of a biocatalyst according to this invention.
[041] Figure 2 is an SEM image of a portion of a cross-section of another biocatalyst according to this invention.
[042] Figure 3 is a schematic representation of a photobioreactor with the use of biocatalysts according to this invention.
[043] Figure 4 is a schematic representation of an apparatus suitable for treating urban wastewater with the use of the biocatalysts of this invention.
[044] Figure 5 is a schematic representation of a bioreactor used for wastewater nitrification that also contains a zone to collect solid debris for hydrolysis in wastewater treatment.
[045] Figure 6 is a schematic representation of an apparatus that uses five sets of fluidized bioreactors that contain the biocatalysts of this invention suitable for removing the phosphate anion from water.
[046] Figure 7 is a schematic representation of a set of bioreactors contained in the apparatus illustrated in Figure 6.
[047] Figure 8 represents the sequencing of the operating modes of each set of bioreactors contained in the apparatus illustrated in Figure 6.
[048] Figure 9 is a schematic representation of an apparatus containing the biocatalysts of this invention which is suitable for treating water to minimize macro-organism growth and which contains an optional self-cleaning water supply system.
[049] Figure 10 is a schematic representation of an apparatus suitable for the use of biocatalysts of this invention to produce succinic acid, and such apparatus uses sequential reactors.
[050] Figure 11 is a schematic representation of another apparatus suitable for producing succinic acid in which the reactors are subjected to PEP generation cycles to succinate anion generation with the use of carbon dioxide.
[051] Figure 12 is a schematic representation of an apparatus for producing butanol, in which butanol is phase separated for recovery. DETAILED DESCRIPTION OF THE INVENTION
[052] All patents, published patent applications and articles referenced in this detailed description are hereby incorporated in their entirety by way of reference. DEFINITIONS
[053] As used in this document, the following terms have the meanings set forth below, unless otherwise stated or when clear from the context of their use.
[054] The use of the terms "a" and "an" is intended to include one or more of the elements described. Exemplary element lists are intended to include combinations of one or more of the elements described. The term "may" as used in this document means that the use of the element is optional and is not intended to provide any implications regarding operability.
[055] Adhering to the solid structure of the biocatalyst means that the microorganisms are located in cavities within the biocatalyst and are substantially irreversibly retained therein, despite extraordinary conditions and treatments (ie, bioconversion conditions not normal for bioconversion with the use of microorganisms) can, in some cases, cause the microorganism to exit the biocatalyst. Adhering includes surface attachment to the polymer to form the walls of the porous matrix as well as where the trapped microorganisms are close to a polymeric surface, for example, at about 10 or 20 microns, but are not in direct contact with the surface. . Adhering thus includes physical and electrostatic adhesion. In some cases, the polymer used to produce the biocatalyst can become embedded in the extracellular polymeric substance around a cell or even inside or on the microorganism's cell wall.
[056] Ammonium cation includes ammonium cation and dissolved ammonia. One test to determine the ammonium cation concentration is the Salicylate Method Test Tube N, Hach Method 10031, DOC316.53.01079, 7th Edition.
[057] The BTT test is a batch toxicity tolerance test. The BTT test compares the tolerance of a free suspension of the microorganism with a toxin in an aqueous medium under metabolic conditions with the tolerance of the same microorganism, however, provided in substantially the same aqueous medium under substantially the same metabolic conditions, but , in porous matrices to provide substantially the same initial cell density. The toxin of matter is added to the aqueous medium at a concentration such that the bioconversion of the substrate after 24 hours is approximately 50 percent of that in the absence of the toxin (or in the case of a substrate that may be toxic, a concentration which has substantially no adverse effect on the microorganism). It is not essential that the bioconversion be precisely 50 percent smaller, but it should be in a range between about 35 and 65 percent of that in the absence of the toxin. The same concentration of toxin is added to the aqueous medium that contains the microorganisms in the porous matrices and the bioconversion of the substrate, after 24 hours, is determined. It is understood that metabolic conditions can, in some cases, influence the effect that the toxin has on the microorganism. In such cases, the metabolic conditions must be selected so that they are, in general, an average of those suitable for bioconversion. Furthermore, it is understood that the degree of enhancement provided by this invention may vary with different toxins. Consequently, the toxin used must be the toxin in question for the specific metabolic process. For example, if the process is to produce isobutanol as the byproduct, the toxin used must be contained in the aqueous medium for bioconversion and must not be a toxin such as sodium hypochlorite, which is not expected to be in the medium. For phage like toxins, the added toxins can be infected cells.
[058] Biochemical oxygen demand (BOD) is the amount of oxygen required for the metabolic conversion of organic carbon, in water, into carbon dioxide and is an indication of the organic compounds available for food. BOD is reported in milligrams per liter. BOD can be determined by Standard Method 5210B, revision 1 1/16/1999, as published by the US Environmental Protection Agency
[059] The bioconversion activity is the rate of substrate consumption per hour per gram of microorganism. Where an increase or decrease in bioconversion activity is referenced herein, such increase or decrease is ascertained under similar bioconversion conditions, including the concentration of substrate and product in the aqueous medium. Bioconversion activity to bioproduct is the rate of bioproduct production per hour per gram of microorganism.
[060] Biofilm means an aggregate of microorganisms embedded within an extracellular polymeric substance (EPS), in general, composed of polysaccharides and may contain other components, such as one or more of proteins, extracellular DNA and the polymer used to produce the biocatalyst. The thickness of a biofilm is determined by the size of the aggregate contained in a continuous EPS structure, however, a continuous EPS structure does not include fibrils that might extend between separate biofilms. In some cases, the biofilm extends in a random, three-dimensional way and the thickness is determined as the maximum, straight-line distance between the distal ends. A thin biofilm is a biofilm that does not exceed about 10 microns in any specific direction.
[061] The term bioproduct means a product of a bioconversion that can be an anabolic product or a catabolic product and includes, but is not limited to, the primary and secondary metabolites.
[062] Contamination microorganisms are microorganisms that compete with microorganisms for substrate bioconversion and may be fortuitous or from an upstream bioconversion process. With reference to the biocatalysts of this invention, contaminating microorganisms also include those that can damage the surface of the biocatalyst, although they may not compete for the substrate.
[063] Chemical Oxygen Demand (COD) is the amount of oxygen required to convert organic carbon, in water, into carbon dioxide and thus is an indication of the organic compound content of water. COD is reported in milligrams per liter. One procedure for determining COD is Hach Method 8000, February 2009, Ninth Edition.
[064] A state of essential stasis means that a population of microorganisms has undergone a substantial cessation of all metabolic bioconversion activity, but can be revived. The existence of an essential stasis condition can be ascertained by measuring bioconversion activity. The essential stasis condition can be aerobic, anoxic or anaerobic, which may or may not be the same as the normal operating conditions for the microorganism.
[065] Where stasis is targeted, the temperature is typically in the range of about 0 °C to 25 °C, approximately 4 °C to 15 °C, which may be different from the temperatures used under normal operating conditions .
[066] An exo-network is a community of separate microorganisms that can be in the form of biofilms or individual cells that are interconnected by the extracellular polymeric substance in the form of filaments. The spacing between microorganisms or biofilms in the exo-network is sufficient to allow the passage of nutrients and substrates between them and is often at least about 0.25, approximately, at least about 0.5 microns, and can be up to 5 or 10 microns or more.
[067] The outer skin is an outer layer of polymer in the biocatalyst that is less open than the main channels in the inner structure of the biocatalyst. A biocatalyst may or may not have a skin. Where skin is present, it may or may not have surface pores. Where no surface pores are present, fluids diffuse through the skin. Where pores are present, they often have an average diameter between about 1 and 10 microns.
[068] The term fully hydrated means that a biocatalyst is immersed in water at 25 °C until no further expansion of the surface volume of the biocatalyst is perceived.
[069] The "Hydration Expansion Volume" (HEV) for a biocatalyst is determined by hydrating the biocatalyst in water at 25 °C, until the volume of the biocatalyst has stabilized and measuring the surface volume of the biocatalyst (Vw), removing the biocatalyst from the water and removing excess water from the outside, however, without drying and immersing the biocatalyst in ethanol at 25 °C, for a time sufficient for the volume of the biocatalyst to have stabilized and then measuring the surface volume of the biocatalyst (Vs).
[070] HEV in percent volume is calculated as the amount of [Vw/Vs] x 100%.
[071] To ensure dehydration with ethanol, either a large volume ratio of ethanol to biocatalyst is used or successive immersions of the biocatalyst in fresh ethanol are used. Ethanol is initially dehydrated ethanol.
[072] The terms irreversibly retained and substantially irreversibly retained mean that microorganisms adhere to polymeric structures, so as to define cavities, open, porous. Irreversibly retained microorganisms do not include microorganisms located on the outer surface of a biocatalyst. The microorganisms are irreversibly trapped, even if the biocatalyst has outer pores large enough to allow the microorganisms to escape.
[073] Highly hydrophilic polymers are polymers to which water is attracted, that is, they are hydroscopic. Polymers often exhibit, when cast as a film, a contact angle with water less than about 60° and sometimes less than about 45° and, in some cases, less than about 10°, as measured by sessile drop method using a 5 microliter drop of pure distilled water.
[074] The term highly hydrated means that the volume of the biocatalyst (without the volume of microorganisms) is at least about 90 percent water.
[075] An isolated enzyme is an enzyme removed from a cell and may or may not be in a mixture with other metabolically active or inactive materials.
[076] Macro-organisms include, but are not limited to, molluscs such as bivalve molluscs including mussels and oysters; cirripedias; bryozoans; polychaetes; and macroalgae.
[077] A matrix is a polymeric structure, open and porous and in an article of manufacture that has an interconnected plurality of channels or cavities (in this document "larger cavities") defined by the polymeric structures, said cavities having between about 5 and 100 microns in the smallest dimension (excluding any microorganisms contained therein), where fluid can enter and exit the larger cavities to and from the matrix. The porous matrix may contain larger and smaller channels or cavities than the larger cavities and may contain channels and cavities not open to the outside of the matrix. Larger cavities, that is, open interconnected regions that are between about 5 or 10 to 70 or 100 microns in the smallest dimension (excluding the microorganisms contained in them) have larger nominal dimensions less than about 300, preferably smaller than about 200 microns and sometimes a smaller dimension that is at least about 10 microns. The term open and porous, thus, refers to the existence of channels or cavities that are interconnected by openings between them.
[078] Metabolic conditions include conditions of temperature, pressure, oxygenation, pH and nutrients (including micronutrients) and necessary or desired additives for the microorganisms in the biocatalyst. Nutrients and additives include growth promoters, buffers, antibiotics, vitamins, minerals, nitrogen sources and sulfur sources and carbon sources, which are not otherwise provided.
[079] A metallate is an oxyanion, hydroxyl or salt of a metal or semiconductor element.
[080] Urban wastewater is wastewater collected from two or more sources where the wastewater is generated by human activity including, but not limited to, human and animal excreta; domestic, commercial, agricultural, mining and industrial waste and drainage; storm runoff; food products; and disposal of raw materials, intermediates and product.
[081] Organic oxygenated product means a product that contains one or more oxygenated organic compounds that have 2 to 100 and often 2 to 50 carbons and at least one chemical moiety selected from the group consisting of hydroxyl, carbonyl, ether and carboxyl.
[082] The term permeable means that a component can enter or exit larger cavities from the outside or outside of the biocatalyst.
[083] The population of microorganisms refers to the amount of microorganisms in a given volume and includes cultures, substantially pure (axenic) and mixed cultures.
[084] A phenotypic change or phenotypic alternation or deviation is an alteration in the traits or characteristics of a microorganism from environmental factors and is, therefore, different from an alteration in the microorganism's genetic makeup.
[085] Quiescent time means that the aqueous medium in a biocatalyst is stopped; however, flows of nutrients and substrates and bioproducts can occur through the aqueous medium through diffusion and capillary flow.
[086] The term retained solids means that solids are retained within the biocatalyst. Solids can be retained by any suitable mechanism, including, but not limited to, restricted, but not able to pass through the pores in the skin of a biocatalyst, through capture in a biofilm or a polysaccharide structure formed by microorganisms, through retention in the polymeric structure of the biocatalyst or by sterile entanglement within the structure of the biocatalyst or microorganisms.
[087] The term smallest dimension means the maximum dimension of the smallest of the maximum dimensions that define the length, width and height of a larger cavity. Typically, a preponderance of the largest cavities in a matrix is substantially symmetrical in width and height. For this reason, the smallest dimension can be approximated by the maximum width of a cavity observed in a two-dimensional cross-section, for example, using optical or electron microscopy.
[088] A solubilized precursor for the polymer is a monomer or prepolymer or the polymer itself that is dissolved or dispersed so that the solids cannot be visualized with the naked eye and is stable. For example, a solid can be highly hydrated and can be suspended in an aqueous medium even though the solid is not dissolved.
[089] The term sorption means any physical or chemical attraction and can be adsorption or absorption and can be relatively weak, for example, about 10 kilojoules per mol or a chemical interaction with a sorbent. Preferably, the sorption attraction for the sorbent is greater than that between water and the substrate, but not so great that undue energy is required to desorb the substrate. Often, the sorption force is between about 10 and 70, approximately 15 and 60 kilojoules per mole. A sorbent is a solid that is capable of sorption over at least one substrate.
[090] A stable microorganism population means that the microorganism population does not decrease by more than 50 percent nor increase by more than 400 percent. The stability of the population of microorganisms can, in general, be ascertained from the alteration, or lack of alteration, in the bioconversion activity of the biocatalyst without its intended use.
[091] The term sterilization means any process that kills all forms of microbial life and a sterilizing agent means one or more chemicals or processes that can carry out sterilization. Sterilization is thus a non-selective process due to the fact that it affects all forms of microbial life. Disinfection can be sterilization or it can affect microbial life without killing microorganisms. A disinfecting agent means one or more chemicals or processes that can affect disinfection.
[092] Substrates are carbon sources, electron donors, electron acceptors and other chemicals that can be metabolized by a microorganism, and such chemicals may or may not provide sustaining value to the microorganisms.
[093] The term sugar means carbohydrates that have 5 to 12 carbon atoms and include, but are not limited to, D-glyceraldehyde, L-glyceraldehyde, D-erythrose, L-erythrose, D-terose, L-terose, D- ribose, L-ribose, D-lixose, L-lixose, D-allose, L-allose, D-altrose, L-altrose 2-keto-3-deoxy D-gluconate (KDG), D-mannitol, guluronate, mannuronate, mannitol, lyxose, xylitol, D-glucose, L-glucose, D-mannose, L-mannose, D-gluose, L-gluose, D-idose, L-idose, D-galactose, L-galactose, D- xylose, L-xylose, D-arabinose, L-arabinose, D-talose, L-thalose, glucuronate, galacturonate, rhamnose, fructooligosaccharide (FOS), galactooligosaccharide (GOS), inulin, mannanoligosaccharide (MOS), oligolginate, mannuronate , alpha-keto acid, or 4-deoxy-L-erythrohexocellulose uronate (DEHU).
[094] Typical Bioreactor Systems are those operated in a continuous, semi-continuous or batch mode of operation and include bioreactor designs such as, but not limited to, lakes (in the case of photosynthetic processes), bubble column reactors, stirred reactors, fixed bed reactors, filled bed reactors, fluidized bed reactors, plug flow reactors (tubular) and membrane reactors (biofilm). In conducting photosynthetic bioconversions, reactors can be designed to allow the transfer of photoenergy. The biocatalyst can be freely mobile in the aqueous medium or fixed, for example, to a structure in the reactor vessel or it can itself provide a fixed structure. More than one reactor vessel can be used. For example, reactor vessels can be in parallel or in series of sequential flow.
[095] Typical Mesophilic Conditions are metabolic conditions that include a temperature in a range between about 0 °C and 50 °C or more, depending on the temperature tolerance of the microorganism, more often about 5 °C or 10 °C at 40°C or 45°C; a pressure in the ranges of about 70 to 500, approximately 90 to 300 kPa, is absolute due to equipment configurations, although upper and lower pressures may find applicability; and a pH in the range between about 3 and 9. Typical Mesophilic Conditions can be aerobic or anaerobic.
[096] Typical Separation Techniques for chemicals include phase separation for gaseous chemicals, use of a still, a distillation column, phase separation (liquid and liquid and solid and liquid), gas extraction, centrifuge through-flow, Karr column for liquid and liquid extraction, mixing and settling system or expanded bed absorption. The separation and purification steps can take place from different approaches that combine different methodologies, which can include centrifugation, filtration, reduced pressure evaporation, liquid and liquid phase separation, membranes, distillation and/or other methodologies mentioned in this document. The principles and details of standard separation and purification steps are known in the art, for example, in "Bioseparations Science and Engineering," Roger G. Harrison et al., Oxford University Press (2003) and Membrane Separations in the Recovery of Biofuels and Biochemicals--An Update Review, Stephen A. Leeper, pages 99 to 194, in Separation and Purification Technology, Norman N. Li and Joseph M. Calo, Eds., Marcel Dekker (1992).
[097] The wet weight or wet mass of cells is the mass of cells from which free water has been removed, ie they are at the incipient moisture point.
[098] References to organic acids in this document should be considered to include the corresponding salts and esters.
[099] References to the dimensions and volumes of the biocatalyst in this document are in relation to the fully hydrated biocatalyst, unless otherwise stated or when it is clear from the context. BIOCATALYST
[0100] A. Biocatalyst Overview
[0101] The biocatalysts of this invention have a polymeric structure (matrix) that defines the larger interconnected cavities, that is, they are open porous matrices, in which the microorganisms are metabolically retained within the matrices, that is, the microorganisms promote adhesion in rather than being physically constrained by an external structure. In the biocatalysts of this invention, microorganisms and their communities, among others, regulate their population. Furthermore, together with the detected nature of the microenvironment in the matrices, it is believed that microorganisms establish a spatial relationship among community members.
[0102] Community communication among microorganisms and the behavior of microorganisms, thus, are important to reach and maintain the microorganisms metabolically retained. Communication between microorganisms is believed to occur through the emission of chemical agents, including, but not limited to, autoinducers, and communication includes communications for community behavior and for signaling. Often, the preparation of biocatalysts used in the processes of this invention can result in a population of microorganisms that are initially located within the biocatalyst that is substantially one that exists at the steady-state level. At such microorganism densities in biocatalysts, community communications are facilitated, and it is believed that they are initiated during biocatalyst formation and phenotypic shifts occur to enable metabolic retention and modulate the microorganism population.
[0103] The environment to achieve the metabolically retained stable population of microorganisms is characterized by a highly hydrated hydrophilic polymer structure, such structure defines a plurality of interconnected cavities between about 5 and 100 microns in the smallest dimension and has a Volume of Hydration Expansion (HEV) of at least about 1,000. Structure, therefore, defines the microenvironments for microorganisms. Such microenvironments not only facilitate communication between microorganisms, but also, in some cases, modulate environmental stresses on microorganisms and modulate the supply of substrate and nutrients for microorganisms. The highly hydrated and expanded structure of porous matrices and their opening can also accommodate the metabolic retention of a large population of microorganisms and accommodate the community behaviors associated with metabolic retention.
[0104] Without wanting to be limited to theory, it is believed that the very high HEV of the matrices means that there is water inside the solid structure itself. The absorbed water is believed to act through van der Waals or hydrogen bonding interactions with the hydrophilic polymer to interconnect the polymer chains and strengthen the polymeric structure in the expanded state. When water is removed through dehydration, the polymer filaments can collapse in such a way as to allow for significant shrinkage of the structure. The hydrated polymeric structure is believed to have a medium-low surface energy while still providing sites for attachment by microorganisms. In some cases the polymer surface is highly hydrated which, in itself, can be a source of water and nutrients due to hydration.
[0105] The microorganisms that are retained in the matrices have the ability to form an exo-network. The quiescent nature of the cavities facilitates the formation and then maintenance of any formed exo-networks. A discernible exonet is not believed to be essential to achieve phenotypic changes in the microorganism population such as population modulation and metabolic shift. Where an exonetwork develops, EPS filaments often interconnect with nearby microorganisms and connect the microorganisms to the surface and form the exonetwork. In some cases, microorganisms form thin biofilms and such thin biofilms are engulfed in the exo-network. The biocatalysts of this invention have a substantial absence of biofilms in their interiors that are larger than thin biofilms. For that reason, any biofilms that may primarily form on the biocatalysts are relatively thin, for example up to about 10 and preferably up to about 2 or 5 microns thick and stable in size. In this way, each thin biofilm often has only a few cells and is connected to an exonet.
[0106] Figures 1 and 2 are SEM images that illustrate two potential configurations of microorganisms within the larger cavities within the biocatalysts of this invention. Such images do not limit the broader aspects of the invention. Each biocatalyst is used in a bioconversion for extended periods of time before being prepared for SEM analysis. The bioconversion activity of each biocatalyst remains substantially constant over the duration of the bioconversion. "The biocatalyst in Figure 1 comprises Saccharomyces cerevisiae and was used to produce ethanol from sugar over a period of about 2 weeks. The biocatalyst during its use showed that the microorganisms were irreversibly retained and a metabolic shift towards a Superior conversion efficiency to ethanol occurred. The biocatalyst in Figure 2 comprises Achromobacter denitrificans and was used to degrade nitrate and perchlorate during about 1 month of continuous flow operation. Such biocatalyst also evidenced that the microorganisms were irreversibly retained in the biocatalyst and degraded effectively nitrate and perchlorate anions without generating solids. Each image represents that the microorganisms are at a high population density, but have a spatial configuration that does not evidence overgrowth or thick biofilm formation. observable network in Figure 1 additionally evidences an adi phenotypic. tional occurred in the sense that the interconnection of microorganisms is not characteristic of yeast used in bioconversion processes. Figure 2 illustrates the formation of an exo-network. In general, the most extensive exonetworks, when the microorganism generates EPS, occur over the duration of the use of the biocatalyst.
[0107] It is believed that, in some cases, the spatial configuration of the interior of the biocatalyst and any exo-network promotes communication between microorganisms. Communications can extend to separate thin biofilm and exo-network units. As a general rule, the strength or concentration of autoinducers is amplified by microorganisms in response to the fact that such autoinducer is emitted by another microorganism. Such amplification is enhanced by the spatial configuration of the microenvironment within the biocatalyst and, in some cases, the chemical composition of the polymer to form the biocatalyst. The import of the spatial configuration of larger cavities for phenotypic change and population stability has been demonstrated by examining biocatalysts that contain large cavities, for example, larger than about 1,000 microns in the smallest dimension. Although the biocatalyst exhibits bioconversion activity, the surface of the large cavities appears to be substantially devoid of any microorganisms, as opposed to a large stable population of microorganisms in smaller cavities.
[0108] It is believed that communications result in the community of microorganisms maintaining a relatively constant population within the biocatalyst. Another phenotypic change that occurs in the biocatalysts of this invention, which is believed to result from such communication, is a metabolic shift, i.e., community metabolic functions for reproduction are diminished and targeted bioconversion continues. The population of microorganisms in the biocatalyst may tend to have an advanced mean age due to such a shift in metabolic activity. Older microorganisms also tend to provide more robust and sustainable performance compared to younger cells due to the fact that older cells have adapted to operating conditions.
[0109] Additional benefits of such communication may be an increase in community-level strength or aptitude exhibited by the community in protecting against fortuitous microorganisms and in maintaining strain type uniformity. In some cases, microorganisms, during use of the biocatalyst, may undergo natural selection to make the type of strain in the community more substantial or to provide another benefit for the survival of the microorganism community. In some cases, communication between microorganisms may allow the population of microorganisms to exhibit multicellularity or multicellular-like behavior. Thus, the population of microorganisms in a biocatalyst of this invention may have microorganisms that adapt to different circumstances, yet work in sync for the benefit of the community.
[0110] In some cases, the porous matrix can provide substrate and nutrient modulation for microorganisms to optimize metabolic pathways involving substrates that are available and such pathways may or may not be the pathways used primarily, in which ample substrates and other nutrients are available. Consequently, microorganisms in biocatalysts may exhibit enhanced bioactivity for a primarily used trajectory or a metabolic activity that is normally repressed.
[0111] It is also believed that microenvironments can promote genetic exchange or horizontal gene transfer. Bacterial conjugation or matching can also be facilitated, including the transfer of plasmids and chromosomal elements. Furthermore, microorganisms lyse, DNA and RNA strands in microenvironments are more readily accessible to be taken up by microorganisms in such microenvironments. Such phenomena can enhance the functional abilities of microorganisms.
[0112] Biocatalysts exhibit increased tolerance to toxins. In some cases, communications between microorganisms and any exonetwork can facilitate the population to establish defenses against toxins. The community response to the presence of toxins was observed in the biocatalysts of this invention. For example, biocatalysts survive the addition of toxins such as ethanol and sodium hypochlorite and the original bioconversion activity is quickly recovered, thus indicating the survival of essentially the entire community.
[0113] If desired, biocatalysts can be treated to enhance the formation of the exo-network and, if desired, thin biofilms, prior to use in the metabolic process. However, the performance of the biocatalyst is generally not dependent on the extent of exonet formation and often bioconversion activities remain relatively unchanged between the time before the microorganisms have attached to the polymeric structure and the time when the extensive exo-network structures were generated.
[0114] B. Physical Description of Porous Matrices
[0115] The biocatalysts of this invention comprise a matrix that has an open and porous interior structure with microorganisms metabolically irreversibly retained at least in the larger cavities of the matrix.
[0116] The matrices can be a self-supporting structure structure or can be placed or in a realized structure such as a film, fiber or hollow fiber or shaped article. The realized structure may be constructed of any suitable material including, but not limited to, metal, ceramic, polymer, glass, wood, composite material, neutral fiber, stone and carbon. Where self-supporting, the dies are often in sheet, cylinder form, various lobal structures such as trilobal extrudates, hollow fibers or microspheres which can be spherical, oblong or free-form. The dies, whether self-supporting or placed or in an realized structure, preferably have a thickness or axial dimension less than about 5, preferably less than about 2, approximately between about 0.01 to 1 centimeter.
[0117] Porous matrices can have an isotropic structure or, preferably, an anisotropic structure, with the outer portion of the cross-section the denser structure. Larger cavities, even if an anisotropic structure exists, may be relatively uniform in size across the interior of the matrix or the size of larger cavities and their frequency may vary along the cross section of the biocatalyst.
[0118] The biocatalyst of this invention has larger cavities, that is, interconnected regions open between about 5 or 10 to 70 or 100 microns in the smallest dimension (excluding any microorganisms contained in them). For the purposes of dimensioning, the dimensions of microorganisms include any mass in the exo-network. In several cases, the larger cavities have larger nominal dimensions less than about 300, preferably less than about 200 microns, and sometimes a smaller dimension of at least about 10 microns. The biocatalyst often contains smaller channels and cavities that are in open communication with the larger cavities. Smaller channels often have a maximum cross-sectional diameter between about 0.5 to 20, for example 1 to 5 or 10 microns. The cumulative volume of the larger cavities, excluding the volume occupied by the microorganisms and the mass associated with the microorganisms, for the biocatalyst volume is generally in the range of about 40 or 50 to 70 or 99 percent volume. In many cases, the larger cavities constitute less than about 70 percent of the volume of the complete catalyst, with the remainder constituting the smaller channels and pores. The volume fraction of the biocatalyst that constitutes the larger cavities can be estimated from its cross section. The cross section can be viewed using any suitable microscopic technique, eg scanning electron microscopy and high power optical microscopy. The total pore volume for the matrices can be estimated from volumetric measurements of the matrices and the amount and density of polymer and any other solids used to produce the matrices.
[0119] The biocatalyst is characterized by having high internal surface area, often in excess of at least about 1, and sometimes at least about 10, square meters per gram. In some cases, the volume of water that can be trapped by a fully hydrated biocatalyst (excluding the volume of microorganisms) is in the range of 90 to 99 or more percent. Preferably, the biocatalyst exhibits a Hydration Expansion Volume (HEV) of at least about 1,000, often at least about 5,000, preferably at least about 20,000, and sometimes between 50,000 and 200,000 percent.
[0120] Typically, the polymer type selected and the void volume percent of the matrices are such that the matrices have adequate strengths to enable handling, storage and use in a bioconversion process.
[0121] Porous matrices may or may not have an outer skin. Preferably, the matrices have an outer skin to assist in modulating the inflow and efflux of components to and from the interior of the porous matrix channels. Furthermore, due to the fact that the skin is highly hydrophilic and the added benefit is gained as contaminants or incidentals, microorganisms have difficulty establishing a strong biofilm on the outside of the biocatalyst. Such contaminating microorganisms are often subjected to removal under even lower physical forces such as through fluid flow around the biocatalysts. In this way, damage to the biocatalyst can be substantially eliminated or alleviated through washing or through fluid flows during use.
[0122] Where present, the skin typically has pores of an average diameter between about 1 and 10, preferably 2 to 7 microns in average diameter. Pores can comprise about 1 to 30, approximately 2 to 20 percent outer surface area. The outer skin, in addition to providing a barrier against the entry of chance microorganisms into the interior of the biocatalyst, is preferably relatively soft to reduce the adhesion of microorganisms to the outer side of the skin through physical forces such as fluid flow and contact. with other solid surfaces. Often, the skin is substantially devoid of abnormalities, other than pores, larger than about 2 or 3 microns. Where a skin is present, its thickness is typically less than about 50, approximately between about 1 and 25 microns. It should be understood that the thickness of the skin can be difficult to discern where the porous matrix has an anisotropic structure, with the denser structure being on the outside of the matrix.
[0123] Porous matrices provide a plurality of unique microenvironments and nanoenvironments in their interiors. Such unique microenvironments and nanoenvironments result in enzymes or microorganisms located in different regions within the biocatalyst that is subjected to different metabolic conditions. Metabolic conditions may differ in one or more of composition, oxidation or reduction potential, and pH. For example, the composition may vary based on electron donor, other nutrients, contaminants, bioconversion products and the like and thus may affect metabolic processes within the microorganism in such an environment. For this reason, it is possible to have, in the same matrix, aerobic and anaerobic metabolism and have intensified bioconversion of a less preferential substrate as the more preferential substrate is metabolized. Such ability to have multiple enhanced bioconversions can occur with the use of a single strain of microorganism or with the use of two or more different strains. In some cases, different phenotypic changes can occur depending on the microenvironment in which the microorganisms are located.
[0124] Several factors contribute to the existence of such unique microenvironments. For example, concentration gradients are a major driving force for the ingress and egress of liquid phase components in such channels. As microorganisms in the biocatalyst bioconvert the components, concentration gradients occur, especially along the channels extending from the larger cavities. Changes in component concentration, therefore, result in variations in component concentrations within the biocatalyst. In some situations, microorganisms that have a reduced supply of electron donor or nutrients in one or more regions within the biocatalyst may be deprived or close to deprivation, which can result in phenotypic changes that lead to stress resistance. Bioconversion and consequent gradient changes also affect the rate of outward ingress and egress of the component biocatalyst.
[0125] A high density of microorganisms can exist in steady state operation within the biocatalysts. The combination of the flow channels and the high permeability of the polymeric structure that defines the channels enables a viable population of microorganisms throughout the matrix, albeit with a plurality of unique microenvironments and nanoenvironments. In some cases, cell density based on biocatalyst volume is preferably at least about 100 grams per liter, preferably at least about 200, and often between about 250 and 750 grams per liter. BIOCATALYST CONTAINING POLYSACCHARIDES
[0126] In a preferred aspect of the biocatalyst of this invention, it was found that through the incorporation of polysaccharide inside the biocatalyst, the viability of the population of microorganisms can be maintained. Typically, polysaccharides are not usable by most microorganisms. Often, the polysaccharide is provided in an amount of at least about 0.1, approximately, at least about 0.2 to 100 grams per gram of cells retained in the biocatalyst, and sometimes the biocatalyst contains between 25 and 500 grams of polysaccharide per liter of volume of fully hydrated biocatalyst. The polysaccharide particles used in preparing the biocatalysts preferably have a larger dimension of less than about 50, preferably less than about 20, often between about 0.1 to 5 microns. Solid polysaccharide particles are preferably granular and often have an aspect ratio of minimum cross-sectional dimension to maximum cross-sectional dimension of between about 1:10 to 1:1, approximately 1:2 to 1:1 .
[0127] Due to the ability of the polysaccharide to maintain the viability of microorganisms in the biocatalyst, storage, handling and processes for using the biocatalyst can be facilitated. For example, biocatalysts can be used in bioconversion processes that are operated in a carbon-deficient manner. In metabolic processes where the carbon source is added to maintain microorganisms and not used in the targeted bioconversion of substrate to bioproduct, such as in nitrate, nitrite and perchlorate anion catabolism and the metabolic reduction of metallates, the polysaccharide can serve as the only carbon source and thus eliminating the need to add the carbon source or it can reduce the amount of carbon source added, ie, enable carbon deficient operation. One advantage is that bioprocesses can be operated so that the effluent has essentially no COD. Biocatalysts also have enhanced abilities to tolerate disturbances in the presence of substrate and to quickly regain bioconversion activity. Furthermore, biocatalysts can be remotely manufactured and shipped to the place of use without undue detrimental effect on the biocatalyst's bioconversion activity. Biocatalysts can enter a state of essential stasis for extended durations of time in the absence of a supply of substrate and other nutrients to the microbial composites, even where excursions to desired storage conditions, such as temperature, occur. Bioactivity can be rapidly revived in a bioreactor, even after episodic extended occurrences of shutdown, feedstock disturbance, or feedstock variability. Biocatalysts can be bundled and shipped in sealed barrels and tanks and the like.
[0128] The polysaccharide can be from any suitable source, including but not limited to cellulosic starches or polysaccharides. Polysaccharides are carbohydrates characterized by repeating units linked together by glycosidic bonds and are substantially insoluble in water. Polysaccharides can be homopolysaccharides or heteropolysaccharides and typically have a degree of polymerization between about 200 and 15,000 or more, preferably between about 200 and 5,000. Preferred polysaccharides are those in which about 10, more preferably, at least about 20 percent of the repeating units are amylose (D-glucose units). More preferably, the polysaccharide has at least about 20, more preferably, at least about 30 percent of the repeating units being amylose. Polysaccharides may or may not be functionalized, for example, with acetate, sulfate, phosphate, cyclic pyruvil acetal and the like, however, such functionalizations should not produce the water-soluble polysaccharide at temperatures below about 50°C. A preferred class of polysaccharides are starches.
[0129] Sources of polysaccharides include naturally occurring and synthetic polysaccharides (eg, polydextrose). Various plant-based materials that provide polysaccharides include, but are not limited to, woody plant materials that provide cellulose and hemicellulose and wheat, barley, potato, sweet potato, tapioca, corn, maize, cassava, sorghum, rye, and bran which typically , provide starches. BIOCATALYST CONTAINING SOLID sorbent
[0130] In another preferred aspect of the biocatalysts of this invention, the biocatalysts comprise a solid sorbent. The solid sorbent can be the hydrophilic polymer that forms the structure or can be a particulate, i.e., a distinct solid structure, regardless of shape) contained within the solid structure. The sorbent can be any solid sorbent suitable for the substrate or nutrients or other chemical that influences the targeted metabolic activity, such as, but not limited to, comabolites, inducers and promoters or for components that may be adverse to microorganisms, such as, but without limitations, toxins, phages, by-products and by-products. The solid sorbent is typically an adsorbent in which sorption takes place on the surface of the sorbent. Solid particulate sorbents are preferably nanomaterials that have a larger dimension less than about 5 microns, preferably between about 5 nanometers to 3 microns. Where the solid sorbent is composed of polymer, the solid structure may be essentially composed entirely of polymer or it may be a block copolymer or polymer mixture that constitutes between about 5 and 90 weight percent of the solid structure (excluding the water). Where the solid sorbent is a particulate separated in the biocatalyst, the biocatalyst may comprise between about 5 to 90 mass percent of the biocatalyst's mass (excluding water and microorganisms, but including both hydrophilic polymer and particulates). More than one solid sorbent can be used in a biocatalyst. Preferably, the solid sorbent is relatively evenly dispersed throughout the interior of the biocatalyst, although the solid sorbent may have a variable distribution within the biocatalyst. Where distribution varies, regions with the highest concentration of solid sorbent are often found towards the surface of the biocatalyst.
[0131] Where a particulate sorbent is used, the sorbent comprises an organic or inorganic material that has the intended sorption capacity. Examples of solid sorbents include, without limitation, polymeric materials, especially with polar chemical moieties, carbon (including, but not limited to, activated carbon), silica (including, but not limited to, fumed silica), silicates, clays, molecular sieves, and similar. Molecular sieves include, but are not limited to, zeolites and synthetic crystalline structures that contain oxides and phosphates of one or more of silicon, aluminum, titanium, copper, cobalt, vanadium, titanium, chromium, iron, nickel and the like. The sorption properties can comprise one or more of quasi-chemical or chemical or physical sorption on the surface of the solid sorbent. Thus, surface area and structure can influence the sorption properties of some solid sorbents. Solid sorbents are often porous and thus provide high surface area and physical sorption capabilities. The pores in solid sorbents are often in the range of about 0.3 to 2 nanometers in true diameter.
[0132] The solid sorbent can be incorporated into the polymeric structure in any convenient way, preferably during the preparation of the biocatalyst. PHOSPHORESCENT BIOCATALYST
[0133] Another preferred aspect of the invention relates to biocatalysts that contain phosphorescent material and photosynthetic microorganisms, that is, microorganisms that use light energy in a metabolic process. Preferably, the microorganism is an alga, more preferably a microalgae or cyanobacteria.
[0134] The bioactivity of photosynthetic microorganisms can be enhanced to produce expressed bioproduct using a broad-based light source, such as sunlight. According to the invention, photosynthetic microorganisms are irreversibly retained in biocatalysts in which the The interior of the biocatalyst contains phosphorescent material that can deflect UV light to light having a wavelength between about 400 and 800, preferably between about 450 and 650 nm and can exhibit persistence, with the emission of light often , lasts at least about 5 seconds. A phosphorescent material is a material that has the ability to be excited by electromagnetic radiation so as to reach an excited state, but the stored energy is gradually released. Emissions from phosphorescent materials have persistence, that is, emissions from such materials can last for seconds, minutes or even hours after the source of excitation is removed. A luminescent material is a material that can emit electromagnetic radiation after being excited so as to reach an excited state. Persistence is the time it takes, after discontinuing the irradiation, for the photoluminescent emissions that emanate from a photoluminescent object to decrease to the threshold detection capacity.
[0135] The persistence of radiation allows microorganisms to be cycled in order to enter and exit a region of the culture liquid exposed to the light source and still be productive. With longer persistence durations, photosynthetic microorganisms can continue photobioconversion in the absence or reduction of light intensity. The ability of biocatalysts to maintain photosynthetic activity over extended periods of time, often at least about 30 days and in some cases for at least a year, the cost of phosphorescent materials is often offset by increased production , reduced bioreactor footprint and facilitated bioproduct recovery.
[0136] The biocatalyst, which is highly hydrated, is a significant light radiation distributor for photosynthetic microorganisms trapped within the biocatalyst and also serves to protect the microorganism from photorespiration. Solid debris in the culture liquid (an aqueous solution comprising nutrients for metabolic processes) can be materially reduced, if not essentially eliminated, due to the fact that microorganisms are irreversibly retained in the biocatalyst. In this way, turbidity is reduced and a certain light intensity can thus be found at a greater depth in the culture liquid. Such advantages provided by the biocatalysts of this invention can be realized in any photosynthetic process regardless of whether or not a phosphorescent material is used.
[0137] Examples of phosphorescent materials include, but are not limited to, phosphorescent materials which are metal sulfide phosphors such as ZnCdS:Cu:Al, ZnCdS:Ag:Al, ZnS:Ag:Al, ZnS:Cu:Al as per described in US Patent No. 3,595,804 and metal sulfides which are coactivated with a rare earth element such as those described in US Patent No. 3,957,678. Phosphors that have superior light intensity and longer light persistence than metal sulfide pigments include compositions comprising a host material which is generally an alkaline earth aluminate or an alkaline earth silicate. Host materials generally comprise Europium as an activator and often comprise one or more coactivators, such as elements of the Lanthanide series (eg, lanthanum, cerium, praseodymium, neodymium, samarium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium and lutetium), tin, manganese, yttrium or bismuth. Examples of such matches are described in U.S. Patent No. 5,424,006.
[0138] The phosphorescent materials of high persistence and emission intensity can be alkaline earth aluminate oxides that have the formula MOmAl203:Eu2+, R3+ where m is a number in the range of 1.6 to about 2.2, M is an alkaline earth metal (strontium, calcium or barium), Eu2+ is an activator, and R is one or more coactivators of trivalent rare earth materials of the lanthanide series (eg, lanthanum, cerium, praseodymium, neodymium, samarium, gadolinium , terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium), yttrium or bismuth. Examples of such matches are described in U.S. Patent 6,117,362. Phosphorescent materials also include alkaline earth aluminate oxides that have the formula Mk Al204:2xEu2+, 2yR3+ where k = 1-2x-2y, x is a number in the range of about 0.0001 to about 0.05 , y is a number in the range of about x to 3x, M is an alkaline earth metal (strontium, calcium or barium), Eu2+ is an activator, and R is one or more coactivators of trivalent rare earth materials (eg, lanthanum , cerium, praseodymium, neodymium, samarium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium), yttrium or bismuth. See U.S. Patent 6,267,911B1.
[0139] Phosphorescent materials also include those in which a portion of the Al3+ in the host matrix is replaced with divalent ions such as Mg2+ or Zn2+ and those in which the alkaline earth metal ion (M2+) is replaced with a ion of monovalent alkali metal, such as Li+, Na+, K+, Cs+ or Rb+, as described in US Patent documents 6,117,362 and 6,267,911B1.
[0140] Silicates of high intensity and high persistence have been disclosed in Patent No. 5,839,718, such as Sr.BaO.Mg.MO.SiGe:Eu:Ln where M is beryllium, zinc or cadmium and Ln is chosen from group consisting of rare earth materials, group 3A elements, scandium, titanium, vanadium, chromium, manganese, yttrium, zirconium, niobium, molybdenum, hafnium, tantalum, tungsten, indium, thallium, phosphorus, arsenic, antimony, bismuth , tin and lead. Dysprosium, neodymium, thulium, tin, indium and bismuth are specifically useful. X in such compounds is at least one halide atom.
[0141] Other phosphorescent materials include alkaline earth aluminate of formula MO.A1203.B203:R where M is a combination of more than one alkaline earth metal (strontium, calcium or barium or combinations thereof) and R is a combination of Eu2+ activator and at least one trivalent rare earth material coactivator, (eg, lanthanum, cerium, praseodymium, neodymium, samarium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium), bismuth or manganese. Examples of such matches can be found in U.S. Patent 5,885,483. Alkaline earth aluminates of the MAl204 type, which are described in US Patent No. 5,424,006, may also find application, as can phosphorescent materials comprising a donor system and an acceptor system, as described in US Patent No. 6,953 .536B2.
[0142] As can be seen, several other phosphors can find application. See, for example, Yen and Weber, Inorganic Phosphors: Compositions, Preparation and Optical Properties, CRC Press, 2004.
[0143] The phosphorescent material can be a distinct particle or it can be a particle that has a coating to facilitate incorporation and retention in the polymer that forms the matrix. Particles can be of any suitable shape. In general, the maximum particle size is less than about 1 millimeter, preferably less than about 0.1 millimeter. Particles can be nanoparticles.
[0144] The persistence time exhibited by phosphorescent materials can be in the range of a short duration, for example, about 5 to 10 seconds, to up to 10 or 20 hours or more and will be dependent on the phosphorescent material used. Preferred phosphorescent materials exhibit a persistence of at least about one minute. The intensity of the emitted radiation will, in part, depend on the concentration of the phosphor material in the biocatalyst and the nature of the phosphor material. Typically, the phosphorescent material is provided in an amount of at least about 0.1, approximately, between 0.2 and 5 or 10, by weight, percent polymer (unhydrated) in the biocatalyst. One or more phosphorescent materials can be used in the biocatalyst. Where more than one phosphorescent material is used, the combination can be selected to provide one or more of different wavelength shifts of light contained in the radiation source bandwidth and provide different persistence times. In preferred embodiments, the phosphorescent materials are in the form of nanoparticles, for example, which have a largest dimension between about 10 nm and 10 µm. In some cases, it may be desirable to coat the phosphorescent materials with a compatibilizing agent to facilitate incorporation of the phosphorescent material into the polymer. Compatibilizing agents include, but are not limited to, molecules that have one or more of hydroxyl, thiol, silyl, carboxyl or phosphoryl groups. BIOCATALIZERS THAT CONTAIN ENZYMES
[0145] In another aspect, biocatalysts may contain, in addition to microorganisms, one or more enzymes isolated within the biocatalyst to generate a catalytic change in a component that can be substrate or other nutrients or a bioproduct or by-product or co-product of microorganisms or it can be a toxin, phage or the like. Typically, extracellular enzymes bind or adhere to solid surfaces, such as hydrophilic polymer, additive solids, cell walls and extracellular polymeric substance. It is understood that isolated enzymes can also be located inside the cell of a microorganism. Therefore, enzymes can be substantially irreversibly trapped within the biocatalyst. Due to the structure of the biocatalysts of this invention, microorganisms and enzymes can be in close proximity and thus effective cooperative bioconversions can be obtained. The association of enzymes to the interior surfaces of the biocatalyst typically increases the resistance of the enzyme or enzymes to denaturation due to changes in temperature, pH, or other factors related to the thermal or operational stability of the enzymes. Furthermore, through retention in the biocatalyst, the use of the enzyme in a bioreactor is facilitated and undesirable post-reactions can be alleviated.
[0146] Examples of enzymes include, but are not limited to, one or more of oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Enzymes can cause one or more metabolic conversions. For example, an enzyme can metabolize a component in the feed to provide an intermediate for use by microorganisms in the biocatalyst. An enzyme can be used to metabolize a microorganism's metabolite to provide a target bioproduct. An enzyme can be used to metabolize a component in the food or a comabolite of the microorganism that may be adverse to the microorganism into a metabolite that is less adverse to the microorganism. If desired, two or more different enzymes can be used to carry out a series of metabolic conversions to a component in the food or a metabolite of the microorganism.
[0147] Representative enzymes include, without limitation: cellulase, cellobiohydrolase (eg, CBHI, CBHII), alcohol dehydrogenase (A, B and C), acetaldehyde dehydrogenase, amylase, alpha amylase, gluamylase, beta glucanase, beta glucosidase , invertase, endoglucanase (eg EGI, EGII, EGIII), lactase, hemicellulase, pectinase, hydrogenase, pullalanase, phytase, a hydrolase, a lipase, polysaccharase, ligninase, Accellerase® 1000, Accellerase® 1500, Accellerase® DUET ® TRIO, or cyclic CTec2 enzymes, phosphoglucose isomerase, inositol-1-phosphate synthase, inositol monophosphatase, myo-inositol dehydrogenase, myo-inosose-2-dehydratase, inositol 2-dehydrogenase, deoxy-D-gluconate isomerase, cyanase, 5-dehydro-2-deoxygluconokinase, deoxyfofigluconate aldolase, 3-hydroxy acid dehydrogenase, isomerase, topoisomerase, dehydrase, monosaccharide dehydrogenase, aldolase, phosphatase, a protease, DNase, alginate lyase, laminarinase, endoglucanase L-butanediol ogenase, acetoin reductase, acyl-CoA 3-hydroxyl dehydrogenase, or cis-aconitate decarboxylase. Enzymes include those described by Heinzelman et al. (2009) PNAS 106: 5610 to 5615, herein incorporated by reference in their entirety.
[0148] Enzymes can be linked to the precursor to the hydrophilic polymer of the biocatalyst before the formation of the biocatalyst or can be introduced during the preparation of the biocatalyst, for example, by addition to the liquid medium to form the biocatalyst. There are several methods that are known to a person skilled in the art to deliver enzymes or fragments thereof, or nucleic acids on a solid support. Some examples of such methods include, for example, electrostatic droplet generation, electrochemical means, through adsorption, through covalent bonding, through crosslinking, through a chemical reaction or chemical process. Various methods are described in Methods in Enzymology, Immobilized Enzymes and Cells, part C. 1987. Academic Press. Edited by S.P. Colowick and N.O. Kaplan. Volume 136; Immobilization of Enzymes and Cells. 1997. Human Press. Edited by GF Bickerstaff. Series: Methods in Biotechnology, Edited by J.M. Walker; DiCosimo, R., McAuliffe, J., Poulose, A.J. Bohlmann, G. 2012. Industrial use of immobilized enzymes. Chem. Rev. Soc.; and Immobilized Enzymes: Methods and Applications. Wilhelm Tischer and Frank Wedekind, Topics in Current Chemistry, Volume 200. Pages 95 to 126. C. METHODS FOR PRODUCING BIOCATALYST
[0149] The components, which include microorganisms, used to produce the biocatalysts and the process conditions used to prepare the biocatalysts are not critical to the general aspects of this invention and may vary widely, as is well understood in the art, since it is understood the principles of metabolic retention of the microorganisms described above. In any event, the components and process conditions for producing the biocatalysts with the metabolically irreversible trapped microorganisms should not unduly adversely affect the microorganisms.
[0150] Biocatalysts can be prepared from a liquid medium that contains the microorganism and solubilized precursor to the hydrophilic polymer, which can be one or more of a polymerizable or solidifiable component or a solid that is meltable or bondable to form the headquarters. Aqueous media are used in most cases due to the compatibility of most microorganisms and enzymes with water. However, with microorganisms that tolerate other liquids, such liquids can be used to produce all or a portion of the liquid medium. Examples of such other liquids include, but are not limited to, liquid hydrocarbons, peroxygenated liquids, liquid carboxy-containing compounds, and the like. Mixed liquid media can also be used to prepare the biocatalyst. The mixed media can comprise miscible or immiscible liquid phases. For example, the microorganism can be suspended in a dispersed aqueous phase and the polymerizable or solidifiable component can be contained in a continuous solvent phase.
[0151] The liquid medium used to prepare the biocatalyst may contain more than one type of microorganism, especially in which the microorganisms do not significantly compete for the same substrate, and may contain one or more isolated enzymes or functional additives such as polysaccharide , solid sorbent and phosphorescent materials as described above. Preferably, biocatalysts contain a single type of microorganism. The concentration of microorganisms in the liquid medium used to produce the biocatalysts should be at least about 60 grams per liter. As discussed above, the concentration of microorganisms should preferably approach the desired density of microorganisms in the biocatalyst. The relative amounts of microorganism and polymeric material in forming the biocatalyst can vary widely. The growth of the population of microorganisms after biocatalyst formation is contemplated, as is the potential for damage to some population of microorganisms during the biocatalyst formation process. However, higher concentrations of microorganism are generally preferred, for example, at least about 100 grams per liter, preferably at least about 200, and often between about 250 and 750, grams per liter of the liquid medium used. to produce the biocatalysts.
[0152] Any suitable process can be used to solidify or polymerize the polymeric material or to adhere or fuse particles to form the open porous polymeric matrix with microorganism irreversibly retained therein. Proper process conditions should not unduly adversely affect microorganisms. As microorganisms differ in tolerance to temperatures, pressures and the presence of other chemicals, some matrix formation processes may be more advantageous for one type of microorganism than another type of microorganism.
[0153] Preferably, the polymer matrix is formed from the solidification of a high molecular weight material, through polymerization or through prepolymer crosslinking in a way that a population of microorganisms is provided within the biocatalyst, to measure that the same is formed. Exemplary processes include solution polymerization, slurry polymerization (characterized by having two or more initial stages) and solidification through cooling or solvent removal.
[0154] Biocatalysts can be formed in situ in the liquid medium by subjecting the medium to solidification conditions (such as cooling or evaporation) or by adding a component to cause a polymerization, crosslinking or agglomeration of solids to occur to form a solid structure, such as a catalyst, crosslinking agent or coagulating agent. Alternatively, the liquid medium can be extruded into a solution containing a solidifying agent, such as a catalyst, crosslinking or coagulating agent, or coated onto a substrate and then the composite subjected to conditions to form the solid biocatalyst.
[0155] The polymeric materials used to produce the biocatalysts may have an organic or inorganic backbone, but have sufficient hydrophilic portions to provide a highly hydrophilic polymer that, when incorporated into the matrices, exhibits sufficient water absorption properties to provide the volume of desired hydration expansion of the biocatalyst. Polymeric materials are also intended to include high molecular weight substances, such as waxes (whether or not prepared through a polymerization process), oligomers, and the like, as long as they form biocatalysts that remain solid under the conditions of the intended bioconversion process. for their use and have sufficient hydrophilic properties that the volume of hydration expansion can be achieved. As stated above, it is not essential that the polymeric materials become cross-linked or further polymerized in forming the polymer matrix.
[0156] Examples of polymeric materials include homopolymers and copolymers that may or may not be cross-linked and include condensation and addition polymers that provide high hydrophilic capacity and enable hydration expansion volumes to be achieved. The polymer can be a homopolymer or a copolymer, that is, of a hydrophilic portion and a more hydrophobic portion. The molecular weight and molecular weight distribution are preferably selected to provide the combination of hydrophilic capacity and strength as is known in the art. Polymers can be functionalized with hydrophilic moieties to enhance the hydrophilic capacity. Examples of hydrophilic moieties include, but are not limited to, hydroxyl, alkoxyl, acyl, carboxyl, starch and oxyanions of one or more of titanium, molybdenum, phosphorus, sulfur and nitrogen, such as phosphates, phosphonates, sulfates, sulfonates and nitrates, and the hydrophilic moieties can be further substituted by hydrophilic moieties, such as hydroxyalkoxides, acetylacetonate, and the like. Polymers typically contain carbonyl and hydroxyl groups, especially in some adjacent hydrophilic moieties such as glycol moieties. In some cases, the polymer backbone contains ether oxygens to enhance the hydrophilic capacity. In some cases, the atomic ratio of oxygen to carbon in the polymer is between about 0.3:1 to 5:1.
[0157] Polymers that can find use in forming matrices include functionalized or unfunctionalized polyacrylamides, polyvinyl alcohols, polyether ketones, polyurethanes, polycarbonates, polysulfones, polysulfides, polysilicones, olefinic polymers such as polyethylene, polypropylene, polybutadiene, rubbers , nylons, polytyloxazolin, polyethylene glycol, polysaccharides, such as sodium alginate, carrageenan, agar, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum , guar gum, water-soluble cellulose derivatives and carrageenan, and proteins such as gelatin, collagen and albumin, which can be polymers, prepolymers or oligomers, and polymers and copolymers from the following monomers, oligomers and pre- polymers: monomethacrylates, such as polyethylene glycol monomethacrylate, polypropylene glycol monomethacrylate, polypro pylene glycol monomethacrylate, methoxydiethylene glycol methacrylate, methoxypolyethylene glycol methacrylate, methacryloyloxyethyl hydrogen phthalate, methacryloyloxyethyl hydrogen succinate, 3-chloro-2-hydroxypropyl methacrylate, stearyl methacrylate, 2-hydroxy methacrylate and ethyl methacrylate; monoacrylates such as 2-hydroxy ethyl acrylate, 2-hydroxypropyl acrylate, isobutyl acrylate, t-butyl acrylate, isooctyl acrylate, lauryl acrylate, stearyl acrylate, isobornyl acrylate, cyclohexyl acrylate, methoxytriethylene glycol acrylate, 2-ethoxyethyl acrylate, phenoxyethyl acrylate, nonylphenoxypolyethylene glycol acrylate, nonylphenoxypolypropylene glycol acrylate, silicon modified acrylate, polypropylene glycol monoacrylate, phenoxyethyl acrylate, phenoxydiethylene glycol acrylate, phenoxypolyethylene glycol acrylate, methoxypolyethylene glycol acrylate, acryloyloxyethyl hydrogen acrylate; dimethacrylates such as 1,3-butylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, butylene glycol dimethacrylate, hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polymethacrylate 2-hydroxy-1,3-dimethacryloxypropane, 2,2-bis-4-methacryloxyethoxyphenylpropane, 3,2-bis-4-methacryloxydiethoxyphenylpropane and 2,2-bis-4-methacryloxypolyethoxyphenylpropane; diacrylates such as ethoxylated neopentyl glycol diacrylate, polyethylene glycol diacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene glycol diacrylate, 2,2-bis-4-acryloxyethoxyphenylpropane, 2-hydroxy-1-acryloxy- 3-methacryloxypropane; trimethacrylates such as trimethylolpropane trimethacrylate; triacrylates, such as trimethylolpropane triacrylate, pentaerythritol triacrylate, EO-added trimethylolpropane triacrylate, PO-added glycerol triacrylate, and ethoxylated trimethylolpropane triacrylate; tetraacrylates, such as pentaerythritol tetraacrylate, ethoxylated pentaerythritol tetraacrylate, propoxylated pentaerythritol tetraacrylate and ditrimethylolpropane tetraacrylate; urethane acrylates such as urethane acrylate, dimethyl urethane acrylate and trimethyl urethane acrylate; amino-containing moieties such as 2-aminoethyl acrylate, 2-aminoethyl methacrylate, aminoethyl methacrylate, dimethyl aminoethyl methacrylate, monomethyl aminoethyl methacrylate, t-butylaminoethyl methacrylate, p-aminostyrene, o-aminostyrene, 2-amino-4-vinyltoluene, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, piperidinoethyl ethyl acrylate, piperidinoethyl methacrylate, morpholinoethyl acrylate, morpholinoethyl methacrylate, 2-vinyl pyridine, 3-vinyl pyridine, 2-ethyl-5-vinyl pyridine, dimethylaminopropylethyl acrylate, dimethylaminopropylethyl methacrylate, 2-vinyl pyrrolidone, 3-vinyl pyrrolidone , dimethylaminoethyl vinyl ether, dimethylaminoethyl vinyl sulfide, diethylaminoethyl vinyl ether, 2-pyrrolidinoethyl acrylate, 2-pyrrolidinoethyl methacrylate, and other monomers, such as acrylamide, acrylic acid and dimethylacrylamide.
[0158] Not all polymers listed above will be useful on their own, but may be required to be functionalized or used to form a copolymer with a highly hydrophilic polymer.
[0159] Crosslinking agents, accelerators, polymerization catalysts and other polymerization additives can be employed, such as triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzylamino, N-benzyl ethanolamine, N-isopropyl benzylamino, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, ornithine, histidine, arginine, N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, acrylic acid and 2-allyl- 2-methyl-1,3-cyclopentane dione. For polyvinyl alcohol copolymers and polymers, boric acid and phosphoric acid can be used in the preparation of polymeric matrices. As stated above, the amount of crosslinking agent may need to be limited to ensure that the matrices retain high hydrophilic capacity and the ability to have a high volume of hydration expansion. The selection of polymer, crosslinking agents and other additives to produce porous matrices, which have the physical properties set out above, is within the level of a person skilled in the art of highly hydrophilic and water-soluble polymer synthesis.
[0160] Biocatalysts can be formed in the presence of other additives that can serve to enhance structural integrity or provide a beneficial activity to the microorganism, such as attracting or sequestering components, providing nutrients, and the like. Additives can also be used to provide, for example, a suitable density to be suspended in the aqueous medium rather than tending to float or sink in the broth. Typical additives include, but are not limited to, starch, glycogen, cellulose, lignin, chitin, collagen, keratin, clay, alumina, aluminosilicates, silica, aluminum phosphate, diatomaceous earth, carbon, polymer, polysaccharide, and the like. These additives can be in the form of solids when the polymer matrices are formed, and as such, are often in the range of about 0.01 to 100 microns in the largest dimension.
[0161] If desired, microorganisms can be subjected to stress, as is known in the art. The strain can be one or more of physical, chemical or starvation conditions. Chemical strains include toxins, antimicrobial agents, and inhibitory concentrations of compounds. Physical stresses include light intensity, UV light, temperature, mechanical agitation, pressure or compression, and desiccation or osmotic pressure. Stress can produce regulated biological reactions that protect microorganisms from shock, and stress can allow stronger microorganisms to survive while weaker cells die. MICRO-ORGANISMS
[0162] The microorganisms can be unicellular or they can be multicellular that behaves like a single cell microorganism, such as filamentous growth microorganisms and budding microorganisms. Cells of multicellular microorganisms often have the ability to exist in a unique way. The microorganisms may be of any type, which include, but are not limited to, those microorganisms that are aerobic, anaerobic, facultative anaerobes, heterotrophs, autotrophs, photoautotrophs, photoheterotrophs, chemoautotrophs and/or chemoheterotrophs. Cell activity, which includes cell growth, can be aerobic, microaerophilic, or anaerobic. Cells can be in any phase of growth, which includes latency (or conduction), exponential, transition, stationary, death, dormant, vegetative, sporulation, etc. The one or more microorganisms can be a psychrophile (ideal growth at -10 °C to 25 °C), a mesophile (ideal growth at 20 to 50 °C), a thermophile (ideal growth at 45 °C to 80 °C) or a hyperthermophile (ideal growth at 80 °C to 100 °C). The one or more microorganisms can be a gram-negative or a gram-positive bacterium. A bacterium can be a cocci (spherical), bacillus (stick-like), or spirile (spiral-shaped; for example, vibrios or comma bacteria). Microorganisms can be phenotypically and genotypically diverse.
[0163] The microorganisms can be a wild-type (naturally occurring) microorganism or a recombinant microorganism (which includes, but is not limited to, genetically modified microorganisms). A recombinant microorganism can comprise one or more heterologous nucleic acid sequences (for example, genes). One or more genes can be introduced into a microorganism used in the methods, compositions or kits described herein, for example, by homologous recombination. One or more genes can be introduced into a microorganism with, for example, a vector. The one or more microorganisms can comprise one or more vectors. A vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, for example, a closed circular or linear plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain a means for self-replication. The vector can, when introduced into a host cell, integrate into the host cell's genome and replicate together with the one or more chromosomes into which it has been integrated. Such a vector can comprise specific sequences that can allow recombination at a particular desired site on the host chromosome. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the host cell genome, or a transposon. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can include a reporter gene, such as a green fluorescent protein (GFP), which can be fused in frame to one or more of the encoded polypeptides, or expressed separately. The vector can also include a selection marker, such as an antibiotic resistance gene that can be used to select suitable transformants. Means to genetically manipulate organisms are described, for example, in Current Protocols in Molecular Biology, last updated July 25, 2011, Wiley, Print ISSN: 1934 to 3639. In some embodiments, one or more genes involved in the formation of by-product are deleted in a microorganism. In some embodiments, one or more genes involved in byproduct formation are not deleted. Nucleic acid introduced into a microorganism can be codon-optimized for the microorganism. A gene can be modified (eg mutated) to increase the activity of the resulting gene product (eg enzyme). Desired properties in wild-type or genetically modified microorganisms can often be enhanced through a natural modification process, or self-engineering process, which involves multi-generation selective collection to obtain strain enhancements, such as microorganisms that exhibit enhanced properties, such as robustness in an environment or bioactivity. See, for example, Ben-Jacob, et al., Self-engineering capabilities of bacteria, J.R. Soc. Interface 2006, 3, doi: 10.1098/rsif.2005.0089, February 22, 2006.
[0164] The selected microorganism to be used in a biocatalyst can be targeted in relation to the desired activity. Biocatalysts thus often contain substantially pure strain types of microorganisms and, due to bleaching, enable high bioactivity to be achieved and provide a stable population of the microorganism in the biocatalyst.
[0165] Representative microorganisms to produce biocatalysts of this invention include, without limitation, those presented in patent application published nos. U.S. 2011/0072714, especially, paragraph 0122; 2010/0279354, especially, paragraphs 0083 to 0089; 2011/0185017, especially, paragraph 0046; 2009/0155873; especially, paragraph 0093; and 20060063217, especially paragraphs 0030 and 0031, and those presented in Annex A in that document.
[0166] Photosynthetic microorganisms include bacteria, algae and molds that have biocatalytic activity activated by light radiation. Examples of photosynthetic microorganisms for the production of superior oxygenated organic compound include, but are not limited to, algae, such as strains of Bacillariophyceae, Chlorophyceae, Cyanophyceae, Xanthophyceaei, Chrysophyceae, Chlorella (e.g., Chlorella protothecoides), Crypthecodiniums, Schennocytrium, Nazizodinium Ulkenia, Dunaliella, Cyclotella, Navicala, Nitzschia, Cyclotella, Phaeodactylum and Thaustochytrids; yeasts such as Rhodotorula, Saccharomyces and Apiotrichum strains; and fungal species such as the Mortierella strain. Algae, genetically optimized photoautotrophic cyanobacteria and other photoautotrophic organisms have been adapted to bioconvert internal carbohydrates to the microorganism directly into ethanol, butanol, pentanol and other higher alcohols and other biofuels. For example, genetically modified cyanobacteria that have constructs comprising DNA fragments encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) enzymes are described in patent no. U.S. 6.699,696. Cyanobacteria are photosynthetic bacteria that use light, inorganic elements, water and a carbon source, usually carbon dioxide, to metabolize and grow. Ethanol production using genetically modified cyanobacteria has also been described in published PCT patent application no. WO 2007/084477.
[0167] The following examples are provided as an illustration of the biocatalysts and processes for producing the biocatalysts and not as a limitation. All parts and percentages of solids are by mass and liquids and gases are by volume, unless otherwise indicated or made clear from the context.
[0168] In these examples, the following general procedure is used. The microorganisms for the biocatalyst are grown under suitable planktonic conditions in an aqueous medium for the microorganisms, which includes the presence of nutrients and micronutrients. This medium is referred to in this document as the "Culture Medium". The microorganisms used are as available and thus can be substantially pure strains or mixed cultures. Cell density in the Culture Medium is determined by optical density. If the cell density of the Culture Medium is below that desired to produce the biocatalyst, the Culture Medium is centrifuged or filtered to provide a more dense cell-containing fraction. A separately prepared aqueous solution of solubilized precursor is produced (referred to herein as the "Polymer Solution"). Any solid additive to the biocatalysts is added to the Polymer Solution in amounts that will provide the desired amount in the biocatalyst. The Polymer Solution is mixed with a mechanical stirrer to ensure uniform dispersion of the components in the aqueous medium. Where necessary to solubilize the precursor, the Polymer Solution can be heated as appropriate. In some cases, a micronutrient solution is also added to the Polymer Solution.
[0169] Aliquots of each of the Culture Medium (hi dense phase from centrifugation) and the Polymer Solution are mixed under mechanical agitation at about 30 °C up to a Precursor Solution. Where the microorganism is anaerobic, the Culture Medium and the mixture of Culture Medium and Polymer Solution and all subsequent steps are maintained under anaerobic conditions by purging with nitrogen.
[0170] The Precursor Solution is then extruded through a perforated plate having holes of about 0.75mm in diameter to form droplets of about 3mm in diameter. The droplets fall into a gently stirred coagulant bath of an aqueous boric acid solution which has a pH of about 5. The biocatalyst is recovered from the coagulant bath and washed with distilled water. The biocatalyst, after washing, is placed in a liquid medium that contains micronutrients and the substrate under metabolic conditions suitable for the microorganisms.
[0171] Table I summarizes the examples. Table II shows the microorganisms used in the examples. Table III sets out the hydrophilic polymer(s) that are used in the examples. Table IV presents the solid additive groupings used in the examples.









TABLE II


TABLE III










TABLE IV



[0172] Each of the above biocatalysts exhibit phenotypic changes and the biocatalysts have a stable population of microorganisms and do not generate any noticeable debris from metabolic activity. BIOCONVERSIONS A. OVERVIEW
[0173] As described above, the biocatalysts of this invention can be used for a wide range of anabolic and catabolic bioconversion processes. Substrates can be one or more of normally a gas, liquid or solid. The substrates are preferably capable of being dissolved in the aqueous medium for contact with the biocatalyst, although the biocatalysts of this invention may find advantageous application in processes where the substrate has little, if any, solubility in water, especially in processes gas-phase metabolic compounds made possible by the biocatalysts of this invention. In broad aspects, the processes of this invention pertain to the bioconversion of substrate to bioproduct, such processes comprise (a) placing the substrate in contact with the biocatalyst of this invention, and (b) maintaining the biocatalyst under metabolic conditions for a time sufficient to bioconvert by the minus a portion of the substrate in said bioproduct. Usually, the bioproduct is recovered; however, in some aspects of this invention, the bioproduct may purposely be a chemical that is capable of being sequestered in the biocatalyst, for example, in which it removes water-soluble metal compounds.
[0174] Substrates can be natural or xenobiotic substances in an organism (plant or animal) or can be obtained from other sources. Accordingly, substrates include, but are not limited to, those that can be, or can be derived from, plant, animal, or fossil fuel sources, or can be produced by a chemical or industrial process. Biocatalysts can also be applicable to wastewater cleaning or water supply operations where the substrate is one or more contaminants. Biocatalysts generate metabolites as a result of anabolic or catabolic activity and metabolites can be primary or secondary metabolites. The processes of this invention can be used to produce any type of anabolic metabolite.
[0175] Bioproducts can be degradation products, especially where contaminants are being removed from a fluid, such as for wastewater treatment or water supply. Such degradation by-products include, but are not limited to, carbon dioxide, carbon monoxide, hydrogen, carbonyl sulfide, hydrogen sulfide, water, and salts such as carbonate, bicarbonate, sulfide, sulfite, sulfate, phosphate, phosphite, chloride , bromide, iodide, and ammonium borate salts, or group 1 to 16 metals (IUPAC), such as sodium, potassium, manganese, magnesium, calcium, barium, iron, copper, cobalt, tin, selenium, radium, uranium , bismuth, cadmium, mercury, molybdenum and tungsten.
[0176] Bioproducts can be one or more of aliphatic compounds and aromatic compounds that include, but are not limited to, hydrocarbons of up to 44 or 50 carbons, and hydrocarbons substituted by one or more of hydroxyl, acyl, carboxyl, amine, amide moieties, halo, nitro, sulfonyl and phosphine, and hydrocarbons containing one or more heteroatoms which include, but are not limited to, nitrogen, sulfur, oxygen and phosphorus atoms. Examples of organic products as end products from metabolic processes are those listed in patent application published no. U.S. 2010/0279354 A1 , especially as set forth in paragraphs 0129 to 0149. See also published patent application no. US 2011/0165639 Al. Other bioproducts include p-toluate, terephthalate, terephthalic acid, aniline, putrescine, cyclohexanone, adipate, hexamethylenediamine (HMDA), 6-aminocaproic acid, malate, acrylate, apidipic acid, methacrylic acid, 3-hydroxypropionic acid (3HP), succinate, butadiene, propylene, caprolactam, fatty alcohols, fatty acids, glycerates, acrylic acid, acrylate esters, methacrylic acid, methacrylic acids, fucoidan, muconate, iodine, chlorophyll, carotenoid, calcium, magnesium, iron, sodium , potassium and phosphate. The bioproduct can be a chemical that provides a biological activity in relation to a plant, animal or human being. Biological activity can be one or more of a number of different activities, such as antiviral, antibiotic, depressant, stimulant, growth promoter, hormone, insulin, reproductive, attractant, repellent, biocide, and the like. Examples of antibiotics include, but are not limited to, aminoglycosides (for example, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin); ansamycins (for example geldanamycin, herbimycin); carbacefen (loracarbef); carbapenems (for example ertapenema, doripenema, imipenema/cilastatin, meropenem); cephalosporins (first generation, e.g. cefadroxil, cefazolin, cephalotin, cephalexin); cephalosporins (second generation, e.g. cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime); cephalosporins (third generation, e.g., cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibutene, ceftizoxime, ceftriaxone); cephalosporins (fourth generation, eg cefepime); cephalosporins (fifth generation, e.g. ceftobiprole); glycopeptides (for example teicoplanin, vancomycin, telavancin); lincosamides (for example, clindamycin, lincomycin); macrolides (for example azithromycin, clarithromycin, dirythromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin); monobactams (for example aztreonam); nitrofurans (for example furazolidone, nitrofurantoin); penicillins (for example amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin); penicillin combinations (for example amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate); polypeptides (for example, bacitracin, colistin, polymyxin B); quinolones (for example, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin); sulfonamides (e.g. mafenide; sulfonamidochrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-Syllazine); , doxycycline, minocycline, oxytetracycline, tetracycline); drugs against mycobacteria (eg, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampin, rifabutin, rifapentin, dapsone, streptomycin, and others) , fosfomycin, fusidic acid, linezolid, metronidazole, mupirocin, platensimycin, luinupristin/dalfopristin, rifaximin, thiamphenicol, tinidazole).
[0177] Preferably, an anabolic byproduct is at least one of an oxygenated organic compound and hydrocarbon of up to about 100, often up to about 50, carbon atoms. The most preferred organic oxygenated product includes methanol, ethanol, acetic acid, n-propanol, i-propanol, propionic acid, n-butanol, i-butanol, butyric acid, acetone and methyl ethyl ketone.
[0178] Examples of suitable anabolic or catabolic processes to be practiced by the processes of this invention include, but are not limited to: • Syngas, that is, gas containing carbon monoxide and optionally hydrogen, for conversion to oxygenated organic product and hydrocarbons . In typical prior art processes for converting syngas to oxygenated organic product, a limiting factor on productivity is the mass transfer of carbon monoxide and hydrogen from the gas phase to the liquid phase of the aqueous medium. With the use of the biocatalysts of this invention for syngas bioconversion, mass transfer can be optimized. • Gases containing carbon dioxide for conversion to organic oxygenated product and hydrocarbons. Anabolic conversion can be carried out by algae, cyanobacteria, or other photoactivated microorganisms, for example, to produce alcohols, biodiesel, and the like. Other bioconversion processes that use carbon dioxide to produce by-products include those to produce organic acids and esters and diacids and diesters, such as succinic acid and lactic acid. • Combustion gases, for example, from solid waste disposal or power generation, where the substrate comprises contaminants that must be removed from the gases, such as oxygen halides, sulfoxy moieties, nitrogen oxides, heavy metal compounds, and the like. • Waste gases from the industrial process that contain, for example, volatile organic compounds; solvents, such as chlorine-containing solvents, ketones, aldehydes, peroxygenates, and the like; ammonia or volatile amines; mercaptans and other sulfur-containing compounds; nitrogen oxides; and the like. Waste gases from the industrial process can be air-based, such as exhaust from painting operations, or they can be devoid of air, such as waste or purge gases. The ability to subject these substrates to catabolic degradation can often eliminate the need for a thermal oxidation unit operation, resulting in both energy and capital savings, as natural gas or other fuel is often required to maintain temperature for the thermal oxidation unit. • Natural gas (which includes, but is not limited to, gas recovered by underground fracturing processes, ie fracturing gas), where the substrate for catabolic processing may be one or more of oxygenates, such as nitrogen oxides, oxides of sulfur; perchlorates; sulphides, ammonia; mercaptans; and the like. • Removal of nitrates, perchlorates, taste and odor compounds, organics, chlorinated hydrocarbons, and the like, from water. The source of water can be from a water treatment facility, land sources, surface sources, urban waste processing, and industrial wastewater. The water stream can be derived from other bioconversion processes where the substrate is not completely consumed, such as in corn ethanol processes. • Carbohydrate, which includes, but is not limited to, cellulose, hemicellulose, starches and sugars for conversion to an oxygenated organic product and hydrocarbons. • Oxyanions, hydroxyls or soluble salts of sulfur, phosphorus, selenium, tungsten, molybdenum, bismuth, strontium, cadmium, chromium, titanium, nickel, iron, zinc, copper, arsenic, vanadium, uranium, radium, manganese, germanium, indium, antimony mercury, and rare earth metals for water removal through bioconversion and sequestration.
[0179] The metabolic processes using biocatalysts can be conducted in any suitable manner employing sufficient metabolic conditions for the biocatalyst to convert the substrate into the desired bioproduct. Metabolic conditions include temperature, pressure, oxygenation, pH and nutrient conditions (which include micronutrients) and required or desired additives for the microorganisms in the biocatalyst. Due to the microenvironments and phenotypic changes associated with the biocatalysts of this invention, often a broader range of metabolic conditions can be used effectively than those suitable for planktonic microorganisms. Any bioreactor system can be used, which includes typical bioreactor systems.
[0180] The metabolic processes that use the biocatalysts of this invention provide enough water for the biocatalyst to keep the biocatalyst hydrated. Bioconversion processes can involve direct contact with a gas that contains a substrate or contact with a liquid medium, often an aqueous medium. Water for this aqueous medium may be supplied from any suitable source, which includes, but is not limited to, mains water, demineralized water, distilled water, and waste or process water streams. The aqueous medium can contain nutrients and additives such as comabolites, enhancers, enhancers, inducers, growth promoters, buffers, antibiotics, vitamins, minerals, nitrogen sources and sulfur sources, as are known in the art. If desired, an antifoam agent can be used in the aqueous medium. In some cases, where additives are desired or required for the metabolic process, the biocatalysts of this invention exhibit less equivalent bioconversion activity at less concentration of such additives, as compared to a planktonic free suspension system, all others being substantially equal .
[0181] The bioreactor may or may not be sterilized prior to introduction of the aqueous medium. Due to the use of biocatalysts that contain significant populations of microorganisms, bioreactors can have a fast start-up time.
[0182] Processes can be conducted with all carbon requirements being supplied in the aqueous medium or on a carbon source deficient basis. When operating in a carbon source deficiency, the aqueous medium often provides at least about 50, often at least about 75, supposedly 80 to less than 100 mass percent on a carbon basis of the carbon nutrient. In some cases, the polysaccharide is included in the biocatalyst, where carbon source deficiency operations are predicted. Carbon source deficiency can occur intermittently or continuously during the metabolic process.
[0183] Bioconversion processes can be optimized to achieve one or more goals. For example, processes can be designed to provide high conversions of substrate to bioproduct, or they can be designed to balance capital and energy costs against conversion to bioproduct. As biocatalysts are highly hydrated, their density is generally close to that of water. Consequently, with fluidized bed reactor designs using an aqueous feed stream, the energy consumption is less than where higher density supports are used. In some cases, where metabolic processes generate a gas, for example, in the conversion of sugars to alkanols or in the bioconversion of nitrate anion to nitrogen gas, the gas can accumulate in the biocatalyst to increase buoyancy. This accumulated gas can reduce energy consumption for a fluid bed operation and can facilitate the use of other bioreactor designs, such as recirculating bioreactors.
[0184] The bioproduct can be recovered from the medium in any suitable manner, which includes typical separation techniques. B. METABOLIC DISPLACEMENT
[0185] In a preferred embodiment of the invention, a phenotypic change occurs that results in a metabolic shift of microorganisms in biocatalysts. Metabolic shift can occur in each of the anabolic and catabolic bioconversions. The metabolic shift results in less energy being consumed by microorganisms for growth. Consequently, where a substrate is used for both bioproduct bioconversion and energy by microorganisms, the metabolic shift enhances the bioconversion efficiency in the bioproduct. An additional type of metabolic shift is called carbon flux shift. A carbon flux shift occurs when a microorganism can produce more than one bioproduct from a substrate and the relative amounts of the bioproducts are changed. For example, fermentation of sugars using yeast produces both ethanol and acetate anion. A carbon flux shift occurs when, for example, the ratio of ethanol to acetate anion is increased. Similarly, in the production of butanol from sugars using Clostridia acetobutyricum, ethanol and acetone are co-produced and a shift in carbon flux increases the ratio of butanol produced. A metabolic shift can be of significant economic benefit, especially in large bioconversion plants and where the cost of the substrate is material, such as where syngas or carbohydrate is the substrate. In metabolic systems where a carbon source must be supplied to maintain the microorganisms, metabolic displacement beneficially reduces the amount of carbon source supplied to maintain a given rate of bioproduct production.
[0186] In more preferred embodiments pertaining to anabolic bioconversions of a carbon-containing substrate to a bioproduct, at least about 95, preferably at least about 98, percent of the theoretical maximum bioconversion of the substrate to the desired bioproduct is achieved. For example, in the bioconversion of glucose to ethanol, carbon dioxide and ethanol are produced. The amount of ethanol produced, as compared to the theoretical amount that would be produced if all sugar were bioconverted to ethanol and carbon dioxide produced in the ethanol production trajectory. Similarly, in carbon monoxide bioconversion, the theoretical maximum conversion to ethanol is that 6 moles of carbon monoxide produce 1 mole of ethanol and 4 moles of carbon dioxide (carbon dioxide, of course, can be bioconverted to ethanol in the presence of hydrogen).
[0187] In addition to a metabolic shift, the biocatalysts of this invention ensure a cryptic growth, which is believed to be enabled by phenotypic changes and communication between microorganisms. Cryptic growth helps in providing a biocatalyst that does not generate solid debris. However, the ability of the biocatalysts of this invention to maintain a stable population of microorganisms for prolonged periods of time evidences that a phenotypic metabolic shift occurs in the population of microorganisms. C. INTENSIFIED BIOCONVERSION
[0188] In another preferred aspect of the invention, biocatalysts exhibit an enhanced rate of bioconversion, as compared to that of planktonic microorganisms that have the same cell density per unit volume of bioreactor, all others being substantially the same. This aspect of the invention provides improved anabolic and catabolic bioconversion processes due to increased bioactivity. In some cases, microorganisms can undergo a phenotypic change such that a bioconversion that does not occur in planktonic growth is observed.
[0189] Furthermore, since higher cell densities can often be provided by the biocatalysts of this invention than with planktonic growth in sustained or free suspension biocatalysts, even greater increases in bioconversion activity can be obtained per unit volume of bioreactor or by certain unit of hydraulic dwell time. Therefore, reduced residence times for batch or continuous processing can be achieved per bioconversion unit.
[0190] The use of the biocatalysts of this invention also enables the substrate in feed streams to be reduced to very low concentrations and also enables very low substrate concentrations to be metabolically bioconverted. In some applications, a bioconversion process is desired to reduce a substrate to very low concentrations, for example, for effective use of the substrate or such that the bioconversion effluent does not need to be further treated to remove the substrate. Examples of the latter are urban wastewater, where the effluent should contain few, if any, biodegradable carbon compounds, and reducing toxic materials such as 1,4-dioxane, N-nitrosodimethylamine (NDMA) and perchlorate anion and disruptors endocrines contained in water at concentrations of parts per billion or less. Other examples are components that affect taste and odor in drinking water affected by algal blooms, such as methyl isoborneol (MIB) and geosmin that may only be present in microconcentrations. Thus, one embodiment of the processes of this invention pertains to reducing the concentration of ultra-low contaminants (contaminants at a concentration less than about 50 micrograms per liter) in a stream of water comprising: a. continuously passing said stream of water to a bioreactor, said bioreactor being maintained under metabolic conditions that include the presence of the biocatalyst of this invention that contains microorganisms capable of bioconversion of said ultra-low contaminants irreversibly retained therein; B. contacting said stream of water with said biocatalyst for a time sufficient to reduce the concentration of said ultra-low contaminants; and c. withdrawing from said bioreactor a stream of treated water having a reduced concentration of said ultra-low contaminants.
[0191] Preferably, each of the ultra-low contaminants is present at a concentration in the water stream that passed through the bioreactor in an amount of at least about 10, that is, at least about 50, nanograms per liter (ng/L) and less than about 50, often less than about 20, micrograms per liter (mcg/L). Preferably, at least about 50, and sometimes at least about 80 or 90, percent of the contaminant in the water stream is bioconverted.
[0192] The internal microenvironments and phenotypic changes in the biocatalysts of this invention, in another preferred aspect of this invention, also provide enhanced simultaneous bioconversion of two or more substrates through a single species of microorganism. Microorganisms usually prefer or metabolize one substrate over another in a phenomenon known as diauxie. In accordance with this aspect of the invention, the bioconversion rate of the least preferred substrate is less disadvantaged at the same molar ratio between the most preferred and least preferred substrate than that in a planktonic process using the same microorganism and cell density and substantially the same process conditions. An example of diauxie is the treatment of water containing nitrate and perchlorate anions, where nitrate anions are the preferred substrate.
[0193] The biocatalysts of this invention contain microenvironments that may have conditions different from those external to the biocatalyst. Thus, the microenvironments inside the biocatalysts enable both aerobic and anaerobic bioconversion processes to occur, even with the use of the same microorganism. In this way, for example, the ammonium cation can be oxidized and the resulting nitrate anion reduces to nitrogen in an aerobic aqueous medium. Metabolic conditions in a given microenvironment can be affected by other metabolic activity within biocatalysts. For example, metabolizing an electron donor, such as a carbon source, can consume oxygen and thus provide a reducing environment.
[0194] Biocatalysts can serve to provide self-modulation and enable metabolic activity that would not be possible in planktonic growth in free suspension. This phenomenon is readily observed for redox-type bioconversions. Consequently, metabolic processes that are substrate reductions can proceed in the presence of oxygen or oxidizing components in an aqueous medium surrounding the biocatalyst. By way of example, and not limitation, biocatalysts can be used for the catabolition of hydrocarbons, such as aliphatic and aromatic hydrocarbons of 1 to 50 or more, carbons, which include alkanes, alkenes and alkynes, and aromatics, such as benzene, toluene and xylene; ethers, ketones, aldehydes, alcohols, carboxylic acids and esters of 1 to 50 or more carbons; halogenated hydrocarbons, such as chlorinated and brominated hydrocarbons which include perchlorethylene, dichlorethylene, vinyl chloride, trichloroethane, trichlorethylene, methylene chloride, chloroform, carbon tetrachloride and polychlorinated biphenyls (PCB's), and soluble metal and semimetal compounds which include nitrates, nitrites, sulfates, sulfites, phosphates, phosphites and other metalates. D. TOLERANCE TO INTENSIFIED TOXIN
[0195] Surprisingly, the biocatalysts of this invention exhibit an increased tolerance to toxins. Without sticking to theory, it is believed that some potential reasons for this increased tolerance, in addition to providing an environment where microorganisms are metabolically retained and physically protected, could lie in the fact that the biocatalyst provides an environment where microorganisms have time to react to the presence of toxins to develop internal resistance; the ability of microorganisms to have cell geometric stability and increased cell wall stability; and communication among the microorganism population to enhance the community's ability to react and develop resistance mechanisms to toxins. The tolerance exhibited is greater than that exhibited by planktonic microorganisms and is sometimes greater than that exhibited by conventional immobilized biofilms. Therefore, a phenotypic shift by microorganisms and their community can also contribute to heightened tolerance to toxins.
[0196] In some processes, especially anabolic processes, the bioconversion product itself (byproduct) is toxic to microorganisms or a co-product or by-product that is toxic to microorganisms is produced. For example, bioconversion of sugars to ethanol with yeast in a free cell batch fermentation bioreactor is typically limited to an ethanol concentration of about 15 percent. With the bioproduction of isobutanol or n-butanol from sugars, the maximum concentration of fermentation broth is generally about 2.5 percent. Thereby, the bioproduct titer in the fermentation broth has to be limited to avoid harmful bioproduct concentrations, and the energy required for bioproduct separation increases. Furthermore, with batch processes, the limitation on title results in shorter cycle times. Therefore, increased costs per unit volume of bioproduct are incurred, which include those associated with bioreactor downtime, bioreactor cleaning, and replacement of the microorganism population. The increased resistance of microorganisms to toxicity, according to the processes of this invention, enables higher concentrations of bioproduct to be produced and, for batch fermentation processes, reduces the frequency of interruptions.
[0197] In other processes, toxins can be included as contaminants in the raw materials that provide the substrate for microorganisms. Although pretreatment of raw materials to reduce the concentration of these toxins to tolerable levels can be done, such pretreatment results in additional capital and operating costs. A particularly attractive application for the biocatalysts of this invention is in the treatment of brackish water, saline water or brine to metabolize other components in water. Water can be from surface, ground or industrial sources. Examples of components that may require degradation in such waters include, but are not limited to, nitrates, nitrites, chlorates, perchlorates, halogenated organics including, but not limited to, chlorinated solvents and PCB's, hydrocarbons including, but not limited to, aliphatic hydrocarbons and aromatics and oxygenated hydrocarbons such as 1,4-dioxane, carboxylic acids, ethers, ketones and aldehydes.
[0198] In some cases, the substrate itself can have an adverse effect on microorganisms when present in a very high concentration. Reducing substrate concentration, for example through dilution, increases the operating and capital costs of the metabolic process. Examples of substrates that may be toxic include carbon monoxide, hydrogen cyanide, hydrogen sulfide, permanganate, and oxygenated organic compounds, where used to produce other bioproducts.
[0199] Another toxin that affects microorganisms includes viruses or phage. The treatment of aqueous media that suffer from phage is problematic. The bioreactor can be emptied, sterilized and then repopulated, which incurs significant downtime as well as operating expense. In some cases, additives can be supplied to aqueous media as antiviral agents. This again increases the costs of the metabolic process.
[0200] In broad aspects, the processes to bioconvert a substrate contained in an aqueous medium into a bioproduct under bioconversion conditions, which include the presence of microorganisms for said bioconversion, in which the aqueous medium contains at least one toxin in a sufficient amount to have a deleterious effect, if said microorganisms are freely suspended in said aqueous medium under said bioconversion conditions, they comprise attenuating the effect of the toxin through the use of a biocatalyst of this invention in the bioconversion process.
[0201] In some cases, the rate of bioconversion of substrate to bioproduct is not significantly reduced, all other parameters remain the same, when the concentration of bioproduct in the fermentation broth is around 20, often around 30, supposedly about 50, or more percent greater than that achievable using a cell-free suspension of the microorganism. When used to bioconvert sugar to ethanol, fermentation broth ethanol concentrations of 20 percent by mass or more can often be achieved. For the bioconversion of sugar to isobutanol or n-butanol, the concentration of bioproduct in the fermentation broth of at least about 3 and sometimes between about 3.5 and 5 or more percent by mass can be achieved.
[0202] In many cases, microorganisms have the ability to resist concentrations of toxins at least about 20 and preferably at least about 30 percent greater than said microorganisms in free suspension in said aqueous medium. In many cases, the increased tolerance of processes to the presence of toxins can be observed using a batch toxin tolerance test (BTT test) defined above. The processes of this invention exhibit in a BTT test a bioconversion of at least about 20 percent, preferably at least about 30 or 50 percent, greater than that using the free suspension. In some cases, the microorganisms are able to resist concentrations of toxins at least about 10, and preferably at least about 20, percent greater than said microorganisms in the form of a biofilm on a bone char support, all other conditions being equal.
[0203] In many cases, even when concentrations of chemicals reach levels where the rate of bioconversion exhibited by the biocatalyst is materially affected, the processes of this invention tend to provide protection for at least a portion of the microorganisms metabolically retained in the biocatalyst. Thus, upon termination of excursion in regimes in which microorganisms are adversely affected, the bioconversion activity resumes showing the survival of at least a part of the population of microorganisms that provide the desired metabolism. Where a part of the population is adversely affected during the excursion, the population of microorganisms in the biocatalyst may increase after the termination of the excursion to a steady state level.
[0204] In examples 205 to 207, a continuous stirred tank bioreactor that has a working volume of 7 liters is used for the batch experiments. The bioreactor is equipped with controls to maintain the temperature. The pH of the fermentation broth is controlled, typically at a pH of about 5. Fermentations are conducted in a batch mode. A 1,000 milliliter solution of 223 grams of sugar in the form of honey per liter is loaded into the batch bioreactor. Honey has a composition of about 38 percent by weight fructose, 31 percent by weight glucose, 7.31 percent by weight maltose, 1.3 percent by weight sucrose, 1.5 percent by weight. mass of higher sugars and about 17 percent water with the remainder being non-sugar components. Fermentations are conducted at about 30 °C to 35 °C. In all examples, the same strain of Saccharomyces cerevisiae is used. The biocatalyst used is substantially that prepared in accordance with Example 25 and has a hydration expansion volume of about 70,000. The biocatalyst is placed in contact with dilute aqueous ethanol (ranging between about 5 and 10 percent by mass) for about two days before use. EXAMPLE 205
[0205] In this example, the batch bioreactor is used, and industrial grade ethanol is added to the fermentation broth to provide a 20 mass percent concentration of ethanol. One procedure uses a suspension free of S. cerevisiae to deliver about 150 grams of yeast per liter, and another procedure uses enough biocatalyst to deliver about 150 grams of yeast per liter. After 72 hours under fermentation conditions, the fermentation broth containing the free suspension does not yield ethanol, whereas the biocatalyst produces about 75 percent of the theoretically possible amount of ethanol. In the absence of added ethanol, the free suspension provides, after 72 hours, an ethanol production that is about 78 percent of the theoretically possible amount of ethanol. The biocatalyst produces an amount of ethanol between about 96 and 98 percent of what is theoretically possible. The high conversion evidences that a metabolic shift occurs. EXAMPLE 206
[0206] In this example, over a period of about 24 hours, the biocatalyst is exposed to a 20 weight percent aqueous ethanol solution. The biocatalyst is then washed and then used in the batch bioreactor to provide a theoretical yeast content of about 150 grams per liter. After 72 hours, the cell free batch reaction did not generate any ethanol. The batch reaction using the biocatalyst yields about 98 percent of the theoretically possible ethanol. EXAMPLE 207
[0207] In this example, the amount of honey added to the fermentation broth is increased to provide about 288 grams per liter of sugar. Several batch reactions are conducted using about 150 grams per liter of S. cerevisiae contained in the biocatalyst. After 72 hours, batch reactions using the biocatalyst yield between about 96 and 99 percent of the theoretically possible ethanol. EXAMPLE 208
[0208] This example demonstrates the resistance of Rhodococcus to various concentrations of ethanol, in which the microorganism is in a biocatalyst of this invention. Approximately 20 grams of biocatalyst, substantially as prepared in example 169, is placed in a serum bottle for each batch test. A total of 7 bottles of serum are prepared. About 80 milliliters of the ethanol-containing solution is placed in each serum bottle. The solution for each bottle is in a different ethanol concentration. Each solution is prepared using absolute ethanol and the various solutions contain 0, 10, 20, 35, 50, 80 and 100 volume percent ethanol. The contact between the solution and the biocatalyst is at room temperature (about 22 °C) for 24 hours. After that time, the biocatalyst in each serum bottle is washed off. The biocatalyst from each serum bottle is evaluated for oxygen absorption, thus indicating the viability of the microorganisms. This evaluation is conducted by placing the biocatalyst in a 100 milliliter flask and pouring approximately 80 milliliters of distilled water that has been aerated to saturation in the flask. Oxygen probe measurements are taken every 15 minutes. All biocatalysts survived the immersion in ethanol and, compared to the no ethanol control, all substantially recovered their bioactivities, as evidenced by oxygen uptake. In a control experiment, no microorganism survives the addition of 5 percent volumes of ethanol to a free suspension that contains the microorganism. E. IN-SITU STERILIZATION
[0209] It may be desirable to add toxins to the medium containing the microorganisms to control the population of unwanted microorganisms that may compete for nutrients or may provide unwanted metabolites. In some processes, such as converting corn sugar to ethanol, the presence of incidental or undesirable microorganisms is addressed by sterilizing batch reactors at the conclusion of each procedure. Continuous bioconversion processes, however, are not sensitive to frequent sterilization. Therefore, continuous processes are typically monitored for unwanted metabolites or are monitored for the productivity of the desired bioproduct, and stopped for sterilization when required.
[0210] The removal of random microorganisms from water or other raw material for catabolic or anabolic processes can be done, however, with increased capital and operating costs. The alternative is to periodically replace and replenish the desired microorganisms in the bioreactor used for continuous processes. Another alternative is disclosed by Sumner, et al., in published patent application no. U.S. 20090087897, wherein a stabilized chlorine dioxide is preventively added, in an amount effective to prevent the growth of bacteria, to a fermentation process to produce ethanol.
[0211] The biocatalysts of this invention enable the control of the presence of contaminating microorganisms due to enhanced toxin resistance exhibited by biocatalysts. These processes pertain to the bioconversion of a substrate contained in an aqueous medium into a bioproduct, under metabolic conditions, which include the presence of microorganisms for said bioconversion, in which a toxin is supplied to the aqueous medium in an amount sufficient to control contaminating microorganisms , in which microorganisms are retained in a biocatalyst of this invention.
[0212] The toxin used to control contaminating microorganisms can be a bacteriostatic agent, a bactericidal agent or a bacteriolytic agent. Preferably, the toxin is a disinfecting agent, and most preferably, it is an oxidizing agent. These preferred disinfecting agents are relatively inexpensive and include hydrogen peroxide, peracetic acid, aldehydes (especially glutaraldehyde and ophthalaldehyde), ozone and hypochlorite. The introduction of the toxin into the aqueous medium may occur in response to an unwanted buildup of the contaminating microorganism population, or it may be on a periodic or continuous schedule to control the accumulation of contaminating microorganisms. The concentration of disinfecting agent added to the aqueous medium should be sufficient to reduce or maintain the population of contaminating microorganisms at a desired level.
[0213] Where hydrogen peroxide is the sterilizing agent, it is usually introduced such that it is present in the aqueous medium at a concentration of between about 0.1 to about 5, preferably 0.5 to 3, percent in large scale. Where peracetic acid is used as the sterilizing agent, the amount introduced into the aqueous medium is generally sufficient to provide a concentration of between about 0.1 to about 3, preferably 0.2 to 2, percent by weight. Glutaraldehyde and o-phthalaldehyde are often used to provide a concentration in the aqueous medium of between about 0.01 to about 0.5 weight percent. Ozone can be bubbled through the aqueous medium in order to effect a reduction in the population of contaminating microorganisms. The hypochlorite anion is typically available as an aqueous solution of sodium hypochlorite. Typically, the concentration of hypochlorite anion in the aqueous medium is between about 0.1 to 3, supposedly, between about 0.2 to 2, percent by mass.
[0214] The duration of the presence of the sterilizing agent in the aqueous medium should be sufficient in order to effect the desired reduction in the population of contaminating microorganisms. In one embodiment, a concentration of the sterilizing agent can be maintained on a continuous basis in the aqueous medium and thereby act as a preventive measure for the accumulation of a population of contaminating microorganisms. In other preferred embodiments, the sterilizing agent is added intermittently as needed to control the population of contaminating microorganisms. In the latter case, the duration of maintaining microorganism-killing concentrations of the sterilizing agent is typically less than about 20 hours and often is in the range of 0.1 to 10 hours.
[0215] The population control of contaminating microorganisms is conducted more advantageously under the same conditions in which the metabolic process would be conducted. If desired, it is possible to use conditions different from those that would normally be used for the metabolic process, provided the conditions are not unduly deleterious to the microorganisms contained in the biocatalyst. Conditions typically include temperatures in the range between about 5°C to 60°C, and a pH in the range between about 5.0 and 8.5, supposedly 6 to 8. Preferably, the presence of nutrients and other adjuvants remain. at a concentration in the aqueous medium that is substantially the same as that used for the bioconversion process. EXAMPLE 209
[0216] In this example, water containing about 50 parts per million by mass of nitrate anion per liter is continuously passed through a bioreactor containing biocatalyst with Paracoccus denitrificans ATCC® 17741 trapped inside. The biocatalyst is prepared substantially as shown in Example 101 and has a hydration expansion volume of about 70,000. The biocatalyst delivers about 150 grams of microorganism per liter in the bioreactor. The hydraulic residence time is about 600 minutes and the reactor is operated at about 25 °C. Effluent water contains less than about 1 part per million by mass of nitrate anion per liter. Sodium hypochlorite is added to water to provide a concentration of 0.5 grams per liter, and sodium acetate is added in an amount approximately equivalent to 1.1 times the theoretical demand. The bioreactor is operated for 2 hours with this composition. The effluent water continues to contain less than about 5 parts per million by mass of nitrate anion per liter. In additional procedures, the biocatalyst is immersed for 24 hours at about 25 °C in an aqueous solution containing sodium hypochlorite in concentrations of 1.0 and 2.0 grams per liter, before being used in the instrument. These sodium hypochlorite concentrations are considered lethal to planktonic microorganisms in free suspension, as shown by a control experiment.
[0217] After immersion in the 1.0% concentration solution, the performance of the microorganisms is not affected and the water effluent continues to contain less than about 1 parts per million by mass of nitrate anion per liter. The biocatalyst is removed and again immersed in the 1.0% solution for 24 hours. After the second immersion in the 1.0% concentration solution, the performance of the microorganisms is unaffected and the water effluent continues to contain less than about 1 part per million by mass of nitrate anion per liter.
[0218] After immersion in the 2.0% concentration solution, the performance of microorganisms is reduced to about 62 percent and the effluent water contains less than about 20 parts per million by mass of nitrate anion per liter . The biocatalyst is removed and again immersed in the 2.0% solution for 24 hours. After the second immersion in the 2.0% concentration solution, the performance of the microorganisms is improved and the water effluent contains less than about 4 parts per million by mass of nitrate anion per liter. F. CEPA STABILITY
[0219] In the preferred embodiments, the biocatalysts of this invention contain essentially in their interiors a single strain of microorganisms, that is, they are axenic. This provides consistency in bioconversion performance, which includes selectivity and bioconversion rate, and thus enhances the feasibility of commercial-scale processes. Furthermore, having a single strain of microorganism often optimizes sociobiological behavior, such as horizontal gene transfer, production of public goods, altruistic behavior, DNA collection from lysed cells of the same strain, and the like, all of which can be beneficial to the biocatalyst and its performance.
[0220] If a metabolic process uses a wild-type, modified-wild-state, or genetically-modified microorganism, there are several concerns, which include that self-mutation of the microorganism strain could lead to an adverse change in the microorganism population. Although self-mutation can inherently occur, the sociobiological behavior of microorganisms, such behavior that is intensified in the biocatalysts of this invention, can attenuate or prevent inconvenient genotypic changes for the population. Additionally, because the phenotypic changes of microorganisms in the biocatalysts of this invention usually result in a metabolic shift, the reproduction rate is reduced. Therefore, automutation is more readily modulated by the population of microorganisms. Additionally, biocatalysts tend to attenuate external inputs that can induce unwanted automutations
[0221] The biocatalysts of this invention are produced with a high concentration of microorganisms, often substantially at the steady state density of the microorganisms in the biocatalyst. Numerous advantages result from this method. First, essentially fully active biocatalysts can be produced under conditions that ensure strain purity. Second, the step-up is simplified as a plurality of batches can be made and then accumulated to the volume required for a commercial-scale bioreactor. Quality checks can be done with each batch. Third, the population of microorganisms in the biocatalyst can be sufficiently concentrated so that sociobiological behaviors exist that prevent inconvenient self-mutation. Fourth, as the microorganisms are substantially irreversibly retained within the biocatalyst, any contaminating microorganisms would be limited to one biocatalyst and would not adversely affect the microorganisms in the other biocatalysts. Fifth, within a single biocatalyst structure, any contaminating microorganism will tend to be metabolically trapped in a region with a limited population, and the intended microorganism strain community is believed to communicate or interact to enhance competitive strength against invasive microorganisms (territorial competitiveness) and maintain the uniformity of the strain. Sixth, the exo-network of microorganisms on biocatalysts facilitates horizontal gene transfer. And seventh, biocatalysts provide a microenvironment that tends to ensure a stable microbial constitution.
[0222] In some embodiments, the desired microorganism is less robust and, in fact, some, such as syntrophic microorganisms, may only be able to thrive relative to another microorganism and are typically difficult to obtain and maintain a pure culture, even if the syntrophic microorganism may have the ability to effect the desired bioconversion into a bioproduct. In some cases, microorganisms in the biocatalyst can undergo adaptation and potential genetic alteration during use in a metabolic process. The potential horizontal gene communication and transfer provided by the biocatalysts of this invention and the exo-network facilitates the uniformity of the microorganism strain within the biocatalysts. G. STASIS CAPACITY
[0223] The biocatalysts of this invention provide an internal environment that allows microorganisms metabolically retained in them to communicate effectively. Communication also serves to ensure that the microorganism community survives during periods when little or no nutrients are supplied to the biocatalysts, ie the biocatalyst can enter an essential state of stasis. The essential state of stasis, as described in this section, is in relation to the biocatalyst itself. It should be understood that with the high populations of microorganisms in a biocatalyst, there may be microenvironments in which microorganisms occasionally do not obtain nutrients. Therefore, even when the biocatalyst exhibits bioconversion activity, zones within the biocatalyst may be in essential stasis and regain bioactivity under an increase in the supply of nutrients and substrates for the biocatalyst.
[0224] Until now, stasis of microorganisms has been achieved through storage at cold temperatures with reduced nutrient supply or freezing. Maintaining such freezing or cold conditions can be costly, especially for large volumes of microorganisms, and is subject to loss of potency or mechanical breakdown, and can be deleterious to the population of microorganisms.
[0225] The biocatalysts of this aspect of the invention can move to a state of stasis that does not require the supply of nutrients and no costly storage conditions, yet with the microorganisms having an ability to quickly achieve the desired biological activity upon initiation in a bioreactor . Keeping microorganisms in an essential state of stasis for extended periods of time, not only can the time lag between manufacture and initiation be tolerated, but also microbial composites can be placed in simple containers for transport, such as sealed barrels, tanks, and the like, without the addition of nutrients during the storage period. Planned and unplanned interruptions of a bioconversion process using the biocatalysts of this invention can be accommodated without loss of bioactivity. Due to the fact that microorganisms themselves modulate stasis, no equipment or control system is required to protect the population of microorganisms or restart metabolic activity. In yet another further aspect of the invention, the biocatalyst contains solid polysaccharide within it to further enhance the ability of the biocatalyst to remain in a state of stasis for longer durations.
[0226] The conditions required for entry into stasis fall within a wide range. The temperature can be substantially that used for the metabolic process. The biocatalyst should have some degree of hydration during storage, although it is not essential that the biocatalyst be immersed in an aqueous medium. The temperature often ranges between about -10°C to 50°C or more, and most preferably, between about 5°C or 10°C to 30°C for the purposes of energy savings and convenience. Although lower temperatures are generally preferred, higher temperatures still provide significant durations of stasis of microorganisms in the biocatalysts of this invention. Where the microorganism is sensitive to oxygen, it is preferred, but sometimes not essential, that oxygen be excluded during storage.
[0227] Typically, the biocatalyst may remain in a stasis state for prolonged periods of time, for example, at least about 1, often at least about 20, and sometimes longer than about 50 or 100 weeks. The bioactivity of biocatalysts is recovered by subjecting the biocatalysts to metabolic conditions. Often, essentially complete bioactivity is recovered in less than 5, and sometimes less than 3, days. EXAMPLES 210 TO 215
[0228] In the following examples, several biocatalysts, according to the invention, are subjected to storage during the periods shown in the table below and are then used for the intended metabolic process. Before being stored, biocatalysts are used for the metabolic reaction for at least 2 days. The results are summarized in Table V. TABLE V
H. PHOTOSYNTHETIC PROCESSES
[0229] The biocatalysts of this invention can be used in photosynthetic processes. The biocatalyst contains one or more suitable photosynthetic microorganisms, which include bacteria, algae, yeasts and molds with biocatalytic activity activated by light radiation. Preferably, the microorganism is an alga, most preferably a microalgae, or cyanobacteria. In some cases, Botryococcus is desired due to the productivity of organic compost. The biocatalyst may contain luminescent components as described above, but such components are not critical to the use of a biocatalyst in a photosynthetic process.
[0230] Photo-bioconversion conditions are maintained for the conversion of at least one substrate into the desired bioproduct that include the conditions of temperature, pressure, oxygenation, pH, nutrients and additives. Bioconversion can be in a continuous, semi-continuous or batch mode of operation. Reactor designs include, but are not limited to, typical bioreactor systems, provided access is provided to provide light energy for the biocatalyst. The biocatalyst is freely mobile in the culture liquid. More than one reactor vessel can be used. For example, reactor vessels can be in parallel or in series of sequential flow. The processes and apparatus of this invention can use land-based reactors or the reactors can be adapted to float on a body of water, such as a reservoir, river, lake or ocean. Floating reactors can be adapted to take advantage of the natural temperature moderation of the water body and, in some cases, the natural movements of the water body can help with agitation.
[0231] The bioreactor can be open, for example, as a pond or gutter, or closed. The light source can be any suitable light source, but preferably sunlight provides at least a major part of the light radiation from step (c), often at least about 75, supposedly at least about 90 percent, and sometimes essentially all light radiation is from sunlight. Since biocatalysts provide at least some UV protection for microorganisms, lenses, mirrors, moving arrangements that follow the sun, and the like, can be used to intensify the intensity of light radiation that comes in contact with the culture liquid. . Furthermore, the biocatalyst can provide protection for microorganisms that are susceptible to bleaching or death in the presence of high light intensity.
[0232] The photo-bioconversion conditions, the substrate supply rate and the density of microorganisms in the culture liquid can influence the bioconversion. Consequently, for a given system of biocatalysts, substrates and bioproducts, the productivities can vary widely. In some cases, the biocatalysts used in the processes of this invention can facilitate the maintenance of desired microorganism densities in the culture liquid and thereby facilitate high productivities per surface area exposed to light radiation. In some cases, it may be desired to provide periods of darkness for the photosynthetic microorganisms, where such periods enhance the productivity of the microorganisms.
[0233] The culture liquid in the bioreactor can be substantially stagnant, but preferably it is subjected to forces to provide movement for the culture liquid. Most preferably, the movement of the culture liquid is sufficient to cause movement of the biocatalyst to and from the region receiving the light radiation ("direct contact area") of the culture liquid.
[0234] Examples of substrates include, but are not limited to, carbon dioxide, carbon monoxide, hydrogen, methane, ethane, propane, hydrogen sulfide, carbonyl sulfide, mercaptans, ammonia, lower alkylamines, phosphines, and mixtures thereof . Syngas (synthesis gas) is often a proposed gaseous substrate for anaerobic bioconversions. Carbohydrates, which include sugars and polysaccharides, may find application as substrates. Lipids may also find utility as substrates. Other substrates include, but are not limited to, aliphatic and aromatic molecules. Aliphatic (which include cycloaliphatic) and aromatic substrates include, but are not limited to, hydrocarbons of, for example, about 1 to about 44 or 50 carbon atoms, which may contain heteroatoms, for example, oxygen, sulfur, phosphorus and nitrogen, and which may be substituted, for example, by acyl, halogen, hydroxyl, amine, amide, thiol, nitro or phosphine groups.
[0235] Photo-bioconversion conditions, substrate supply rate and density of biocatalysts in the fermentation broth can influence the productivity of the culture liquid to produce bioproducts. Consequently, for a given system of biocatalysts, substrates and bioproducts, the productivities can vary widely. In some cases, the irreversibly retained biocatalysts used in the processes of this invention can facilitate the maintenance of desired biocatalyst densities in the culture liquid and thereby facilitate high productivities per surface area exposed to light radiation. In some cases, it may be desired to provide periods of darkness for the photosynthetic microorganisms, where such periods enhance the productivity of the microorganisms.
[0236] The recovery of the bioproduct from the culture liquid can be carried out by any unit operation or suitable unit operations that include typical separation techniques. Culture liquid can be removed from the reactor for bioproduct recovery or bioproduct recovery can be carried out in the reactor. In the latter case, separation can be through evaporation, for example, with lower vapor pressure organic compounds, such as ethanol, or phase separation as with, for example, higher molecular weight organic compounds, such as aromatic hydrocarbons or aliphatics, alcohols, ethers and esters (eg glycerides) of 6 or more carbons. Where the culture liquid is removed from the reactor for bioproduct purge or recovery, any suitable unit operation can be used to retain the biocatalyst in the reactor, such as, but not limited to, decantation (where the density of the biocatalyst is greater or lesser than that of the culture liquid), filtration, centrifugation, and the like. If desired, especially where the biocatalytic activity of the biocatalyst is observed to be decreasing, a portion of the biocatalyst can be removed and replaced to provide a continuous operating capability.
[0237] For the purposes of facilitating the understanding of the processes and apparatus of this invention, and not as a limitation thereof, reference is made to Figure 3, which depicts a cross-section of a portion of a reactor 300. The reactor 300 comprises a vessel of reactor 302 has a transparent polymeric coating on its top represented by the dotted line and contains culture liquid 304. A plurality of biocatalyst particles 306 that contain cyanobacteria and phosphorescent material are dispersed within the culture liquid 304. Carbon dioxide is used as the substrate to produce ethanol in reactor 300.
[0238] As described, the culture liquid and the effluent gas are recycled. Screen filter 308 is provided to prevent biocatalyst removal and allow effluent gas and culture liquid to be withdrawn from reactor vessel 302. Fluid passes through line 310 for recycling through line 312 and distributor 314 for return to reactor vessel 304. Manifold 314 is adapted to provide movement of biocatalyst 306 to culture liquid surface 304 to receive radiation 316 from a radiation source. Although an external radiation source is described, alternatively or additionally, internal radiation sources could be used. A drag stream to recycle the culture liquid is taken through line 318 for product recovery. Constitution culture liquid is supplied through line 320. Constitution culture liquid contains dissolved carbon dioxide. If desired, ammonium carbonate can be used to supply both carbon and nitrogen to microorganisms. I. DISCUSSIONS OF THE REPRESENTATIVE METABOLIC PROCESS
[0239] Biocatalysts due to the microenvironments in the biocatalyst, the communication between the microorganisms and the phenotypic changes undergone by the microorganisms provide a number of process-related advantages, which include, but are not limited to, • no solid debris that is generated, • the potential for high densities of microorganisms in a bioreactor, • adequate population of microorganisms and bioactivity for prolonged periods of time, • metabolic shift towards production rather than growth and carbon flux shift, • ability to undergo essential stasis for durations • ability to respond quickly to changes in substrate supply rate and concentration, • diauxie attenuation, • modulation and optimized control of pH and redox balances in the biocatalyst microenvironment, • greater tolerance to substrate, byproduct and contaminants, • ability to bioconvert substrate at ultra-low concentrations, • ability to use less robust and slower growing microorganisms and increased resistance to competitiveness, • optimized strain purity capabilities, • ability to undergo antimicrobial treatment in situ, • ability to rapidly start a bioreactor once the population of microorganisms required in total operation is contained in the biocatalyst, • ability to bring the biocatalyst into contact with the gas phase substrate, and • ease of separation of bioproduct from the biocatalyst, thus facilitating continuous operations.
[0240] In the following discussions pertaining to certain uses among the diverse uses of the biocatalysts of this invention, part or all of these process-related advantages provide significant improvements over existing processes. A recitation of these process-related advantages, as they pertain to each of the processes described below, is not repeated for each and must be imputed to each of the processes.
[0241] Additionally, it should be understood that biocatalyst options such as the incorporation of sorbents, polysaccharide and phosphorescent materials (for photosynthetic processes) can be used with any of the processes described below. Additionally, process steps such as in situ sterilization, gas phase bioconversion and photosynthetic reactor setup (for photosynthetic processes) can be used with any of the processes described below. It should be understood that, for the discussions below, cell concentrations in a bioreactor will depend on the concentration of the biocatalyst in the bioreactor, as well as the concentration of cells within the biocatalyst.
[0242] The unique properties of the biocatalysts of this invention enable many metabolic processes. Below are described some of the processes that provide beneficial bioconversions. The figures used to describe the processes are non-limiting and omit smaller equipment such as pumps, compressors, valves, instruments and other devices, the placement and operation of which are well known to those skilled in chemical engineering and omit auxiliary unit operations. URBAN WASTE WATER
[0243] Urban wastewater typically contains dissolved organic compounds (BOD and COD), solids (total suspended solids, TSS), and various ions that include ammonium cation and phosphorus-containing anions. The release of urban wastewater into the environment results in numerous adverse effects. Nitrogen and phosphorus are the predominant contributors to surface water eutrophication. These nutrients can also lead to algal blooms. Algae blooms can cause taste and odor problems when water is to be used for drinking purposes. Other biochemical activities that can be stimulated by the overenrichment of surface waters include the stimulation of microbes that can pose risks to human health.
[0244] Urban wastewater treatment systems generally have a plurality of operations that include three stages of processing treatment. The primary treatment separates solids from liquids. Where the wastewater contains low-density and immiscible liquids, such as fats and oils, these liquids are removed from the surface, and the remaining liquid passes to a secondary treatment that usually uses microorganisms to substantially degrade, under aerobic conditions, biodegradable soluble organic contaminants . On completion of secondary treatment, solids from these microorganisms are separated by decantation and the liquid passes to tertiary treatment before discharge. Not all urban wastewater treatment facilities employ tertiary treatment. Consequently, the effluent can contain significant amounts of nitrogen compounds.
[0245] Tertiary treatment may include nitrogen removal. The ammonium cation may first be subjected to nitrification to produce nitrite and then nitrate, for example, in the presence of Nitrosomonas spp. And then Nitrobacter spp. Denitrification requires anoxic and electron donor conditions. Therefore, some facilities add a donor, such as methanol, or even raw sewage. Often over 4 kilograms of oxygen is consumed per kilogram of ammonium nitrogen removed, and nitrification and denitrification processes increase the energy consumption of a typical installation by 30 percent or more. Solids are generated by microorganisms and are removed upon completion of tertiary treatment, prior to discharge of treated water.
[0246] Typical urban wastewater treatment facilities need to treat 4 or 5 million liters of raw sewage per day, and some facilities treat more than 1 billion liters of urban wastewater per day. Due to these large volumes and the time required to effect the desired biodegradation, a plurality of parallel treatment units are required to handle this substantial flow of urban wastewater. Secondary and tertiary treatments require an aerobic environment. Efforts to reduce treatment residence time for these aerobics include bubbling air through the wastewater. The use of air enriched with oxygen or oxygen provides additional reductions in cycle time, that is, the duration required to achieve the desired reduction in ammonium cation and organic carbon. Still, the hydraulic retention time of water that is treated in a secondary or tertiary treatment operation often exceeds at least 16 hours, supposedly between about 18 and 30, hours with a slurry retention time that can be the from 10 to 30 days. This significant hydraulic retention time necessitates the use of large reactors. Generally, reactors are operated in a batch mode. Consequently, multiple reactors are required in order to sequence connections such that the urban wastewater treatment facility can handle a continuous incoming stream of wastewater. A plurality of settling ponds is required to effect the separation of solids (sludge) from secondary and tertiary treatments. And the sludge needs to be disposed of in an environmentally sound way.
[0247] An additional challenge facing tertiary treatments is that microorganisms are sometimes not stable, due to the presence of fortuitous competitive microorganisms or changes in conditions or composition of the wastewater.
[0248] Hiatt in patent no. U.S. 6,025,152 discloses a mixture of bacteria that is reported to be able to oxidize ammonia and nitrites, organic amines and organonitriles, and aerobically reduce nitrates to molecular nitrogen. The anammox process has been proposed for the removal of ammonia under anaerobic conditions. In this anammox process, bacteria oxidize ammonium under anoxic conditions, with nitrite as the electron acceptor. Typically, the anammox process is conducted with a low carbon to nitrogen source ratio in order to retard the population growth of heterotrophic denitrification bacteria. Anammox bacteria are very sensitive to the presence of oxygen, thus presenting another challenge for use in urban wastewater treatment.
[0249] Processes are provided, in this aspect of the invention, for the treatment of urban wastewater, in which water from the primary treatment continuously passes through a bioreactor that effectively catabolizes organic carbon to carbon dioxide and cation of ammonium to nitrate anion. The bioreactor generates essentially no solids that pass into the treated water, thereby eliminating the need for any additional sludge separation and sludge disposal operations from such operations. Furthermore, the desired bioconversion of organic carbon and ammonium cation can often be accomplished with a relatively short hydraulic retention time, often less than about 12, and in preferred respects, less than about 6 hours. Short hydraulic retention times allow a bioreactor with a small footprint to be used, and once in continuous processes, a plurality of large bioreactors otherwise required to operate in batch mode is not required. These relatively short hydraulic residence times can be achieved without the need to use oxygen- or oxygen-enriched air. Preferably, the dissolved oxygen concentration in the wastewater stream during contact with the biocatalyst is at least about 1 milligram per liter, and preferably between about 2 to 3 milligrams per liter, to save aeration costs. Additionally, the oxygen concentration in the wastewater does not need to be high to achieve the short hydraulic retention times, and the short hydraulic retention times reduce the amount of energy required to aerate the wastewater to a given reduction in ammonia and organic carbon .
[0250] In the broad aspects of this aspect of the invention, the processes to catabolize organic carbon and ammonium cation dissolved in a wastewater stream comprise: • . continuously passing said wastewater stream to a bioreactor containing the biocatalyst, of this invention, with microorganisms substantially irreversibly retained in it, with the ability to catabolize dissolved organic carbon into carbon dioxide and ammonium cation into nitrate anion, of preferably, an ammonia oxidizing microorganism; • . placing in said bioreactor said wastewater stream in contact with said biocatalyst in the presence of oxygen for a time sufficient to provide an oxidized effluent containing less than about 5, preferably less than about 1 ppm, by mass of ammonium cation and which has a reduced biochemical oxygen demand (BOD), preferably less than about 10, preferably less than about 4, milligrams per liter, in which substantially no solids pass from the biocatalyst to the oxidized effluent.
[0251] If desired, the oxidized effluent is subjected to subsequent unit operations, eg for the bioconversion of nitrate to nitrogen and for the removal of phosphorus. Preferably, the denitrification is conducted using the biocatalyst of this invention, with microorganisms substantially irreversibly retained therein, capable of denitrifying nitrate anion, preferably a heterotrophic denitrifying microorganism. By substantially irreversibly retaining denitrification microorganisms in the biocatalyst of this invention, the wastewater that is treated does not need to be de-aerated to obtain high denitrification bioactivity.
[0252] An advantage of sequential nitrification and denitrification unit operations, in accordance with this aspect of the invention, is that the carbon source for denitrification can be controlled to meet the stoichiometric requirement for denitrification, without resulting in an increase in COD in the effluent. The organic carbon from the nitrification operation can vary depending on the composition and rate of introduction of waste water into the nitrification operation and the nitrification operation to achieve the desired reduction in ammonium cation concentration. Consequently, the COD in the water from the nitrification operation can be above about 5, preferably less than about 20, milligrams per liter. This organic carbon thereby offsets any carbon source required for denitrification.
[0253] In another preferred embodiment of this aspect of the invention, at least a portion of the solids contained in the wastewater that is processed, for example, waste from native microorganisms, is hydrolyzed and degraded to further reduce BOD and TSS in the effluent. Typically, these solids tend not to adhere to the biocatalyst, especially biocatalysts that have a coating, due to water currents passing through the biocatalyst bed. By reducing the speed of the water that is treated, for example, as would happen as water emerges from the biocatalyst bed or by providing an expanded section, at least some solids are released from the water and can be subjected to hydrolysis for extended periods of time. Carbon values from waste hydrolysis become dissolved in treated water and pass to a subsequent bioreactor for degradation of carbon values. Solids retention can occur at a number of points in the processes of this invention. For example, the nitrification operation may contain two or more bioreactors in series and hydrolysis takes place between the biocatalyst beds in these bioreactors. Similarly, retention of solids for hydrolysis can occur between the nitrification and denitrification operations.
[0254] Since the processes of this invention use biocatalyst in which microorganisms are retained and have an ability to delay or exclude the entry of native microorganisms, the selected microorganism can be targeted for the desired activity, and the biocatalyst often contains a substantially pure strain of microorganisms, thus enabling greater bioactivity than can be achieved using native microorganisms or activated sludge.
[0255] The raw wastewater can be from any source, although the processes of this invention are particularly useful for urban wastewater treatment. Raw wastewater typically has a BOD between about 50 or 100 and 600 or more milligrams of oxygen per liter. The COD of wastewater is greater than the BOD and is often substantially higher, for example up to 5,000 or more milligrams per liter. The ammonium cation content of raw wastewater can also vary over a wide range and is often between about 10 and 700, more often between about 25 and 200 milligrams per liter. Raw wastewater may contain other components including, but not limited to, sulfur compounds, phosphorus compounds, inorganic salts and solubilized metals.
[0256] Preferably, the waste water to be treated contains less than about 200 and often less than about 100 grams per liter of solids that have a main dimension greater than about 10 microns. If desired, the wastewater can be subjected to ultrafiltration to remove substantially all of the competitive microorganisms before passing through the aerobic bioreactor.
[0257] The waste water passes to at least one aerobic bioreactor that contains biocatalyst for the bioconversion of organic carbon to carbon dioxide and ammonium cation to nitrate anion. The water in the aerobic bioreactor contains dissolved oxygen. Preferably, the concentration of dissolved oxygen in the wastewater stream during a contact with the biocatalyst is at least about 2, say at least about 3 or more milligrams per liter. Conveniently, oxygen is supplied by air or oxygen-enriched air. Oxygen can be supplied by any convenient means including by bubbling or spraying oxygen-containing gas by water or agitation or otherwise mechanical treatment of water such as spraying to facilitate water-gas contact. Oxidizing components include, but are not limited to, peroxide and percarbonate. The environment provided by the biocatalyst can serve to protect the microorganisms trapped in it from the effects of peroxide, percarbonate and other oxidizing components. Where such oxidizing components are used, the active oxygen concentration is preferably in the range of between about 1 and 10, more preferably, between about 1 and 5 milligrams per liter. In general, Common Mesophilic Conditions are used. For most municipal wastewater installations, the other aerobic treatment conditions are usually those defined by the environmental conditions.
[0258] The aerobic bioreactor can be in any suitable configuration that includes Common Bioreactor Systems, preferably suitable for continuous operation. Often the cell density is at least about 100, preferably at least about 200, and sometimes between about 400 and 800 grams per liter.
[0259] The duration of contact between the wastewater and the biocatalyst during the aerobic treatment in the bioreactor is sufficient to provide the desired reduction of metabolizable organic carbon and ammonium cation. The duration will thus depend on the concentration of organic carbon and ammonium cation in the wastewater, the desired reduction and density of microorganisms in the bioreactor, as well as the conditions employed. Relatively low average hydraulic retention times can be realized. The average hydraulic retention time, in some cases, is less than about 6 and more preferably less than about 4 hours. Thus, the bioreactor can be relatively compact, that is, provide small dimensions, but handle high volumes of wastewater to be treated.
[0260] If desired, the oxidized effluent can be filtered. Oxidized effluent can be discharged from the wastewater treatment system, but since it contains nitrate anion, it typically undergoes a process to reduce nitrate anion to nitrogen. Any suitable denitrification unit operation can be used. A particularly advantageous denitrification unit operation uses a biocatalyst of this invention that has substantially irreversibly retained denitrifying microorganisms in it so that no slurry is generated that needs to be removed from the process.
[0261] As mentioned above, denitrification can be conducted in the same bioreactor in which a nitrification takes place or in a separate bioreactor. Denitrification unit operation can be incorporated into aerobic treatment to catabolize organic carbon and ammonium cation. One process uses a biocatalyst of this invention that contains a microorganism capable of both the nitrification and denitrification discussed elsewhere herein. Alternatively, the biocatalyst of this invention which contains denitrifying microorganisms can be intermixed with the biocatalyst for oxidation. The microenvironments provided by the biocatalyst are believed to generate anaerobic conditions and thus allow denitrification to occur. Most often, nitrate anion removal is conducted in a separate unit operation. Where the nitrate anion concentration is low, the use of ion exchange resins may be possible. Chemical reduction processes have also been proposed, for example, to use sulfur dioxide or another reducing agent. However, most municipal wastewater facilities that remove nitrate anion use metabolic processes under anoxic conditions or anaerobic conditions and activated slurry. The denitrified effluent typically contains less than about 1, preferably less than about 0.01 milligrams of nitrate anion per liter.
[0262] Common denitrifying microorganisms include a species of Pseudomonas, Achromobacter, Bacillus and Micrococcus such as Paracoccus denitrificans, Thiobacillus denitrificans and Micrococcus denitrificans. Denitrifying microorganisms require the presence of metabolizable organic carbon as well as anoxic conditions. Normally, denitrifying microorganisms are less sensitive to toxic chemicals than nitrifying microorganisms and recover from toxic shock more quickly than nitrifying microorganisms, especially autotrophic microorganisms. Common bioreactors include those that have a suspension free of microorganisms and those that have supported microorganisms that can be in a fixed bed or filled or fluidized bed.
[0263] In these processes, Common Mesophilic Conditions can be used. The pH of the water to be treated will depend on its source. In general, the pH is maintained between about 4 and 8.5, for example between 6.0 and 8.0. Buffers, if desired, can be used to keep the water at a given pH value during the process. In some cases, it may be possible to use metabolizable organic carbon left over from the aerobic treatment. Generally, metabolizable organic carbon is added separately in a controlled manner to ensure that the denitrified effluent has a low BOD. Any suitable metabolizable organic carbon can be used such as methanol, acetate anion and the like.
[0264] A general understanding of this process can be facilitated by referring to Figures 4 and 5.
[0265] An apparatus 400 is a schematic representation of a municipal wastewater treatment plant using the processes of this invention. Municipal waste water enters apparatus 400 through line 402. For purposes of ease of understanding and not limitation of the invention, waste water contains about 50 parts per million by mass of ammonium cation and has a BOD of about 150 milligrams per liter. The waste water passes to the 404 centrifuge for solids separation. It should be understood that instead of the 404 centrifuge, a filter or settling ponds can be used. A thick slurry containing solids is drawn from the 404 centrifuge through line 406. A supernatant liquid passes from the 404 centrifuge through line 408 to a bioreactor 410.
[0266] The bioreactor 410 contains biocatalyst of this invention suitable for the catabolic conversion of organic carbon into carbon dioxide and ammonium cation into nitrate anion. For purposes of this discussion, the biocatalyst is substantially that of Example 148 which contains Rhodococcus sp. Air enters bioreactor 410 through line 412 and off-gas is extracted through line 414. The average hydraulic residence time in bioreactor 410 is sufficient to provide an oxidized effluent that has an ammonium cation concentration of less than 5 parts per million in mass and a BOD of less than 20 milligrams per liter. Bioreactor 410 is a downflow bed reactor for the purposes of this illustration. Because substantially no solids are generated, no solids separation unit operation is required. However, if desired, the oxidized effluent can be passed through a filter to remove at least a portion of any solids present.
[0267] A preferred bioreactor is illustrated in Figure 5 in which similar components have the same identification number as those in Figure 4. The supernatant liquor passes through line 408 to a bioreactor 410 on top of a bed of biocatalyst 502. The air is introduced into bioreactor 410 through line 412 and manifold 504 in a bottom portion of reactor 410. Air flows upward through the bed of biocatalyst. An off-gas is extracted through line 414 at the top of bioreactor 410 and an oxidized effluent is extracted through line 416 from the bottom of bioreactor 410 through line 416. Bioreactor 410 is revealed to have retention zone 506 below the bed of the biocatalyst . This holding zone serves to retain at least a portion of the solids, at least some of which are hydrolyzed to organics that can be further oxidized to carbon dioxide during subsequent processing of the effluent. The metabolic oxidation of organics can be carried out by the microorganisms used in the biocatalyst, for example, for nitrification, denitrification or phosphate removal. Sometimes, native microorganisms in the nitrification bioreactor and any subsequent reactor using the biocatalyst of this invention contribute less than about 5, often between about 1 and 3 or 4 percent of the observed bioactivity.
[0268] The oxidized effluent passes from a 410 bioreactor through line 416 to a 420 anaerobic bioreactor. The 420 anaerobic bioreactor serves to reduce the nitrate anion to nitrogen and operates under anaerobic conditions. Advantageously, anaerobic bioreactor 420 contains biocatalyst substantially as shown in Example 168 which contains microorganisms from Paracoccus denitrificans. The presence of some oxygen, for example up to about 2 or 4 parts per million by mass, can be tolerated in the oxidized effluent being treated in an anaerobic bioreactor 420. Since organic carbon is substantially reduced in the bioreactor 410, the carbon Additional organic such as acetate anion is added via the 418 line or to the oxidized effluent in the 416 line or the 420 anaerobic bioreactor. The 420 anaerobic bioreactor can be any suitable type of reactor. For the purposes of the discussion, it is a downward flow fixed bed bioreactor. The average hydraulic residence time is often less than about 1 hour.
[0269] Nitrogen and other gases exit the anaerobic bioreactor 420 through line 422. An additional advantage of using the biocatalyst is that relatively few of the sulfur compounds contained in the oxidized effluent are reduced to hydrogen sulfate or other sulfhydryl compounds. The 420 anaerobic bioreactor provides a denitrified effluent extracted through line 424. The effluent typically contains less than about 1 part per million by mass of nitrate anion. EXAMPLE 216
[0270] An apparatus containing a nitrifying bioreactor and a denitrifying bioreactor similar to that described in connection with Figure 4 is used for this example. The example is conducted with wastewater temperatures within the range of about 20°C to 25°C.
[0271] Effluent from a primary treatment at the municipal wastewater plant in Union City, California is used as the feed to the apparatus. Fresh effluent samples are typically taken daily both to ensure that fresh raw wastewater is used and to observe the effect, if any, of variations in the raw water composition being fed to a municipal wastewater facility. Changes in use, rainfall runoff and operation of the primary treatment can all have an effect. The primary effluent is kept in a holding tank that is aerated to control odor. The COD of the primary effluent treatment ranges from about 80 to 440 milligrams per liter and the BOD ranges from about 50 to more than 160 milligrams per liter. The ammonium cation concentration varies between about 25 and 55 milligrams per liter.
[0272] The primary effluent is fed to the aerobic nitrification bioreactor which contains biocatalyst substantially as shown in Example 148. During the first 17 days of operation, the primary effluent is fed to the bioreactor without filtration. After that, the effluent is filtered. The aerobic nitrification reactor is a downflow bioreactor with air being fed to the bottom which causes a suspension of the biocatalyst. A perforated plate is used to retain the biocatalyst bed and distribute the air in the bioreactor. The bioreactor influent has a dissolved oxygen concentration of about 5 to 8 milligrams per liter. Various hydraulic holding times are used ranging from about 3 hours to about 5 hours. The aerobic nitrification bioreactor has a bottom volume where solids are observed to settle. The effluent then passes to an anoxic fluidized bed bioreactor which contains the biocatalyst substantially as shown in Example 101. No unit operation is used to remove oxygen from the nitrification reactor effluent prior to introducing it to the denitrification reactor. The effluent from the denitrification bioreactor contains about 2 to 5 milligrams of oxygen per liter. The hydraulic residence time in the denitrification bioreactor is about 20 minutes.
[0273] After 3 days of operation, effluent analysis from the denitrification bioreactor begins and continues for approximately 8 weeks. After 3 days, BOD is less than 20 milligrams per liter and ammonia is about 1 milligram per liter at a hydraulic retention time in the nitrification bioreactor of about 5 hours. After about 14 days, the BOD remained at about 10 milligrams per liter regardless of whether the hydraulic retention time in the nitrification bioreactor was 3, 4, or 5 hours. The effluent ammonium cation concentration also remained relatively constant at 1 or less milligram per liter, except for a few days, but always below about 10 milligrams per liter. The total nitrate and nitrite in the denitrifying bioreactor effluent are typically below 10 and on most days below about 5 milligrams per liter, although an occasional excursion of up to about 15 milligrams per liter is observed. EXAMPLE 217
[0274] Substantially the same apparatus described in Example 216 is used for this example. The wastewater is obtained from the municipal wastewater plant in Union City, California and has a COD of about 350 to 400 milligrams per liter and BOD of about 130 to more than 160 milligrams per liter. The ammonium cation concentration ranges from about 40 to 55 milligrams per liter.
[0275] The aerobic bioreactor contains biocatalyst substantially as described in example 134 and is operated substantially as shown in the previous example. The average hydraulic residence time is about 3 hours in the aerobic bioreactor. The effluent from the aerobic bioreactor contains both nitrate and nitrite anion. The effluent then passes to the anaerobic bioreactor which contains biocatalyst substantially as described in Example 52. The concentration of dissolved oxygen in the aerobic bioreactor effluent is about 4 milligrams per liter. The average hydraulic residence time in the second bioreactor is about 24 minutes and the effluent contains a total nitrogen below about 10 milligrams per liter. II. PHOSPHATE REMOVAL
[0276] As mentioned above, phosphorus can lead to eutrophication. Thus, in some cases, government regulations have required the removal of phosphate from wastewater streams, often to below about 1 part per million by mass (ppm-m). Regardless of the presence of phosphorus in surface water, ironically phosphorus is a limited resource. Many of the commercial fertilizers that contain phosphorus derive that phosphorus from phosphorus rock mining and phosphorus rock reserves become depleted.
[0277] Numerous processes for water treatment to reduce phosphate concentration have been proposed such as chemical precipitation and biological treatment. A disclosed process for both removing phosphate from water and providing a phosphorus-containing fertilizer is disclosed by Britton in United States Patent Application Publication 2012/0031849. In the disclosed process, struvite is produced by adding magnesium to phosphate-containing water. Struvite is precipitated as pellets that can be used as a fertilizer or for other applications. In 2007, the US Environmental Protection Agency issued a "Biological Nutrient Removal Processes and Costs" report. This study considered both the chemical precipitation route and the biological treatment route to remove soluble phosphates which include the cost of installing and operating selected commercial units.
[0278] By this invention, biological processes are provided for the removal of soluble phosphate from water with the use of biocatalysts of this invention. These processes for the biological reduction of water-soluble phosphate comprise: a. contacting said water in a bioreactor with a biocatalyst of this invention that has substantially irreversibly retained in it phosphate accumulating microorganisms (PAOs) under phosphate accumulating conditions for a time sufficient to reduce the phosphate concentration in said water and provide a biocatalyst containing phosphate loaded microorganism wherein said phosphate accumulating conditions comprise the presence of polyhydroxyalkanoate (PHAs), especially poly-β-hydroxybutyrate within said microorganisms and the presence of aerobic or anoxic conditions in the water: b. subjecting said phosphate-laden microorganism-containing biocatalyst to anaerobic conditions in an aqueous medium sufficient to release phosphate from said microorganisms in said aqueous medium to provide an aqueous medium rich in phosphate; and c. separating said biocatalyst from the phosphate-rich aqueous medium for use and step (a).
[0279] Examples of phosphate accumulating microorganisms include, but are not limited to, Acintobacter spp., Actinobacteria, Candiidatus Accumulibacter phosphatates, α-Proteobacteria, β-Proteobacteria, and Y—Proteobacteria.
[0280] The processes of this invention treat water to remove soluble phosphate (soluble phosphate, as used herein, is intended to include monobasic phosphate, dibasic phosphate, tribasic phosphate and pyrophosphate anions). The water to be treated (herein referred to as "raw water stream") may be derived from any suitable source including, but not limited to, surface and ground water, municipal waste water, industrial waste water and generated water. by mining operations. Due to the robustness provided by the biocatalyst, the water to be treated may contain a number of components which include the presence of components that would, if the microorganisms were in a free suspension, adversely affect the removal of phosphate. The raw water feed can be subjected to unit operations to remove one or more components before being subjected to phosphate removal or it can be fed directly to the phosphate removal process.
[0281] Raw water feed often contains at least about 2, say at least about 4 milligrams of phosphate (calculated as PO4+3) per liter. Municipal wastewater often contains between about 4 and 20 milligrams of phosphate per liter; however, the processes of this invention can be used to treat raw water streams that contain high concentrations of phosphate, for example, 500 or more milligrams per liter.
[0282] The processes of this invention serve to reduce the concentration of soluble phosphate in the raw water feed to provide treated water. The reduction in soluble phosphate concentration is often at least about 50, preferably at least about 70 percent. In preferred aspects of this invention, the treated water contains less than about 1 and often less than about 0.1 milligram of phosphate per liter. Advantageously, low concentrations of phosphate in treated water can be achieved without the need to use a chemical precipitant. Treated water may be suitable for discharge, recycling or further processing. Since essentially no microbial debris slurry is generated, the treated water does not need to undergo post-treatment operations such as settling ponds.
[0283] Phosphate removal is carried out under oxidizing conditions. Oxidizing conditions can be provided by the supply of oxygen or an oxidizing component. Conveniently, oxygen is supplied by air or enriched air. Generally, the dissolved oxygen content of the raw water feed during phosphate removal is at least about 1, preferably at least about 2, say between about 2 and 20 milligrams per liter, oxygen can be supplied by any convenient means including bubbling or spraying oxygen-containing gas by water or agitation or other mechanical treatment for water to facilitate water-gas contact. Oxidizing components include, but are not limited to, nitrate, peroxide and percarbonate. Where such oxidizing components are used, the active oxygen concentration is preferably in the range of between about 1 and 10, more preferably between about 1 and 5 milligrams per liter.
[0284] As most phosphate accumulating microorganisms are mesophilic, Common Mesophilic Conditions can be used. The pH of the raw water stream being treated is preferably more basic than about 6 and is often in the range of between about 6 or 6.5 and 9.
[0285] The duration of contact between the raw water stream and the biocatalyst during the phosphate removal step is sufficient to provide the desired reduction of water soluble phosphate. The duration will thus depend on the concentration of soluble phosphate in the raw water stream, the desired reduction in phosphate concentration and the density of phosphate accumulating microorganisms in the bioreactor, as well as the conditions employed for phosphate removal. Due to the high concentration of phosphate accumulating microorganisms that can be provided by the use of the biocatalyst, a relatively low batch cycle or hydraulic retention times can be realized.
[0286] Phosphate accumulating microorganisms retain phosphorus in excess of the amount needed for biological processes in the form of polyphosphate within the cell. In some embodiments, concentrated phosphate-containing water has a phosphate concentration at least 10 times greater than that of raw water feed. In some cases, such concentrated water will have at least about 100, preferably at least about 500 milligrams of phosphate per liter. Typically higher concentrations of phosphate in concentrated water result in less energy being needed to obtain a product that contains solid phosphate.
[0287] A transition between the aerobic and anaerobic stages is required and can be effected in any suitable way. For example, the supply of oxygen or other oxidizing compound to the raw water being processed in the bioreactor can be terminated. Residual oxygen can be consumed in accumulating additional amounts of phosphate and then the water being treated can be displaced with the separate aqueous medium designed to accumulate the released phosphate.
[0288] In the processes of this invention, the release of phosphate is within a separate aqueous medium than in treated water. In preferred operations, phosphate release allows a concentrated phosphate-containing water to be obtained that is relatively free from the presence of other contaminants that may be contained in the raw water stream. Thus, concentrated water has enhanced utility in providing phosphate suitable for industrial or agricultural use. If desired, phosphate can be recovered from concentrated phosphate-containing water. Any suitable process can find application to perform such recovery. Unit operations to provide even more concentrated water include chemical precipitation, evaporation and reverse osmosis.
[0289] Phosphorus can be released by maintaining microorganisms under anaerobic conditions. Often, the dissolved oxygen concentration in water (or the oxidizing value of an oxidizing compound that is used instead of oxygen) is less than about 0.5, preferably less than about 0.2 milligrams per liter. It should be understood that some microenvironments within the biocatalyst may have higher or lower concentrations of oxygen. In fact, in a continuous operation it is possible to sequence between the aerobic phosphate removal stage and the anaerobic phosphate release stage so that only a portion of the microorganisms trapped in the biocatalyst are used to remove and release phosphorus. It is believed that the remainder of the microorganisms are not only available to accommodate changes in the raw water stream volume flow rate and phosphate concentration in the raw water stream, but also serve to transfer oxygen and phosphate within the biocatalyst.
[0290] As the temperature, pressure, and pH conditions for phosphate release can be the same as those for phosphate recovery from raw water, there is often no need to purposefully induce a change. Normally, it is not necessary to add nutrients, which include micronutrients, to the separate aqueous medium.
[0291] For some microorganisms, the kinetic rate of phosphate release from microorganisms is faster than for phosphate accumulation. Preferably, the phosphate release stage is operated to provide a high concentration of phosphorus in the concentrated phosphate containing product. If desired, the phosphate release stage can be operated in two or more steps, the first being to provide the maximum concentration of phosphate in the aqueous medium and the later steps to provide the reduction of phosphate contained in the microorganisms, despite providing a product which contains concentrated phosphate which has a lower concentration of phosphate than that of the phosphate removal stage.
[0292] During phosphate accumulation, it is believed that PHA and oxygen or oxidizing compounds are bioconverted to carbon dioxide and water. In general, between about 2 and 20, preferably between about 4 and 10, PHA carbon atoms are bioconverted per soluble phosphate phosphorus atom accumulated in the microorganism. PHA is formed by phosphate accumulating microorganisms under anaerobic conditions in the presence of a carbon-containing substrate. Volatile fatty acids of 2 to 5 carbon atoms have generally been preferred as the carbon-containing substrate. However, with the use of the biocatalysts of this invention, more complex carbon sources such as sugars and acetic acid can be used to generate the PHA.
[0293] The processes of this aspect of the invention, by retaining the phosphate accumulating microorganisms in the biocatalyst, provide significant flexibility of when PHA production occurs. For example, the carbon-containing substrate can be provided for at least a portion of the duration of the phosphate release or a separate anaerobic stage can be specifically employed for the production of PHA. In the later case, the microenvironments within the biocatalyst and the metabolic state of the microorganisms allow the microorganisms to remain viable for the duration of the phosphate release stage. This later case is beneficial where the product containing concentrated phosphate is desired to have an essential absence of added organic compounds.
[0294] The kinetic rate for the formation of PHA depends, in part, on the substrate that contains carbon used and the concentration of the substrate. In most cases, the biological reaction rate for PHA is faster than that for phosphate accumulation. Additionally, the biocatalyst may allow retention of carbon-containing substrate beyond the duration of the PHA generation stage and more occluded microorganisms in the biocatalyst may have a sufficient absence of oxygen and a sufficient presence of carbon-containing substrate to generate additional PHA.
[0295] Processes can be conducted on a batch, semi-continuous and continuous basis and are preferably conducted on a continuous basis. The bioreactor can be in any suitable configuration that includes Common Bioreactor Systems. A bioreactor can be employed and the batch cycled through all stages of phosphate removal and deliberation. For most business operations, operating on an ongoing basis is preferred. The biocatalyst can be moved from one bioreactor to another, with each of the bioreactors being adapted to perform a different function, for example, a bioreactor for removing phosphate from raw water; a bioreactor for phosphate release; and, optionally, a separate bioreactor for PHA generation. In this way, countercurrent biocatalyst and water flows can occur in each bioreactor to facilitate the removal of soluble phosphate from the raw water stream and maximize the concentration of phosphate in the phosphate-containing product. Another approach is to cycle each bioreactor containing biocatalyst from, for example, a phosphate removal stage to a phosphate release stage, to a PHA generation stage. Combinations of these two approaches can be used. For example, a bioreactor can be used to remove phosphate from the raw water stream and the phosphate remover stage is then stopped with the biocatalyst being then supplied to a countercurrent flow bioreactor operating under anaerobic conditions to deliver a product which contains highly concentrated phosphate. The biocatalyst is then supplied to another bioreactor with a first operation under PHA forming conditions and is then transitioned to phosphate removal conditions.
[0296] A general understanding of the invention and its application can be facilitated by referring to Figures 6, 7 and 8.
[0297] Figure 6 is a schematic representation of a type of apparatus generally designated as 600 suitable for practicing the processes of this invention. As shown, the apparatus comprises 5 sets of bioreactors A, B, C, D and E. Each set will be described in further detail in relation to Figure 7.
[0298] For the purposes of the discussion, bioreactors are fluidized bed reactors, although it can be readily recognized that other types of bioreactors, such as fixed bed and filled bed, can be used. As shown, the apparatus is a number of fluid transport leads which may comprise one or more fluid lead lines. The water flow conductor assembly is indicated by element 602 and provides transport of raw water to the bioreactor assemblies, as well as water between the bioreactor assemblies and water being recycled within a bioreactor assembly. Line 604 is adapted to supply carbon source, if necessary, to conductor assembly 602. Air conductor 606 is adapted to supply air or other oxygen-containing gas to each of the bioreactor assemblies. Drain conductor 608 is adapted to transport training water from one bioreactor assembly to another. Conductor assembly 610 is adapted to provide fluid communication within a bioreactor assembly, from one bioreactor assembly to another bioreactor assembly, and for the removal of a stream containing concentrated phosphate. Conductor assembly 612 is adapted to extract treated water from the apparatus. Conductor assembly 614 is adapted to escape gases from the apparatus. In Figure 6, circular elements generally indicate valve assemblies. Valve assemblies and operation will be discussed further in connection with Figure 7.
[0299] Figure 7 is a more detailed representation of the bioreactor assemblies of Figure 6. As shown, the bioreactor assemblies comprise a 702 bioreactor. The 702 bioreactor contains biocatalyst comprising Candidatus Accumulibacter phosphatis. Line 704 is adapted to direct aqueous streams to bioreactor 702. As will be discussed below, the aqueous stream may be a raw water stream, a stream from another bioreactor, or a recycle stream. Line 706 delivers oxygen-containing gas, typically air, to the bioreactor. Valve 708 controls the flow there and manifold 710 serves to distribute the oxygen-containing gas in bioreactor 702. Line 712 is provided at the bottom of bioreactor 702 for the purge or drain purposes of bioreactor 702. Valve 714 controls flow of water through line 712.
[0300] At the top of bioreactor 702 line 720 is provided for the purposes of extracting gases such as waste gas containing oxygen supplied through line 706 and carbon dioxide resulting from metabolic activity. Valve 722 is supplied in line 720 and is adapted to control the flow of gases through line 720. These extract gases are normally discharged to atmosphere; however, they can undergo a treatment to insert or remove components such as methane. As shown, in an upper portion of bioreactor 702, line 716 is provided to extract treated water from the bioreactor. As will be discussed below, this treated water line is used to remove water from a bioreactor operating in polishing mode or if no bioreactor operates in polishing mode then from a bioreactor operating in primary phosphate removal mode. Valve 718 controls the flow of water through line 716. In addition, line 724 is provided in an upper portion of bioreactor 702 for removing water for recycling or transport to another bioreactor. Both lines 716 and 724 are provided with screens or other devices to prevent biocatalyst contained in bioreactor 702 from passing on these lines. Line 724 is provided with valve 726 which is adapted to control the flow of water from bioreactor 702 in line 724. Line 724 is also provided with diverter valve 728 which is adapted to direct water in line 730 which carries an aqueous stream which contains concentrated phosphate for removal and/or to line 732. Line 732 contains valve 734 which is adapted to recycle water through line 738 and line 704 to bioreactor 702 or to pass water in line 736 for passage to another bioreactor.
[0301] As shown, the 704 line can also provide other aqueous streams. These streams may include raw water supplied through line 740 and water from another bioreactor through line 744. Valve 742 is provided to control the relative volumes of a quantity of these streams as will be discussed below. The 746 line is adapted to supply carbon source, if necessary, to a 702 bioreactor. The 748 valve controls the flow from the carbon source to the 702 bioreactor.
[0302] Only for the purposes of illustration and not limitation of the invention, the 5 sets of bioreactors depicted in Figure 6 are adapted to be sequenced by various modes of operation. One skilled in the art can immediately recognize that a small or large number of bioreactor sets can be used and the sequencing altered.
[0303] The following summarizes the five modes of operation used for the purposes of this illustration: Anaerobic PHA Generation Mode — in this mode, the bioreactor is operated under anaerobic conditions that include the presence of a carbon source that may be a carbon source added to or contained in the raw water to be treated; Primary Aerobic PO4 Removal Mode — in this mode, oxygen-containing gas passes through the bioreactor to effect phosphate removal from the water; Polishing Aerobic PO4 Removal Mode — in this mode, water treated by another bioreactor operating in the Primary Aerobic PO4 Removal Mode is additionally subjected to contact with biocatalyst in the bioreactor to remove additional phosphate from the water; Purge Mode — in this mode, the bioreactor transitions from an aerobic or anoxic environment to an anaerobic environment to release phosphate from the biocatalyst; and Anaerobic PO4 Release Mode — in this mode, the bioreactor is operated under an anaerobic environment to release phosphate from the biocatalyst to provide a phosphate-rich effluent stream.
[0304] The following discussion describes the operation of the apparatus of Figures 6 and 7 using the sequencing outlined in Figure 8. As can be seen in Figure 8, each of the bioreactor sets, A, B, C, D and E sequence through the same modes. This discussion will therefore refer to the operation of a single bioreactor set with the understanding that the discussion will be equally applicable to each of the other bioreactor sets. Each of the operating modes has the same duration of time for the purposes of this illustration.
[0305] The discussion begins with the operation of a bioreactor in Anaerobic PHA Generation Mode which is a reactor that has been, in the immediately preceding period of time, used in Anaerobic PO4 Release Mode. Thus, at the beginning of the cycle, the bioreactor contains an aqueous medium that, although anaerobic, is rich in phosphate. At the beginning of this cycle, valve 742 and valve 714 are closed. First, valve 714 is opened to prevent the aqueous medium in bioreactor 702 from passing through line 712 and then being directed to phosphate recovery. During the drain, the raw water feed to the appliance can be terminated or the raw water feed can be routed to a bioreactor operating in Primary Aerobic PO4 Removal Mode. Alternatively, the apparatus can be fitted with pressure tanks. Any suitable fluid can be used to replace the volume of bioreactor 702 that occurs from the drain. Often an air is adequate even though the bioreactor is desired to be operated and be aerobically in this mode. Alternatively, displacement of the phosphate rich medium in bioreactor 702 at the beginning in this way can be accomplished by passing raw water through line 704 in bioreactor 702 while extracting the phosphate rich aqueous medium from the top of the bioreactor through line 724.
[0306] Once bioreactor 702 is drained, valve 714 is closed and valve 742 is positioned to allow a raw water feed stream containing a soluble phosphate to pass from line 740 to line 704 for introduction into bioreactor 702 The raw water feed stream refluidizes the biocatalyst. Valve 722 and valve 718 remain closed. Bioreactor 702 is operated under metabolic conditions that include anaerobic or anoxic conditions such that the biocatalyst bioconverts the carbon source in the raw water feed to PHA. The carbon source can be contained in the raw water feed stream. If necessary, a carbon source can be supplied during this mode through line 746 to the desired concentration by setting valve 748. The raw water feed stream, after passing through the fluidized bed of the biocatalyst in bioreactor 702, exits through the line 724 and passes through valve 728 to line 732. Valve 734 directs water through line 736 to a bioreactor operating in Primary Aerobic PO4 Removal Mode. If necessary, a portion of this water can be directed through valve 734 through line 738 back to bioreactor 702 to maintain a desired degree of fluidization of the biocatalyst.
[0307] Near the end of Anaerobic PHA Generation Mode, in bioreactor 702, the transition to Aerobic Polishing PO4 Removal Mode begins. This transition comprises initiating the flow of oxygen-containing gas in bioreactor 702 through line 706 by opening valve 708. At this time, valve 722 is opened to allow gases to exit bioreactor 702 through line 720. bioreactor 702 remains unchanged during this transition and switches to a bioreactor operating in Primary Aerobic PO4 Removal Mode. This transition allows the bioreactor to serve as the bioreactor operating in Aerobic Polishing PO4 Removal Mode for the next period.
[0308] At the conclusion of Period 1, valve 742 terminates the raw feedwater flow and begins an effluent flow from the bioreactor operating in Line 744 Primary Aerobic PO4 Removal Mode. In addition, valve 726 is closed and valve 718 are opened to allow reduced phosphate water to pass from the apparatus through line 716. However, if a recycle stream is desired to provide sufficient flow to fluidize the biocatalyst in bioreactor 702, valve 726, valve 728 and the valve 734 can be set to supply the sought flow rate of water back to the bioreactor 702. Since the bioreactor has been transitioned earlier 702 into an aerobic environment, the microorganisms have an enhanced PHA content and phosphorus content. reduced and can thus effectively remove water soluble phosphate being treated to desirably low concentrations. In addition, the use of a bioreactor in the Aerobic Polishing PO4 Removal Mode allows for fluctuations in the concentration of soluble phosphate in, as well as fluctuations in the flow rate of the raw water feed to be accommodated while still supplying the desired flow of concentration of phosphate in treated water.
[0309] In general, no transition is required to cycle an operating bioreactor from Polishing Aerobic PO4 Removal Mode to Primary Aerobic PO4 Removal Mode. Bioreactor 702, at the conclusion of Period 2, begins operation in Primary Aerobic PO4 Removal Mode by changing the water source and line 744 from another bioreactor operating in Primary Aerobic PO4 Removal Mode to a bioreactor effluent which operates in Anaerobic PHA Generation Mode. Valve 718 is closed and valve 726 is opened allowing aqueous medium to be directed through line 724 to valve 728 and to line 732. Valve 734 directs effluent through line 736 to a bioreactor operating in the Removal of Aerobic Polishing PO4. A portion of the effluent may be directed through valve 734 to line 738 for recycling to a bioreactor 702 to provide the desired fluidization of the biocatalyst.
[0310] Either before the end of Period 3 in which the 702 bioreactor operates in Primary Aerobic PO4 Removal Mode or at the beginning of Period 4 in which the 702 bioreactor will operate in the Purge Mode, valve 708 is closed to cease the oxygen-containing gas stream in bioreactor 702. Sufficient residual oxygen remains in the aqueous medium in bioreactor 702 and biocatalyst to allow for absorption of additional dissolved phosphate. The effluent from bioreactor 702 can continue to be directed to a bioreactor operating in Aerobic Polishing PO4 Removal Mode or to a bioreactor operating in Primary Aerobic PO4 Removal Mode (line 736 would thus be in fluid communication with line 744 from another bioreactor operating in Primary Aerobic PO4 Removal Mode). Typically, in Purge Mode, the aqueous medium in bioreactor 702 is continuously recycled by positioning valves 726, 728, and 734. If desired, all or a portion of the aqueous medium contained in bioreactor 702, which medium contains dissolved oxygen, can be drained through line 712. The drained aqueous medium can pass to another bioreactor operating in either the Primary Aerobic PO4 Removal Mode or the Polishing Aerobic PO4 Removal Mode. In Purge Mode, the process for releasing phosphorus retained by microorganisms begins. Purge Mode also facilitates dissipation of oxygen concentration gradients within the biocatalyst.
[0311] In Period 5, bioreactor 702 is operated in Anaerobic PO4 Release Mode. In this mode, the phosphate-filled water in bioreactor 702 is recycled through lines 724, 732, 738, and 704 with a portion of the water being routed through valve 728 to line 730. Combining the use of a Purge Mode with the Anaerobic PO4 Release Mode tends to provide the highest possible concentration of phosphate in tap water drawn through line 730. In an alternative embodiment, phosphate filled water in bioreactor 702 can be drained through line 712. Phosphate filled water can be disposed or subjected to chemical treatment or further processed to recover phosphate. For example, struvite can be formed and the phase separated with the return of water to the apparatus. Alternatively, phosphate-filled water can be subjected to evaporation, distillation or reverse osmosis to provide a more concentrated phosphate-containing stream that can find industrial or agricultural use.
[0312] Although the illustration has depicted the Purge Mode and the Anaerobic PO4 Release Mode occurring in separate periods, the modes can be combined into a single period. III. MITIGATION OF BIODANIFICATION OF AQUATIC MICROORGANISMS
[0313] Water obtained from sources that contain aquatic organisms, especially macro-organisms such as cirripedia (such as barnacles and barnacles); marine mussels; freshwater mussels; zebra mussels; bryozoans; tubular worm; polychaetes, tunicates, sponges; and sea anemones is used for numerous purposes. For example, water can be sought to be used as potable water, a water source for desalination, cooling water such as for power plants and manufacturing facilities, for sanitary facilities and ballast for ships. Water sources that contain the macro-organisms can cause biodamage to surfaces such as pipes, tanks and process equipment such as pumps, valves, heat exchangers, filtering devices, reactors and the like. Periodic maintenance is required to remove deposits or replace damaged equipment. Removal of deposits from these macro-organisms can be problematic due to the strength of the adhesion of these organisms to the surface and the hardness of the shell bodies.
[0314] According to this aspect of the invention, water that contains or can make contact with aquatic macro-organisms is first placed in contact with the biocatalysts of this invention that contain microorganisms that can do catabolic conversion of dissolved metabolizable organic carbon (organo-carbon) in the water. The microorganism selected must be tolerant of other raw water components including, but not limited to, salinity, other anions and cations, any organics or pollutants present, and pH. Examples of microorganisms that can convert organocarbon to carbon dioxide include, but are not limited to, Acinetobacter Johnsonii, Alcanivorax dieselolie, Azoarcus sp, Bacillus globiformis, Bacillus mojavensis, Bacillus subtilis, Escherichia coli, Eubacterium biform, Lactosphaera pasteurii. Microthirx parvicella, Moraxella cuniculi, Nocardia asteroids, Pseudomonas pseudoalcaligenes, Rhococccus rhodnii, Rhodcoccus coprophilus, Rhodoferax fermentans, Rhodococcus jostii, Saccharophagus degradans, Skermania piniformis, Sphingomonas capsulate, Zooglovora paradoxus.
[0315] Contact is for a time sufficient to reduce the concentration of metabolizable organo-carbon to a level where the survival of macro-organisms is inhibited. A water supply can be continuously contacted with a biocatalyst; however, intermittent or periodic water treatment may be sufficient to interrupt macroorganism growth downstream of the biocatalyst.
[0316] Consequently, biodamage by aquatic macro-organisms can be obtained without the addition of chemicals. Furthermore, biocatalysts do not alone generate food sources for the macro-organisms, they do not increase the mass of solids to be removed by any downstream filtration. In preferred aspects of the invention, the concentration of organo-carbon downstream from contact with the biocatalyst is insufficient to maintain the viability of suspended microorganisms.
[0317] Water is often, but not necessarily always, obtained from surface sources and perhaps salt, brackish or fresh water. Water contains food and nutrients to support aquatic macro-organisms and usually contains micro-organisms.
[0318] Conditions for water and biocatalyst contact can vary over a wide range and are typically under Common Mesophilic Conditions. Typically, the contact temperature is substantially the ambient temperature of the water. In some cases, the oxygen dissolved in water is sufficient for the metabolic bioconversion of the organo-carbon to carbon dioxide; however, water aeration may be desired in some cases. Generally, the dissolved oxygen content in the water to be brought into contact with biocatalysts is in the range of about 1 to 50 or more, say 1 to 10 parts per million by mass. Normally, no nutrients need to be added to water.
[0319] The bioreactor can be in any suitable configuration that includes Common Bioreactor Systems. With the high cell densities and bioactivities obtainable using the biocatalysts of this invention, the average hydraulic residence time of water in bioreactors is usually less than about 24, more often less than about 6 or 10 hours and, in some cases, it can be in the range of about 0.5 to 4 hours.
[0320] Since the biocatalyst provides environments in which microorganisms can survive for extended periods of time without the addition of additional food sources, the biocatalyst can be cycled between environments that contain organo-carbon ("metabolization cycle") and environments that do not contain essentially organo-carbon ("cleaning cycle"). This cycling often retards any growth of organisms on the surface of the biocatalyst. In general, the duration of the cleaning cycle, if used, is at least about 2, say between about 6 and 48 hours.
[0321] Referring to Figure 9, the apparatus 900 is a set for desalination of seawater that uses reverse osmosis membranes. The seawater passes into a filled space 902. The arrows indicate the flow of sea water in the filled space 902. The filled space 902 as a plurality of screens, two of which are illustrated, screen 904a and screen 904b. A movable flap 905 is provided in the filled space 902 and is adapted to stop the flow of water to the screen 904a and then moves as indicated by the dotted line to stop a flow of water to the screen 904b. It should be understood that movable flap 905 can be positioned so that water can flow to both screen 904a and 906b.
[0322] Each screen has a dedicated conductor which for screen 904a is conductor 906a and for screen 904b it is conductor 906b. Each conductor has lines that go to each bioreactor. For the purposes of this illustration, two bioreactors are shown, the 912 bioreactor and the 914 bioreactor. The 908a line provides fluid communication between the 906a conductor and the 912 bioreactor and the 908b line provides fluid communication between the 906b conductor and the 912 bioreactor. line 910a provides fluid communication between conductor 906a and bioreactor 914 and line 910b provides fluid communication between conductor 906b and bioreactor 914.
[0323] Each bioreactor 912 and 914 is represented as fluid bed bioreactors containing biocatalyst which, for the purposes of discussion, are the biocatalyst of example 113. Bioreactor 912 is shown as having effluent lines 916 and 912 and bioreactor 914 is shown as having effluent lines 918 and 924. Effluent lines 916 and 918 are in fluid communication with the recycling conductor 920. The effluent lines 922 and 924 are in fluid communication with a treated water conductor 926. The conductor of treated water 926 directs treated water to ultrafiltration membrane unit 928. Filtrate from ultrafiltration membrane unit 928 passes through line 930 to reverse osmosis unit 932. Desalinated water exits through line 934 and a reject stream exits through line 936 of reverse osmosis unit 932.
[0324] Returning to the recycling conduit 920, the water passes to the pressure tank 938. The water from the pressure tank 938 can be routed through the line 942 of the filled space 902. The water from the line 942 enters the filled space 902 in the region occluded by the movable flap 905 and upstream of the occluded screen.
[0325] There are several modes of operation of the apparatus, all within the broad aspects of this invention. In one mode, bioreactors 912 and 914 operate independently, and in another mode, bioreactors 912 and 914 operate in the water flow sequence.
[0326] As an example, in a first mode of operation, raw water enters the filled space 902 and is directed by screen 904b to conductor 906b. Line 908b is valve closed and the water in conductor 906b passes through line 910b to bioreactor 914. In bioreactor 914, the organo-carbon is converted to carbon dioxide to provide a stream of treated water that contains essentially no organo-carbons. carbon. This treated water stream exits through line 924 and passes to treated water conduit 926 where it finally passes through the reverse osmosis unit to provide a desalinated water. At this point in time, line 918 is valve-closed.
[0327] The pressure tank 938 which was previously filled with treated water that contains essentially no organo-carbon supplies water through line 940 in the occluded region of filled space 902 defined by movable flap 905 and screen 904a. The water then passes through the screen 904a on conductor 906a. Line 910a is valved so that water does not enter bioreactor 914, but line 908a is valved so that treated water passes through bioreactor 912. The water then exits bioreactor 912 through line 916 to be returned by recycle conduit 920 to pressure tank 938. At that point in time, line 922 is valve-closed. As can be seen, the cycling of treated water by screen 904a, conductor and line 906a and 908a and bioreactor 912 serves to provide a cleaning cycle. The use of movable flap 905 allows treated water to contact the flap to similarly dampen the growth of macro-organisms on the flap.
[0328] After completion of the cleaning cycle for screen 904a, conductor 906a and bioreactor 912, the movable flap 905 is moved to allow raw water flow through screen 904a and occlude the raw water flow to screen 904b. The same valve positions are maintained for the 906a and 906b conductors which result in a 912 bioreactor that treats raw water. Effluent line 916 is valve closed and effluent line 922 is valve opened to pass treated water to treated water conduit 926. Bioreactor 914 is subjected to a cleaning cycle as is screen 904b, the conduit 906b and line 910b. Effluent line 924 from bioreactor 914 is valve closed and effluent line 918 is valve opened and passes water to recycle conduit 920. Pressure tank water 938 passes through line 940 to the defined occluded region by movable flap 905 and screen 904b in full space. The side of the filled space that was exposed to raw water during the previous cycle is now exposed to water that has an essential absence of organocarbon. Thus, the apparatus facilitates keeping both sides of the movable flap 905 relatively free from macro-organisms.
[0329] In the next cycle, line 908a is valve-closed, line 908b is valve-opened, and bioreactor 912 is subjected to a cleaning cycle. At the same time, line 910a is valve opened, line 910b is valve closed, and bioreactor 914 serves to treat raw water to remove organo-carbon. In this cycle, effluent line 918 is closed by valve and effluent line 924 is opened by valve and passes treated water to treated water conduit 926. In addition, effluent line 922 of bioreactor 912 is closed by valve and water passes through line 916 to recycling conductor 920.
[0330] In the last of the four cycles, the movable flap 905 is moved to occlude the flow of raw water to the screen 904a and allow the flow of raw water to the screen 904b. Thus, the bioreactor 914 treats raw water and the effluent line 918 is closed by a valve and the effluent line 924 is opened by a valve to direct the treated water to the treated water conduit 926. The bioreactor 912 is subjected to a cycle of cleaning with a valve-open effluent line 916 and a valve-closed effluent line 922. Line 940 directs water from pressure tank 938 to the occluded region defined by movable flap 905 and screen 904a.
[0331] The apparatus depicted in Figure 9 can also be used in a sequential bed mode. In a first cycle, the movable flap 905 provides an occluded region upstream of the screen 904a in a filled space 902. Raw water entering the filled space 902 passes through the screen 904b and conductor 906b. Line 908b is valve-closed and water passes through line 910b to bioreactor 914 for treatment to remove organo-carbon. Treated water passes through line 918 to recycle conductor line 920. Line 924 is valve-closed. Thus, pressure tank 938 receives treated water which then passes through line 940 to the occluded region in front of screen 904a. Since treated water has substantially no organocarbons, the water is useful for a cleaning cycle. This water enters conductor 906a and passes to bioreactor 912 through line 908a which is opened by valve. Line 910a is valve-closed. Any residual or additional organo-carbon is subjected to a biocatalyst in the 912 bioreactor for further bioconversion to carbon dioxide. The water from bioreactor 912 passes through line 918 to treated water conduit 926. Effluent line 916 is closed by valve.
[0332] In a manner consistent with the description of the first mode of operation of the apparatus, the movable flap positioning and the valves for each of the bioreactors can be cycled so that all lines and reactors go through a cleaning cycle. IV. AMMONIUM ION AEROBIC CATABOLIS FOR NITROGEN
[0333] As discussed above, biological processes for removing ammonium cation from aqueous streams oxidize ammonium cation normally conducted in an aerobic environment. The oxidation effluent contains nitrate anions and possibly nitrite anions, especially where oxidation is not complete. More than 4 kilograms of oxygen is often consumed per kilogram of ammonium nitrogen removed, and the nitrification and denitrification processes increase power consumption for a typical installation by 30 percent or more.
[0334] The nitrate and the resulting nitrite ions are also contaminants and are preferably removed from the water before discharge to the environment. Bioconversion processes for denitrification are also well known. Typically, the reduction of these oxyanions to nitrogen requires an anoxic or anaerobic, electron-donating environment. So some facilities add a donor like methanol or even raw sewage. The need for fundamentally different conditions for ammonium oxidation and nitrate reduction contributes to the capital and operating expenses of adopting a system to bioconvert ammonium to nitrogen. Anaerobic conditions for nitrate reduction can also lead to the production of hydrogen sulfate and other sulfhydryl compounds.
[0335] Also discussed above is the anammox process.
[0336] By this aspect of the invention, the biocatalysts of this invention allow ammonium cation to be bioconverted in an aerobic environment to nitrogen with the use of microorganisms contained in activated slurry (herein referred to as an "N/D microorganism") . Since the biocatalyst of this invention is used, no solids are generated by the microorganisms in the biocatalyst. In broad respects, the contact between water containing ammonium cation and the biocatalyst is under metabolic conditions long enough to provide a treated water that has an ammonium concentration less than about 50, preferably less than about 90 percent of that. wherein the feedwater nitrate concentration is a nitrate ion concentration less than about 1 milligram per liter. Additionally, this reduction of nitrate and nitrite anions in treated water can occur even in the presence of significant amounts of oxygen, for example, greater than 5 or 8 milligrams of oxygen per liter in water. In preferred embodiments of this aspect of the invention, treated water that contains enough water so that it does not need to be aerated for discharge to the environment, for example, contains at least about 0.5, preferably at least about 1 milligram of oxygen per liter . Also, no hydrogen sulfate or other sulfhydryl compounds are generated by the process.
[0337] Water can be from any source that includes municipal wastewater, groundwater and surface water. The ammonium cation content of the water to be treated can also vary over a wide range and is often between about 5 or 10 and 250, more often between about 25 and 200 milligrams per liter. The water to be treated may contain other components including, but not limited to, sulfur compounds, phosphorus compounds, inorganic salts and solubilized metals. Often, the oxygen concentration in the water to be treated is in the range of about 0.5 to 10 or more milligrams per liter.
[0338] The biocatalyst of this invention used in these processes contains N/D microorganisms, suitable N/D microorganisms may or may not exhibit both nitrification and denitrification metabolic activities when they are in a free suspension in an aqueous medium. While not wishing to be bound by theory, it is believed that a phenotypic change occurs in some cases that contributes to the performance of N/D microorganisms. N/D microorganisms can be obtained from activated slurry. Preferably, the activated slurry is acclimated under aerobic conditions under Common Mesophilic Conditions and fed with bicarbonate anion. In some cases, the pH is kept between about 6 and 8.
[0339] Ammonium biodegradation processes can be conducted in any suitable manner. Processes can be in a continuous, semi-continuous or batch mode of operation and use Common Bioreactor Systems.
[0340]Any suitable metabolic conditions can be used which include Common Mesophilic Conditions. In general, the pH is maintained between about 4 and 8.5, for example between 6.0 and 8.0. Buffers, if desired, can be used to keep the water at a given pH value during the process. The carbon source nutrient may be required and may be any convenient carbon source such as a low molecular weight hydrocarbon or oxygenated hydrocarbon such as ethanol, acetate and sugars.
[0341]The duration of contact of the water and the biocatalyst is long enough to obtain the desired reduction in ammonium. Duration can vary over a wide range depending on the type of reactor, the biocatalyst and the concentration of the microorganism population in the bioreactor. In many cases the duration of contact may be less than 12, preferably less than 8 hours to achieve a reduction in ammonium concentration to less than about 1 milligram per liter and sometimes the contact is less than an hour. A significant advantage of the processes of this invention is that not only is the ammonium converted to nitrogen, but the treated water contains little, if any, nitrate or nitrite anion. EXAMPLE 218
[0342] A continuously stirred aerated tank bioreactor is filled to about 70 percent of its height with a biocatalyst substantially as described in example 92, but using microorganisms derived from acclimatized activated slurry as shown above and at a wet density of cell of about 350 to 400 grams per liter. The biocatalyst is in the form of spheres that have diameters of about 4 millimeters. Effluent from a primary treatment at the municipal wastewater plant in Union City, California is used as feed to the bioreactor. A series of batch tests are conducted in the bioreactor, each using the effluent with different concentrations of ammonium cation: 100, 200 and 1000 milligrams of ammonium cation per liter. Ammonium cation concentrations are adjusted by adding ammonium hydroxide. The pH is adjusted to about 7 at the start of each batch. Each test is conducted until the ammonium cation concentration is below about 0.1 milligram per liter. At the conclusion of each batch, the residual water is analyzed for nitrite and nitrate anion. The total nitrogen in wastewater is below about 1 milligram per liter. V. REMOVAL OF NITRATE AND PERCHLORATE FROM WATER
[0343] The biocatalysts of this invention can be used to remove nitrate and remove perchlorate anion and both when present together from water. Nitrates are a contaminant in water and the US Environmental Protection Agency has set a limit of 10 milligrams of nitrate (based on the mass of nitrogen) in drinking water. Perchlorate anion contamination of a number of groundwater and surface water sources has occurred. The concentration of perchlorate anion in these contaminated waters can vary widely. A health concern that arises from the presence of perchlorate is its interference with the thyroid gland's ability to produce hormones, which in turn can cause problems with metabolism, growth and development. Due to concerns about the adverse effects of perchlorate, efforts are made to reduce perchlorate levels to concentrations down to the very few micrograms per liter. Water that is contaminated with perchlorate anion often contains nitrate anion. The presence of nitrate in perchlorate contaminated water presents a challenge to a metabolic process in which not only the nitrate is preferentially reduced, but the concentration of perchlorate must also be reduced to very low levels.
[0344] In broad aspects, the processes for reducing the concentration of nitrate anion or perchlorate anion or both when present in water comprises placing the water in contact with a biocatalyst of this invention that contains a strain of microorganism capable of reducing said anions under metabolic conditions and for a sufficient time to bioconvert such anion. Nitrate and perchlorate reducing microorganisms, especially bacteria, are readily obtainable from the environment, and some previous workers have described self-inoculation systems for the biodegradation of these anions. See, for example, Published Patent Application no. US 2010/0089825. Representatives of the species of bacteria that are available in nature, especially from streams and wastewater, include Vibrio dechloraticans, Cuznesove B-1168, Wolinella succinogenes, Acinetobacter thermotoleranticus, Ideonella dechloratas and GR-1, a strain identified as belonging to the β subgroup of Proteobacteria. See, for example, Coates, et al, Nature Rev. Microbiol., 2, pages 569 to 80 (2004) and Applied Enviromental Microbiol., 65, pages 5234 to 41 (1999); and Wu, et al, Bioremediation Journal, 5, pages 119 to 30 (2001) for discussions of microorganisms capable of perchlorate respiration. It is understood that the microorganisms used can be wild strains or can be genetically modified recombinant microorganisms.
[0345] The concentration of nitrate in water can vary widely depending on the source and is often in the range of around 0.5, say 1 to 100 or more milligrams per liter. In mining and aquaculture operations, wastewater can often contain 500 or more milligrams of nitrate per liter and reducing such high concentrations of nitrate anion to levels suitable for discharge has been problematic. The concentration of perchlorate in water can vary widely depending on the source. In some reported cases, perchlorate concentrations greater than 10 milligrams per liter have been observed. However, in view of the concerns that arise from perchlorate contamination, it may be desirable to treat water that contains very low concentrations of perchlorate, for example, as low as about 10 micrograms per liter. The water to be treated may contain other components including, but not limited to, sulfur compounds, phosphorus compounds, inorganic salts and solubilized metals. Often, the oxygen concentration in the water to be treated is in the range of about 0.5 to 10 or more milligrams per liter.
[0346] The biocatalyst can contain any suitable microorganism. Biodegradation processes can be carried out in any suitable way. Processes can be in a continuous, semi-continuous or batch mode of operation using suitable bioreactors that include Common Bioreactor Systems.
[0347] Adequate metabolic conditions are maintained as Common Mesophilic Conditions. The pH of the water to be treated will depend on its source. In general, the pH is maintained between about 4 and 8.5, for example between 4 and 8.0. A lower pH tends to intensify the degradation of the perchlorate anion.
[0348] If necessary, electron donors can be added to the water to be treated. Electron donors include, but are not limited to, hydrogen, carbohydrates, hydrocarbons, alkanols, aldehydes, carboxylic acids, ketones, aldehydes, glycerides, and the like. See, for example, paragraph 0055 of Published Patent Application no. US 2006/0263869. If electron donors are needed, they can be added in any suitable way. The addition of electron donors is usually based on achieving the desired reduction in the perchlorate rather than a full electron acceptor in the water to be treated. Consequently, where an electron donor has to be provided, the processes of this invention require less electron donor than those where oxygen is preferentially consumed prior to any significant biodegradation of the perchlorate anion.
[0349] The duration of contact of water with polymeric matrices is for a time sufficient to obtain the desired reduction in nitrate and perchlorate anion. Duration can vary over a wide range. As mentioned above, even though the perchlorate anion concentration can be very low, for example less than 100 micrograms per liter and oxygen is present, the duration of contact can be relatively brief, even to obtain a treated water that contains less than 5 micrograms of perchlorate per liter. In many cases, the duration of contact can be less than several hours to obtain a reduction in the perchlorate anion concentration of less than about 5 micrograms per liter, and sometimes the contact is less than an hour and often less than about 30, even less than about 5 minutes.
[0350] Treated water may contain oxygen since the use of the biocatalyst does not require the oxygen to be consumed before the biodegradation of perchlorate anion. In most cases, the oxygen concentration of the treated water is at least about 0.1, preferably at least about 0.5 and more preferably at least about 10 or even 50 milligrams per liter. Whether the dissolved oxygen concentration in the treated water is high enough or not to be discharged without aeration will, in part, depend on the oxygen concentration in the treated water. Filtration may be desired to remove any solids from, for example, exogenous microorganisms that may be introduced with water to be treated. EXAMPLE 219
[0351] An aqueous solution is prepared that contains perchlorate anion in an amount of 500 micrograms and nitrate anion (calculated as nitrogen) in an amount of 10 milligrams per liter of distilled water. Oxygen is removed to below 0.5 milligrams per liter by spraying the aqueous solution with nitrogen. The volume of the aqueous solution is reduced by about 20 percent volume and contains 410 micrograms of perchlorate anion and 15 milligrams of nitrate anion per liter of aqueous solution. Sodium acetate is then added in an amount of 0.6 parts by mass of sodium acetate per part by mass of total perchlorate and nitrate anion in the aqueous solution. The pH of the aqueous is adjusted to about 7.
[0352] The aqueous solution is then added to a glass vial containing biocatalysts from Example 31 in an amount sufficient to immerse the biocatalyst. The aqueous solution and the biocatalyst are kept at room temperature, about 25°C. After 24 hours, the perchlorate concentration is about 80 micrograms per liter of solution and the nitrate concentration is about 790 micrograms per liter of solution. EXAMPLE 220
[0353] An aqueous solution containing 128 milligrams of nitrate anion, 12 milligrams of nitrite anion and about 9 milligrams of molecular oxygen per liter of water is continuously fed to an upflow bioreactor at a rate that provides a time of 25-minute hydraulic stay. The upflow reactor contains the biocatalyst substantially as shown in Example 52. The treated water of the bioreactor contains less than about 1.3 milligrams of nitrate anion and less than 0.01 milligrams of nitrite anion per liter of water. EXAMPLE 221
[0354] An aqueous solution containing about 12 to 15 milligrams of nitrate anion and about 0.4 milligrams of perchlorate anion and about 4 milligrams of molecular oxygen per liter of water is continuously fed to an upflow bioreactor at a rate that provides a hydraulic dwell time of 25 minutes. The upflow bioreactor contains about 70 percent of the volume thereof with biocatalyst substantially as described in Example 52. The biocatalyst contains Paracoccus denitrificans. The pH of the water passing into the bioreactor is adjusted to about 7 and sodium acetate is added to the water as a carbon source. The nitrate concentration of the bioreactor effluent is less than about 1 milligram per liter and the perchlorate anion is less than about 4 micrograms per liter. EXAMPLE 222
[0355] An aqueous solution containing between about 600 and 800 milligrams of nitrate anion per liter is continuously fed to the two upflow bioreactors in series. Each upflow reactor is the same size and each contains the biocatalyst as substantially shown in Example 52. The hydraulic residence time is varied between 25 minutes and 30 minutes based on the volume of both bioreactors. The treated water from the second bioreactor contains less than 10 parts per million nitrate anion and less than 1 part per million nitrite anion. SAW. METAL REMOVAL
[0356] Soluble semimetallic and metallic compounds can be found as contaminants in various water sources. These contaminants can be naturally occurring or they can be the result of human activities such as manufacturing, mining, metal refining, waste disposal and the like. Some of these compounds pose health hazards and can negatively affect the environment.
[0357] The biocatalysts of this invention are advantageously useful for the treatment of water that contains at least one soluble metal or semimetal compound, since the interior of the biocatalyst provides microenvironments that favor redox conditions to affect metal or semimetal reduction to form a solid. These processes comprise: (a) continuously introducing said water into a reaction zone containing the biocatalyst of this invention; (b) contacting the water with said microorganism-containing biocatalyst capable of reducing said soluble compound for a time sufficient to reduce the concentration of said at least one water soluble compound; (c) maintaining said biocatalyst under metabolic conditions sufficient to metabolically reduce the oxidation state of the metal or semimetal to form semimetal or elemental metal or precipitated compound therefrom; and (d) removing water having a reduced concentration of said at least one soluble compound from the bioreaction zone.
[0358] Often, the metal or semimetal of the soluble compound comprises at least one of sulfur, phosphorus, selenium, tungsten, molybdenum, bismuth, strontium, cadmium, chromium, titanium, nickel, iron, zinc, copper, arsenic, vanadium, uranium , radium, manganese, germanium, indium, mercury, antimony and rare earth metals. The soluble compound will depend on the specific metal or semimetal and may be a hydroxide, carbonate, nitrate, carboxylate (eg formate, acetate or propionate); or a metal or semimetal oxyanion that is soluble in water.
[0359] Metabolic conditions include the presence of a carbon source that is metabolized by microorganisms in the biocatalyst. It is believed that carbon source metabolism provides a gradient within the biocatalyst to enhance the activity of a portion of the microorganisms for metabolic reduction of metals and semimetals. For this reason, metabolic reduction can occur even when the water that is being treated contains oxygen. Metabolic reduction can provide an elemental material or a precipitated compound. The precipitated compound has the metal or semimetal in a reduced oxidation state and the precipitated compound can be one or more of oxides, carbonates, sulfides and hydroxides. The specific nature of the precipitated compound will depend on the metal or semimetal of which it is composed to provide the insolubility properties.
[0360] In some cases, metabolic reduction of the metal or semimetal can delay the accumulation of the soluble compound by porous matrices and microorganisms. In such situations, the bioreaction zone can be modeled to allow a steady state operation or the porous matrices can be alternated between a bioreaction zone, into which the water to be treated passes, and a bioreaction zone that is maintained under conditions where further metabolic reduction occurs.
[0361] The metabolic processes can be conducted in any suitable manner, which include typical mesophilic conditions and with the use of typical bioreactor systems. The carbon source nutrient may be required and may be any convenient carbon source such as a low molecular weight hydrocarbon or oxygenated hydrocarbon such as ethanol, and acetate and sugars.
[0362] The interior of the biocatalyst provides a plethora of microenvironments for microorganisms and these microenvironments can vary within the biocatalyst. Thus, some microenvironments can change the composition of water, so that other microenvironments may be under more favorable conditions for metabolic reduction. For example, when water contains oxygen, microorganisms can metabolize oxygen and provide oxygen-depleted water that passes to other microenvironments where conditions favor metabolic reduction. For this reason, the biocatalyst serves to provide a self-modeling of metabolic reduction. In some embodiments of this invention, the external modulation of metabolic reduction conditions can be affected by the electron donor supply rate. In general, the more electron donors, the more acidic the pH within the biocatalyst. Since modulation is within each biocatalyst structure, the biocatalytic activities of biocatalysts in a reaction zone can be relatively uniform.
[0363] Often at least a portion of the solid metabolic product remains in the cell or otherwise in the biocatalyst. In other examples, the metabolic product is removable from the biocatalyst. If desired, the bioreactor water can be subjected to a solids removal unit operation, such as ultrafiltration, decantation, centrifugation and the like. As described above, with some systems, it is possible to regenerate porous matrices. Alternatively, the biocatalyst can provide a concentrated source of the metal or semimetal for disposal or recovery.
[0364] Representative reducing microorganisms include, but are not limited to, those of the genus Saccharomyes; to sulfur reducing bacteria including the genera Proteus, Campylobacter, Pseudomonas, Salmonella, Desulfuromonas, Desulfovibrio, Desulfonema; phosphorus-reducing bacteria including genera Acinetobacter, Phormidium, Rhodobacter and Staphylococcus; uranium reducing bacteria including Desulfovibrio, Deinococcus, Geobacter, Cellulomonas, Shewanella and Pseudomonas; molybdate-reducing organisms that include the genera Serratia, Enterobacter, and Escherichia; cadmium-reducing bacteria that include the genera Pseudomonas and Klebsiella.
[0365] Some preferred microorganisms for specific types of soluble compounds are, as follows, selenate reducing bacteria that include species from the genome Enterobacter cloacae, Planomicrobium mcmeekinii, Psuedomonas alcaligenes, Psuedomonas denitrificans, Psueomonas stutzeri and Roseomonas; other selenate reducing microorganisms including those disclosed in U.S. patent document 7,815,801, incorporated herein by reference in their entirety; chromate reducing organisms including Enterobacter cloacae, Desulfovibrio vulgaris, Geobacter sulfurreducens, Psuedomonas chromatophilia, Psuedomonas fluorescens, and Swanella alga; ferricion-reducing microorganisms including those of the genera Ferribacterium, Geobacter, and Geothrix; and arsenate reducing bacteria which include those of the Geobacter, Corynebacterium, Pseudomonas, Shewanella and Hydrogenophaga genera. VII. TASTE AND ODOR REMOVAL FROM WATER
[0366] Algae metabolites in drinking water sources can result in a characteristic off-taste and unpleasant odor. The unpleasant taste and odor is believed to be due to the presence of 2-methylisoboreal (MIB) and trans-1,10-dimethyl-trans-decalol (geosmin). Humans can detect MIB levels as low as 5 to 10 parts per trillion. Geosmin is similarly detected at very low levels. Other possibilities for drinking water are halogenated organic substances, such as trihalomethanes, which are derived from a combination of chlorine and bromine, with halogenated organic components in the water. Disinfection by-products such as halogenated organic compounds can also often be present in higher concentrations. Removal of these halogenated organic compounds is desirable for organoleptic and public health reasons. Removal of algae metabolites and halogenated components from drinking water is particularly problematic due to the low concentrations at which these impurities must be reduced.
[0367] The biocatalysts of this invention are able to treat water that contains ultra-low concentrations of contaminants that include these algal metabolites and halogenated organic compounds and reduces their concentrations to acceptable levels. Significantly due to the phenotypic changes of microorganisms in the biocatalyst, a high stable population of microorganisms can exist within, without the need for excessive electron donors to support the microorganism population. Processes for using the biocatalysts of this invention to reduce the concentration of ultra-low contaminants in a stream of water comprise: a. continuously passing said stream of water to a bioreactor, wherein said bioreactor is maintained under metabolic conditions that include the presence of the biocatalyst, wherein the biocatalyst has microorganisms capable of bioconversion of said ultra-low contaminants irreversibly retained therein; B. contacting said stream of water with said biocatalyst for a time sufficient to reduce the concentration of said ultra-low contaminants; and c. withdrawing from said bioreactor a stream of treated water having a reduced concentration of said ultra-low contaminants.
[0368] Preferably, each of the ultra-low contaminants is present at a concentration in the stream of water passed into the bioreactor in an amount of at least about 40, approximately at least about 50 nanograms per liter (ng/l) and less than about 50, often less than about 20 micrograms per liter (mcg/l). Ultra-low contaminants preferably comprise algal metabolites such as MIB and geosmin. Contaminants can also include halogenated organic compounds such as disinfection by-products such as trihalomethanes (THM) and haloacetic acid (HAA), each of which can be presented in amounts from 1 to 1,000 micrograms per liter. After treatment, the concentration of ultra-low contaminants in the treated water is typically below about 40, preferably below about 20, and sometimes below about 10 nanograms per liter. Often, the halogenated organic compounds in treated water are in concentrations of less than about 70, preferably less than 50 micrograms per liter.
[0369] The processes of this invention are suitable for use with any microorganism capable of low concentration bioconversions. Preferred microorganisms are from the genus Rhodococcus. The genus Rhodococcus is a very diverse group of bacteria that has the ability to degrade a large number of organic compounds. They have an ability to acquire a remarkable range of different catabolic genes and have robust cell physiology. Rhodococcus appears to have adopted a hyper-combination strategy associated with a large genome. Notably, they harbor large linear plasmids that contribute to their catabolic diversity, acting as a "mass storage" for a large number of catabolic genes.
[0370] In many instances, metabolic conditions do not require the addition of electron donor in order to maintain the metabolic activity of the porous matrices, as ultra-low contaminants and other contaminants in the water are sufficient to provide the necessary electron donor. When it is desired that the electron donor be added, it is preferably at a concentration that will be essentially completely metabolized by the biocatalyst, i.e. the electron donor will be supplied in an amount insufficient to maintain the population of microorganisms in the bioreactor.
[0371] In some cases, the average hydraulic residence time of the water that is treated in the bioreactor is less than about 5, preferably less than about 2 hours and can be in the range of between about 10 and 50 minutes. Often, the biocatalyst can retain metabolic activity for at least about 50, approximately, at least about 250 days. It is possible that microorganisms can be kept for decades or longer. Preferred biocatalysts can maintain the desired metabolic activity (eg, within about 3 to 5 days of restart) after long periods of downtime, approximately between about 100 and 500 days.
[0372] The metabolic processes can be conducted in any suitable manner and can be under typical mesophilic conditions with the use of typical bioreactor systems. The process can be in a continuous, semi-continuous or batch mode of operation, but is preferably continuous. Oxygenation is preferably at least about 1, more preferably at least about 2, and sometimes between about 2 and 10 or more milligrams of free oxygen per liter. If necessary, electron donors can be added to water to be treated. Electron donors include, but are not limited to, hydrogen, carbohydrates, hydrocarbons, alkanols, aldehydes, carboxylic acids, ketones, aldehydes, glycerides, and the like. See, for example, paragraph 0055 of the Published U.S. Patent Application 2006/0263869. If electron donors are required, they can be added in any suitable way. Typically, the amount added is sufficient to provide the desired biodegradation.
[0373] Degradation products can be removed from the water in any suitable manner, which includes using typical separation techniques. EXAMPLE 223
[0374] A continuously stirred tank bioreactor with a capacity of 4 liters is charged to about 30 percent of its volume with the biocatalyst substantially as described in Example 72. A feed stream of water is continuously passed through the bioreactor at room temperature (22 °C) to a range sufficient to provide an approximate hydraulic residence time of about 30 minutes. The water is from a fresh water reservoir that has not been treated. MIB and geosmin are each supplied in an amount of about 400 nanograms per liter (add MIB and geosmin as required to approximate targeted concentration levels). Fresh water contains algae metabolites as detected by odor and taste. Water has a pH of about 7 and at least 4 parts per million, by mass, of dissolved oxygen per liter. The concentration of MIB is reduced to less than 5 nanograms per liter and geosmin to less than about 20 nanograms per liter. The treated water from the reactor has no detectable odor or taste.
[0375] The reactor is shut down for about 6 months (in the flow of water through the reactor) and kept at room temperature. After restarting, passing a similar stream of water through the bioreactor under substantially the same conditions, within one day, the water discharged from the reactor has no detectable odor or taste. VIII. REMOVAL OF 1,4-DIOXAN FROM WATER
[0376] Animal studies have shown that inhalation and ingestion of 1,4-dioxane can lead to nasal cavity formation and liver carcinomas, along with neurotoxic effects. 1,4-Dioxane entered water sources primarily from use as a solvent stabilizer for solvents used in various cleaning and degreasing applications, especially chlorinated solvents such as trichloroethane (TCA) and trichlorethylene (TCE). 1,4-Dioxane is also detected in consumer products such as shampoos, soaps, waxes and lotions as a result of contamination from ethoxylated compounds such as sodium lauryl sulfate. States such as California, Massachusetts, Florida and North Carolina have set drinking water standards for 1,4-dioxane at low levels of parts per billion (ppb).
[0377] The biodegradation of 1,4-dioxane is problematic. The presence of chlorinated solvents has an inhibitory effect on microorganisms identified to degrade 1,4-dioxane; inducing compounds such as propane or tetrahydrofuran (THF) are required by many microorganisms to degrade 1,4-dioxane, but are contaminants themselves; and microorganisms that do not require inducing compounds tend to be less robust and slow growing.
[0378] The biocatalyst of this invention is particularly attractive for use in processes to reduce the concentration of 1,4-dioxane in a stream of water. In some cases, the use of an induction compound is not required even if the water contains both 1,4-dioxane and a halogenated compound. These processes include: a. continuously passing said stream of water through a bioreactor, wherein said bioreactor is maintained under metabolic conditions that include aerobic conditions and the presence of the biocatalyst of this invention that contains microorganisms adapted to metabolically degrade 1,4-dioxane; B. contacting said water stream with said biocatalyst for a time sufficient to reduce the concentration of said 1,4-dioxane in the water stream; and c. withdrawing from said bioreactor a stream of treated water having a reduced concentration of 1,4-dioxane.
[0379] The preferred microorganisms used in the biocatalyst are of the genus Rhodococci, Pseudonocardia dioxanivorans and Pseudonocardia benzenivorans. Preferred processes include those in which the 1,4-dioxane is present in the water stream in an amount less than about 100 micrograms per liter and the treated water stream has a 1,4-dioxane concentration of less than about of 10 micrograms per liter. In some cases, 1,4-dioxane is present in the water stream in an amount greater than about 10 micrograms per liter and the treated water stream has a 1,4-dioxane concentration of less than about 5 micrograms. per liter.
[0380] In many instances, no additional carbon source is required to maintain the population of microorganisms in the biocatalyst due to microenvironment and phenotypic changes. However, at low concentrations, the 1,4-dioxane in the water to be treated, the addition of small amounts of carbon source can be advantageous to sustain the energetic robustness of the population. However, the metabolic activity of biocatalysts may be sufficient to ensure that substantially no biodegradable carbon is contained in treated water.
[0381] The metabolic processes can be conducted in any suitable way, which includes typical mesophilic conditions and which uses typical bioreactor systems. The process can be in a continuous, semi-continuous or batch mode of operation, but is preferably continuous. Oxygenation is preferably at least about 1, more preferably at least about 2, and sometimes between about 2 and 10 or more milligrams of free oxygen per liter. If necessary, electron donors can be added to the water to be treated. Electron donors include, but are not limited to, hydrogen, carbohydrates, hydrocarbons, alkanols, aldehydes, carboxylic acids, ketones, aldehydes, glycerides, and the like. Acetone or glucose is a convenient electron donor. See, for example, paragraph 0055 of the Published U.S. Patent Application 2006/0263869. If electron donors are required, they can be added in any suitable way.
[0382] Degradation products can be removed from the water in any suitable way, which includes the use of typical separation techniques. EXAMPLE 224
[0383] A 4 liter capacity air lift downflow bioreactor with a perforated plate equipped with diffusers to provide uniform aeration of the bioreactor is loaded with 3000 grams of the biocatalyst of example 48. An air pump supplies air to the bioreactor below the perforated plate in an amount sufficient to keep the biocatalyst suspended. The water to be treated is continuously added to the liquid phase above the perforated plate using a variable speed pump.
[0384] The water to be treated is deionized water to which the components are added. Acetone, ammonium chloride, and dipotassium bisphosphate are added, if necessary, to maintain an atomic ratio of carbon:nitrogen:phosphorus in water of 100:3:1. Atomic carbon is calculated as the total carbon in the components added to water. Dissolved oxygen in the bioreactor is between about 5 and 7 milligrams per liter (as determined using an Oakton D06 Acorn Series probe and meter). The bioreactor is operated at room temperature (about 21°C to 25°C) and an average hydraulic residence time of 5 hours and a pH between about 7 and 8 is maintained in the bioreactor.
[0385] Water is first enriched with about 71,000 micrograms per liter of 1,4-dioxane. Upon completion of this cycle, the water is enriched with 100 micrograms per liter of 1,4-dioxane and 50 micrograms of acetone per liter. The concentration of 1,4-dioxane in the efflux is determined by gas chromatography and is in both cases below the gas chromatography non-detection limit of about 2 micrograms per liter.
[0386] Additionally, the unfiltered effluent from the bioreactor is disposed on agar plates (LBB + glucose). After a 5 day incubation, colonies (if any) were counted. Substantially no colonies are observed to indicate that the microorganisms are substantially irreversibly retained in the biocatalyst.
[0387] The results are indicative that biocatalysts require very little induction time before effective 1,4-dioxane removal occurs and that the 1,4-dioxane concentration can be reduced to non-detection levels at both concentrations higher and lower. IX. SUCCINIC ACID
[0388] The biocatalysts of this invention can be used to convert sugars into succinic acid. In broader aspects, processes for the bioconversion of sugar and, optionally, carbon dioxide with the use of a biocatalyst that contains a microorganism that produces succinic acid, comprises: a. contacting an aqueous medium with said biocatalyst under metabolic conditions that include the temperature and the presence of sugar and other nutrients for the microorganism for a time sufficient to produce succinate anion and provides an aqueous medium containing succinate anion; B. removing at least a portion of said aqueous medium containing succinate anion and said biocatalyst; ç. reusing in step (a) said biocatalyst from which at least a portion of said aqueous medium containing succinate anion has been removed; and d. recovering the anion from said aqueous medium containing succinate anion.
[0389] Examples of microorganisms that produce succinate anion disclosed to date include, but are not limited to, genetically modified or natural microorganisms, such as Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Alcaligenes eutrophus, Aspergili nigerides, Bacillus, , Bacteroides ruminicola, Bacteroides amylophilus, Brevibacterium ammoniagenes, Brevibacterium lactofermentum, Candida brumtii, Candida catenulate, Candida mycoderma, Candida zeylanoides, Candida paludigena, Candid sonorensis, Candida utilis, Candida zeylanoides, Citrobactor freundia, Delicatessen, E. coli (E.coli strains SB550 MG pHL 413, KJ122 and TG400), Fibrobacter succinogenes, Fusarium oxysporum, Gluconobacter oxydans, Glyconobacter asaii, Humical lanuginosa, Kloeckera apiculata, Kluyveromyvercymyos lactices lactices, Kluyveromyces variomyces lactices implicissimum, Pichia anomala, Pichia besseyi, Pichia media, Picha guiliermondii, Pichia inositovora, Pichia stipidis, Rhizobium, Saccharomyces cerevisiae, Saccharomyces bayanus, Schizosaccharommyces, Schizosaccharomyces liposucina, Vecchia stipidis, and Torulops succina succina, Torulops yellomyces poms.
[0390] The metabolic processes can be conducted in any suitable manner. The substrate comprises carbohydrate, which includes C5 and C6 sugars and may include carbon dioxide. Due to the use of the biocatalyst, the less preferred sugars for bioconversion can be used effectively by microorganisms. The concentration of sugars used in the aqueous medium can be in a wide range. In general, sugars are present at a concentration in the aqueous medium of at least about 0.5, approximately, between about 1 and 200 grams per liter. Preferably, the amount of sugar provided in the aqueous medium is such that at least about 90, more preferably at least about 95, by weight percent is consumed during the metabolic process.
[0391] Carbon dioxide can be obtained from any suitable source; however, components that are excessively harmful to microorganisms must be removed before contacting the aqueous medium containing the biocatalyst. Carbon dioxide is usually supplied in gaseous form, although carbonate and bicarbonate salts can be used. When supplied as a gas, the concentration of carbon dioxide in the gas is typically in the range of about 40 to 100, approximately 70 to 100 percent by volume. Sources of carbon dioxide include, but are not limited to, exhaust gases from industrial and fermentation processes, exhaust gases from combustion of fuels and waste material, natural gas streams containing carbon dioxide, streams from gasification biomass, for example, to produce synthesis gas and the like.
[0392] The aqueous medium contains water that can be supplied from any suitable source including, but not limited to, water, demineralized water, distilled water, and waste or process water streams. Any suitable metabolic conditions can be used, including typical mesophilic conditions. Where gaseous substrates are used, higher pressures tend to increase the amount of substrate dissolved in the culture liquid and thus enhance mass transfer. Often the pH is between about 3 and 8.5, approximately 3.5 to 7. The metabolic conditions for the bioconversion of sugars and carbon dioxide to succinate anion are typically anaerobic and, preferably, the aqueous medium has a high concentration. of dissolved molecular oxygen of less than about 0.5 milligram per liter. Where the biocatalyst is subjected to different aqueous media and one of the aqueous media is intended to provide metabolic activities to enhance the viability of the microorganisms, the dissolved molecular oxygen concentration may be in excess of about 2, approximately, about 2 to 10 milligrams per liter.
[0393] The duration of contact between the aqueous medium and biocatalyst can also be in a wide range and will depend, in part, on the substrate concentration, the concentration of succinate anion found in the aqueous medium, the type of bioreactor, other metabolic conditions used, the nature of the microorganism used and the type of microorganism and cell density in the biocatalyst. For batch operations, contact is often in the range of about 5 minutes to 100 hours, approximately about 1 to 50 hours, and in continuous operations, the net hourly space velocity is typically in the range of about 0.01 to 50 h-1.
[0394] Bioconversion can be in a continuous, semi-continuous or batch mode of operation. Any suitable bioreactor design can be used, including typical bioreactor systems.
[0395] In preferred aspects, the biocatalyst is subjected to two or more aqueous environments to enhance the bioconversion of sugars, reduce the presence of unconsumed sugars in the fermentation product containing succinic acid, and enhance the use of carbon dioxide as a co-substrate .
[0396] In one embodiment of the preferred processes, the contact of the aqueous medium with the biocatalyst occurs in at least two reaction zones that have different metabolic conditions. In one aspect of this embodiment, the aqueous medium from a first reaction zone containing the biocatalyst passes to a subsequent reaction zone for further contact with the biocatalyst. Substantially no additional sugar is added to the aqueous medium immediately prior to passing to the subsequent reaction zone or during its residence time in the subsequent reaction zone. Thus, the sugar concentration in the aqueous medium is further depleted in the subsequent reaction zone, preferably to less than about 1, approximately, less than about 0.5, by mass, percent based on mass. of the succinate anion in the aqueous medium. Preferably, the first reaction zone and the subsequent reaction zone are cycled. In some cases, it may be desired to supply the carbon dioxide substrate to the aqueous medium in the subsequent reaction zone to convert additional phosphoenolpyruvate in the microorganisms to the succinate anion.
[0397] In another aspect of this preferred embodiment, a reaction zone containing the biocatalyst is arranged between the use of an aqueous sugar substrate and a gaseous or aqueous carbon dioxide substrate. For example, sugar is provided in a first aqueous medium in contact with the porous matrices in the first reaction zone, which may be under conditions sufficient to generate phosphoenolpyruvate in microorganisms under conditions that do not favor the conversion of phosphoenolpyruvate to succinate anion, as which conditions can comprise micro-aerobic or aerobic conditions. The first aqueous medium is removed. A second aqueous medium or gas is introduced into the reaction zone under anaerobic conditions, which includes the presence of carbon dioxide, sufficient to bioconvert the phosphoenolpyruvate to a succinate anion. While the biocatalyst provides nutrient-retaining environments for the microorganisms, the metabolic activity of the microorganisms can be maintained in the second zone. Preferably the second zone uses an aqueous medium which is substantially water, so that the succinate anion product which passes into the second aqueous medium can more easily be recovered. More preferably, the concentration of the succinate anion in the second aqueous medium is at least about 150, approximately, at least about 200 to as much as 300 or 400 grams per liter of aqueous medium. The second aqueous medium containing succinate anion is removed from the first reaction zone to recover the succinate anion. In some cases, high purity succinic acid can be obtained by reducing the temperature of the aqueous medium enough to crystallize a succinic acid. After removal of the second aqueous medium, the first reaction zone may come into contact with a first aqueous medium. In some cases, a third or even more steps can be used. For example, after removal from the second aqueous medium, the first reaction zone may come into contact with a first aqueous medium from the reaction zone of another reaction zone and be maintained under conditions to reduce metabolites in the aqueous medium and generate more phosphoenolpyruvate . If desired, multiple reaction zones can be used to provide a semi-continuous process.
[0398] The succinic acid product can be recovered from the aqueous medium in any suitable manner. Several methods for succinic acid recovery include membrane separations and precipitation, sorption and ion exchange, electrodialysis, and liquid-liquid extraction. See, for example, Davidson, et al., Succinic Acid Adsorption from Fermentation Broth and Regeneration, Applied Biochemistry and Biotechnology, Spring 2004, pages 653 to 669, for a discussion of sorbents for recovering succinic acid from fermentation broths . See also, Li, et al., Separation of Succinic Acid from Fermentation Broth Using Alkaline Anion Exchange Adsorbents, Ind. Eng. Chem. Res., 2009, 48, pages 3,595 to 3,599. See, for example, a multiple crystallization method for recovering succinic acid disclosed in U.S. Patent Application Publication 2011/0297527. Hepburn, in her master's thesis at Queen's University, The Synthesis of Succinic Acid and its Extraction from Fermentation Broth Using a Two-Phase Partitioning Bioreactor (April 2011), reveals a process where such cynic acid is produced at inhibitory levels and then the system pH was suggested below the PKA2 of succinic acid with the use of dissolved carbon dioxide gas to create an undissociated product. Polymers with an affinity for succinic acid were adsorbed from the solution and then the pH returned to operational levels.
[0399] A general understanding of this process can be facilitated with reference to Figures 10 and 11. With reference to Figure 10, apparatus 1000 is suitable for the biological production of succinic acid on a continuous basis. Apparatus 1000 is depicted as having a primary bioreactor 1002 and a polishing bioreactor 1004. Both bioreactors are fluidized bed reactors that contain biocatalyst, for example, substantially described in Example 175. An aqueous stream containing sugar and other nutrients is supplied via line 1006 to bioreactor 1002. The carbon dioxide-containing gas is supplied to apparatus 1000 via line 1008. A portion of the carbon dioxide-containing gas passes through line 1010 to line 1006 into bioreactor 1002. Bioreactor 1002 is maintained under metabolic conditions sufficient to convert sugar and carbon dioxide to succinate anion. The exhaust gases are removed from bioreactor 1002 through line 1012. A continuous stream of aqueous medium in bioreactor 1002 is withdrawn through line 1014 and passes through bioreactor 1004. Bioreactor 1002 is provided with a screen or other device to essentially prevent allow the biocatalyst to pass into line 1014. A gas containing carbon dioxide from line 1008 and line 1016 is supplied to bioreactor 1004.
[0400] In a typical operation of apparatus 1000, the aqueous medium withdrawn from bioreactor 1002 through line 1014 contains unreacted sugars and metabolites that can be additionally bioconverted by microorganisms. Bioreactor 1004 is operated to further reduce the concentration of unreacted sugars and these metabolites and therefore no additional sugar substrate is supplied to a bioreactor 1004 in such an operation. It should be readily understood that, if desired, additional sugar substrate can be added to the aqueous medium in bioreactor 1004.
[0401] Bioreactor 1004 is operated under metabolic conditions sufficient to convert the substrate to succinate anion. Unreacted gases are withdrawn from bioreactor 1004 via line 1018. A continuous stream of aqueous medium from bioreactor 1004 is withdrawn via line 1020. Bioreactor 1004 is provided with a screen or other device to essentially prevent the biocatalyst from passing into the interior of line 1020. The withdrawn aqueous medium is directed to filter assembly 1022 via line 1020. Since the aqueous medium is substantially devoid of solids, it is practical for filter assembly 1022 to be an ultrafiltration assembly. The aqueous medium then passes from filter assembly 1022 via line 1024 to distillation column assembly 1026. In the case where bioreactors 1002 and 1004 are operated substantially unbuffered, distillation column assembly 1026 it serves to concentrate the aqueous medium to facilitate the crystallization of succinic acid. In case an ammonium hydroxide buffer is used, the 1026 distillation column assembly also serves to convert the ammonium salts from the succinate anion to succinic acid in which the ammonia is released. Distillation column assembly 1026 can comprise one or more unit operations, including neutralization and filtering of precipitates, resolvation, and intermediate crystallization, as described in U.S. Patent Application Publication No. 2011/0297527 and the like.
[0402] As shown, the suspension from distillation column assembly 1026 exits via line 1028. A purge can be obtained via line 1030 and the remaining overhead recycled into one or both of bioreactors 1002 and 1004 via lines 1034 and 1032, respectively. Where ammonium hydroxide buffer is used, recycling reduces the required amount of ammonium hydroxide to be supplied externally. It is also possible to operate bioreactor 1004 at a lower pH than that used in bioreactor 1002. Thus, in a buffer system, the predominant portion of the succinate anion will be the monosalt.
[0403] The bottoms stream from distillation column 1026 passes through line 1036 to crystallization unit 1038. Typically, the concentration of succinic acid in the bottoms stream is greater than about 30%. The bottom stream passing to the crystallization unit is often cooled to a temperature below about 15°C, approximately between about 0°C and 10°C. Crystalline succinic acid is removed from crystallizing unit 1038 via line 1040. Supernatant liquid is removed via line 1042.
[0404] Figure 11 depicts an apparatus 1100 that has three bioreactors 1102, 1104 and 1106 that are operated in a cyclic and sequential routine to bioconvert sugar and carbon dioxide into succinic acid. Each of the reactors contains porous matrices that have succinic acid-producing microorganisms irreversibly retained in them. Bioreactors have an internal liquid recycling system to be operated as fluidized bed reactors.
[0405] Apparatus 1100 is equipped with four collectors: collector 1108 which provides a fresh aqueous medium containing sugar and other nutrients; the collector 1110 which supplies the gas containing carbon dioxide; the collector 1112 which supplies the oxygen-containing gas and the collector 1114 which provides the transport of fluids between the bioreactors. Line assemblies 1116, 1118 and 1120 each connect the four collectors 1108, 1110, 1112 and 1114 to bioreactors 1102, 1104 and 1106, respectively. Each of the bioreactors 1102, 1104 and 1106 are provided with lines 1122, 1124 and 1126, respectively, to allow the outflow of gas and are provided with lines 1128, 1134 and 1138, respectively, for the aqueous medium drained from the bioreactors. Aqueous medium drained from a bioreactor is routed to both collector 1132 and collector 1114. For bioreactor 1102, line 1128 is in fluid communication with line 1130 which is adapted to direct aqueous medium to one of these collectors. For bioreactor 1104, line 1134 is in fluid communication with line 1136 which is adapted to direct aqueous medium to one of these collectors. For bioreactor 1106, line 1138 is in fluid communication with line 1140 which is adapted to direct aqueous medium to one of these collectors.
[0406] Each of the 1102, 1104, and 1106 bioreactors is sequenced between a microaerobic stage, an anaerobic stage, a sugar conversion stage, and a carbon dioxide conversion stage. In the microaerobic stage, the fresh aqueous medium is fed into a bioreactor that has completed the carbon dioxide conversion stage and has been drained from the aqueous medium for succinic acid recovery purposes. In this regard, small amounts of oxygen-containing gas, eg air, are supplied from conductor 1112.
[0407] On completion of the microaerobic stage, the supply of oxygen containing gas from conductor 1112 is ceased and the bioreactor enters the anaerobic sugar conversion stage. The anaerobic sugar conversion stage can be conducted with or without the addition of carbon dioxide from conductor 1110. The anaerobic sugar conversion stage is conducted under suitable metabolic conditions for the production of succinate anion. In some embodiments, metabolic conditions during the microaerobic stage and the anaerobic sugar conversion stage enhance the formation of phosphoenolpyruvate with relatively little succinate anion passing from porous matrices to the surrounding aqueous medium.
[0408] The reactor then goes from the anaerobic sugar conversion stage to the carbon dioxide conversion stage. In the carbon dioxide conversion stage, carbon dioxide is supplied from conductor 1112 in an amount sufficient to improve a portion of the succinate anion that is derived from the carbon dioxide substrate through the conversion of phosphoenolpyruvate. The bioreactor is maintained under metabolic conditions that favor substrate bioconversion to carbon dioxide. On completion of the carbon dioxide conversion stage, the aqueous medium is drained from the bioreactor and passed to conductor 1132 for succinic acid recovery. The reactor is then cycled back to the microaerobic stage.
[0409] As with the apparatus shown in Figure 10, apparatus 1100 passes the aqueous medium withdrawn from the bioreactor having passed through the carbon dioxide conversion stage to the filter assembly 1142 and then through line 1144 to the assembly of distillation 1146. The suspended portion from distillation assembly 1146 exits through line 1148 to recycle in conductor 1108. A purge is taken through line 1150. Bottom current from distillation assembly 1146 passes through line 1152 to the assembly. of crystallization 1154. The succinic acid is removed via line 1156 and the supernatant liquid is removed via line 1158.
[0410] The 1100 can also be operated using a sequence of different stages. Such a sequence uses water that has a substantial absence of sugars and other nutrients in the carbon dioxide conversion stage. For the description of this sequence, reference is made to conductor 1108a that supplies such a stream of water.
[0411] In the microaerobic stage, the aqueous medium is supplied by another bioreactor that has completed the anaerobic sugar conversion stage. On completion of the microaerobic stage, the bioreactor enters the anaerobic sugar conversion stage as described above, but under conditions that minimize the accumulation of succinate anion in the aqueous medium. Sugar and other nutrients are supplied to the bioreactor through conductor 1108. Sugar and other nutrients are preferably dissolved or slurried in an aqueous medium at a concentration sufficient to maintain the imagined amount of aqueous medium in the bioreactor, as well as sufficient concentrations of sugars and other nutrients for the metabolic activity of microorganisms.
[0412] On completion of the anaerobic sugar conversion stage, the aqueous medium is removed and passes through a bioreactor that enters the microaerobic stage. The aqueous medium is replaced by water, which has a substantial absence of sugar and other nutrients. In the carbon dioxide conversion stage, sufficient carbon dioxide is provided to provide, under metabolic conditions, the succinate anion. The succinic anion passes into the aqueous phase which will have reduced concentrations of sugars, other nutrients and other metabolites compared to the aqueous medium in the anaerobic sugar conversion stage. Thereby, the ability to obtain high purity succinic acid is facilitated. Furthermore, by recycling the aqueous medium from the bioreactor that completes the anaerobic sugar conversion stage to the microaerobic stage, certain metabolites such as acetate anion can be consumed by microorganisms for metabolic purposes, thereby improving conversion of sugars in succinate anion.
[0413] In this sequence, a purge current is taken from conductor 1114 through line 1114a to prevent undue accumulation of unwanted components in the aqueous medium, and the suspended part from distillation assembly 1146 can be used to constitute at least part of the water to the water supplied by conductor 1108a via line 1148a. X. BOTYROCOCCI
[0414] Botryococcus, which includes but is not limited to Botryococcus braunii, has been proposed for the photosynthetic conversion of carbon dioxide into various hydrocarbon bioproducts and oxygenated organic compounds, often from 8 or 10 to 50 carbon atoms and, sometimes between about 20 and 40 carbon atoms (“oils”). The Botryococcus species that have been reported have up to 75 percent of the dry mass of microalgae that constitute hydrocarbons, whereas other microalgae may only contain up to about 10 percent by mass of hydrocarbons. Botryococcus species often have high bioproduct productivity. By-products can be expressed from cells and depending on the strain or race of the species can include odd number hydrocarbons, n-alkadiene hydrocarbons, trienes, triterpenes and tetraterpenes. Hydrocarbons can contain oxygen in several functional groups.
[0415] Although Botryococcus species offer significant potential as a source of biochemical elements and biofuels, the practical difficulties associated with supplying and maintaining a sufficient population of Botryococcus species have prevented their adoption on a commercial scale. These difficulties include: a very slow growth rate; an oil secretion is mainly in the non-growth phase, thus proposals were made to harvest the algae once a sufficient population was obtained for oil recovery; sensitivity to bright light that causes chlorophyll degeneration that can be long-lasting or permanent; thick cell walls that are resistant to chemical degradation and prevent oil extraction; sensitivity to hydrodynamic shear; and an impracticality of using a bioreactor large enough to be competitive to supply oils free of contaminating microorganisms that can consume oils, compete with nutrients and produce algaecides.
[0416] The biocatalysts of this invention that contain Botryococcus species take advantage of the metabolic activity of Botryococcus species to provide enhanced process viability. In broad aspects, processes for the bioconversion of carbon dioxide to bioproducts using the biocatalyst of this invention that contain microalgae that comprise a species of Botryococcus comprise: a. keep the biocatalyst in an aqueous medium, said aqueous medium being in metabolic conditions that include temperature and the presence of nutrients for the microalgae; B. putting the aqueous medium in contact with carbon dioxide for bioconversion in which microalgae secrete bioproduct; ç. irradiate the aqueous medium with light at a frequency and intensity sufficient for the microalgae to photosynthesize carbon dioxide into a bioproduct; and d. remove the bioproduct from the aqueous medium.
[0417] Bioconversion can be photosynthetic or heterotrophic in which microalgae have the ability to operate in such an environment, or both. Preferably, the biocatalyst is smaller in size, less than about 15, preferably less than about 2 millimeters, say between about 100 microns and 2 millimeters.
[0418] The processes of this invention use microalgae that comprise species of Botryococcus. Preferably, microalgae consist essentially of Botryococcus species, i.e. there is a monocultural environment for the photosynthetic conversion of carbon dioxide into a byproduct or a multicultural environment with bacteria that can improve the performance of Botryococcus species, as disclosed in Wang, et al., Effect of nutrient conditions on the growth of Botryococcus braunii, Chinese Journal of Process Engineering, 3:141 to 145 (1996), incorporated herein by reference in its entirety. The Botryococcus species can be a wild type (naturally occurring) or a recombinant microalgae. Examples of species of Botryococcus include, but are not limited to, Botryococcus braunii. Numerous strains of Botryococcus braunii are known, such as horridus, minor, perarmatus, validus, Showa and Ninsei. Other Botryococcus species include B. australis, B. calcareous, B. canadensis, B. comperei, B. fernanoi, B. giganteus, B. miromorus, B. neglectus and B. pila. Strains can be further categorized into races such as Botryococcus braunii race A, Botryococcus braunii race B and Botryococcus braunii race L. Botryococcus braunii strains are typically preferred due to production and bioproduct rates, especially those of races A and B , and Botryococcus braunii race B strains are more preferred where the by-products do not contain oxygen atoms are desired.
[0419] An advantageous species of Botryococcus comprises genetically modified Botryococcus containing enzyme to metabolize carbohydrate source such as sugar for heterotrophic growth. This genetic modification facilitates obtaining a large population of Botryococcus to be incorporated into the biocatalyst. Population growth can be facilitated through the use of alternative carbon sources such as carbohydrates where microalgae contain suitable enzymes and transporters. Botryococcus braunii typically has transporters for glucose.
[0420] The biocatalysts of this invention are used in photosynthetic processes to bioconvert carbon dioxide into bioproducts. The composition of the by-products may vary depending on the Botryococcus strain used, and may be branched or cyclic hydrocarbons, which include, but are not limited to 10- to 50-carbon terpenoids, and may be replaced by chemical moieties that contain oxygen, such as , hydroxyl, alkoxy, acyl and carboxyl. Bioproducts can include biodiesel and other glycerides. Bioproducts are expressed from microalgae, and pass from the porous matrices to the aqueous medium containing the porous matrices. A solvent can be used to facilitate the collection of bioproducts. A preferred solvent is one that is immiscible in water, solubilizes hydrocarbons or other bioproducts, has a low boiling point, has a density significantly different from water, is readily available and inexpensive, is reusable and recyclable, and is not extremely toxic for organisms. Heptane is an example of such a solvent.
[0421] The metabolic processes can be conducted in any suitable way. The substrate comprises carbon dioxide and can include carbohydrate, including C5 and C6 sugars. Carbon dioxide can be obtained from any suitable source; however, components that are exclusively harmful to microalgae must be removed before contacting the biocatalyst. Generally, carbon dioxide is supplied in gaseous form, although carbonate and bicarbonate salts can be used but are less preferred. Where supplied as a gas, the concentration of carbon dioxide in the gas is typically in the range of about 40 to 100, say 70 to 100, percent by volume. Sources of carbon dioxide include but are not limited to gaseous effluents from industrial and fermentation processes, exhaust gases from combustion of fuels and waste materials, natural gas streams containing carbon dioxide, streams from gasification of biomass, for example, to produce synthesis gas, and the like.
[0422] Appropriate metabolic conditions that use light radiation of a sufficient intensity to provide photobiocatalytic activity that includes culture liquid can be used including Typical Mesophilic Conditions. The light intensity can vary, but preferably it is relatively strong, for example at least about 20, say between about 20 and 200 or more, microEinsteins per square meter per second, for light within range. 400 to 800 nanometer waveforms. Pressure is not critical and can be ambient, low or high pressure. Where gaseous substrates are used, high pressures tend to increase the amount of substrate dissolved in the culture liquid and thereby improve mass transfer. Often the pH is between about 6.5 and 8.5, say 6.5 and 8.0. Metabolic conditions can include the presence of molecular oxygen and, if present, in an amount between about 5 and 50 percent by volume based on the volume of carbon dioxide fed into the aqueous medium.
[0423] Generally, bioconversion activity can be maintained for at least about 30 and often at least about 300 or more days.
[0424] The chemical can be recovered from the culture liquid in any suitable manner. Frequent continuous or discontinuous removal of the bioproduct is preferred as the bioproduct. XI. BUTANOL
[0425] The biocatalyst of this invention is attractive for converting substrate into butanol which can be isobutanol or n-butanol. Both butanol isomers are toxic at relatively low concentrations to microorganisms that produce butanol, typically less than about 3 percent by mass per liter of aqueous medium. Processes using the biocatalyst of this invention allow higher titers of butanol to be produced, thereby reducing the costs of water/butanol separation. See, for example, Tracy, "Improving Butanol Fermentation to Enter the Advanced Biofuel Market, mbio.asm.org, vol. 3, 6, November/December 2012, and Kaminski, et al., Biobutanol—Production and Purification Methods, Ecological Chemistry and Engineering S, 18:1, pp. 31 to 37 (2011).
[0426] In broad aspects, the processes to bioconvert substrate to butanol comprise: a. placing an aqueous medium in contact with a biocatalyst of this invention, said biocatalyst containing microorganisms capable of bioconverting said substrate to butanol, wherein said aqueous medium is maintained under metabolic conditions that include the presence of nutrients for said microorganisms and contains said substrate; B. maintaining contact between the aqueous medium and the biocatalyst for a time sufficient to bioconvert at least a portion of said substrate to butanol; and c. recovering butanol from said aqueous medium.
[0427] Butanol can be isobutanol or n-butanol depending on the microorganism used in the process. The microorganism to be used will also define the substrate. Substrates that have found application in the production of butanol include carbon dioxide, sugars, glycerol and synthesis gas. The microorganisms capable of producing butanol are butyrogens and include, but are not limited to, wild-type or recombinant Clostridia such as, C. acetobutylicum, C. beijerinckii, C. pasteurianum, C. saccharobutylicurn, C. saccharoperbutylacetonicum; Oeneococcus oeni; and Ralstonia eutropha, and recombinant microorganisms such as E. coli in which pathways to produce butanol were added. See, for example, published U.S. patent application 20100143993 for a more extensive discussion of other microorganisms for producing butanol. Genetically enhanced photoautotrophic cyanobacteria, algae and other photoautotrophic organisms have been adapted to bioconvert carbohydrates within the microorganism directly to butanol. For example, genetically modified cyanobacteria that have constructs comprising DNA fragments that encode pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) enzymes are described in U.S. patent 6.699,696.
[0428] Bioconversion conditions often fall within the Typical Mesophilic Bioconversion Conditions and Typical Bioreactor Systems can be used. Continuous processes are especially preferred since the biocatalysts of this invention can provide high cell densities and thus, together with the improved bioconversion rate, provide high substrate conversion efficiencies with relatively short average residence times in the bioreactor, for example, often less than about 3 or 4 hours and sometimes less than about 30 minutes.
[0429] One aspect of this process is further illustrated in Figure 12 which is a schematic representation of a 1200 bioreactor set for the production of n-butanol. A raw material containing sugar is supplied through line 1202 to the first bioreactor 1204 which is an upflow bioreactor containing an aqueous fermentation medium and biocatalyst for the bioconversion of sugar to n-butanol. The biocatalyst contains. In bioreactor 1204, the supply of sugar occurs so that only a portion is bioconverted to butanol and thus provides an aqueous medium containing about 6 to 8 percent by volume of butanol. The aqueous medium from the first bioreactor 1204 passes through line 1206 to the second bioreactor 1208, where the remaining sugars are bioconverted. The second 1208 bioreactor is a fluidized bed bioreactor. The second bioreactor 1208 contains an aqueous medium with the biocatalyst containing Clostridia acetobutyricutn. In the second bioreactor, some remaining sugar is bioconverted to provide an aqueous medium containing about 10 volume percent butanol. Due to the higher concentration of butanol in the second reactor 1208, the rate of bioconversion to butanol is less than about 50 percent of that in the first bioreactor 1204. upper phase containing n-butanol that passes through line 1216 for product recovery. The high concentration of butanol in the 1216 line facilitates butanol recovery with substantial savings in energy costs.
[0430] An aqueous phase saturated with butanol is returned through line 1218 from decanter 1214 to the second bioreactor 1208 and contains about 7 to 8 volume percent of butanol and unreacted sugars, ethanol, and acetone. A purge is removed via line 1220 to maintain steady state conditions. This stream can be used for product recovery to obtain ethanol, acetone and butanol. The second bioreactor 1208 can be operated so that with the recycling rate of the aqueous medium, only a portion of the sugar is bioconverted, however, that converted to butanol passes to a butanol phase for recovery. If necessary, additional water and nutrients can be supplied to the first bioreactor 1204 through line 1222.
[0431] The bioreactor assembly, although described in connection with the production of n-butanol, is useful for the bioconversion of substrates where the biocatalyst bioconversion activity decreases with increased bioproduct concentration in the aqueous medium and where the bioproduct can form a separate liquid phase. In their broad aspects, these continuous processes for bioconversion of substrate to bioproduct using a microorganism capable of such bioconversion where the bioproduct is toxic to the microorganism comprise: a. continuously supplying substrate and aqueous medium to at least one first bioreactor containing aqueous medium, at least one said first bioreactor having therein the biocatalyst of this invention comprising said microorganism; B. maintaining at least one said first bioreactor under metabolic conditions and continuously withdrawing a first reactor effluent from at least one said first bioreactor at a rate sufficient to maintain steady state conditions and provide sufficient hydraulic residence time to bioconvert a portion of the substrate , a said first bioreactor effluent containing unconsumed substrate and bioproduct, wherein the bioconversion activity for said bioproduct in at least one said first bioreactor is at a first rate; ç. continuously supplying the withdrawn first bioreactor effluent to at least one subsequent bioreactor containing the aqueous medium, said at least one subsequent bioreactor having therein the biocatalyst of this invention comprising said microorganism; d. maintain at least one subsequent bioreactor under metabolic conditions and continuously withdraw a subsequent bioreactor effluent from at least one subsequent bioreactor at a rate sufficient to maintain steady state conditions and provide sufficient hydraulic residence time to bioconvert at least a portion of the substrate, wherein said subsequent bioreactor effluent contains bioproduct, wherein the bioconversion activity for said bioproduct in at least one said subsequent bioreactor is at a second rate that is lower than the first rate; and. continuously separating a rich bioproduct stream from said subsequent withdrawn bioreactor effluent for product recovery and providing a residual aqueous stream; and f. continuously recycle at least a portion of the residual aqueous stream into at least one subsequent bioreactor.
[0432] In many cases, the subsequent bioreactor effluent contains substrate. In preferred aspects of this process, at least one subsequent bioreactor comprises a fluidized bed bioreactor. The separation of step (e) can be any suitable separation technique, which includes, but is not limited to, Typical Separation Techniques. In preferred aspects, the bioproduct is capable of forming a separate liquid phase in the aqueous medium and in at least one subsequent bioreactor, the concentration of the bioproduct forms a separate liquid phase and the subsequent bioreactor effluent is subjected to phase separation to provide a bioproduct-containing phase and the residual aqueous phase. In some preferred aspects, especially where the bioproduct forms a separate liquid phase, the subsequent bioreactor effluent and waste aqueous stream recycling are at rates sufficient to maintain a desired second rate of bioconversion activity and form the second liquid phase . Often, only a portion of the substrate is bioconverted in at least one said subsequent bioreactor, and a sufficient concentration of substrate is maintained in the aqueous medium in at least one subsequent bioreactor to improve the rate of substrate-to-bioproduct conversion. EXAMPLES 225 TO 231
[0433] A series of seven batch fermentation experiments is conducted using the general procedure below. In each experiment, a biocatalyst that is used substantially as described in Example 93 has a nominal diameter of about 4 millimeters and is maintained under an anaerobic nitrogen environment. A batch medium is prepared in accordance with ATCC® 2107 Medium, a modified fortified Clostridial Broth/Agar medium, as follows: Combine 38 grams of BD 218081 fortified Clostridial Medium (ATCC, Manassas, Virginia); 14.5 g of agar and 1000 milliliters of deionized and boiled water to dissolve the agar, Prepare separately a solution of 10 grams of peptone, 10 grams of meat extract, 3 grams of yeast extract, 5 grams of dextrose, 5 grams of Sodium Chloride, 1 gram of Soluble Starch, 0.5 gram of L-Cysteine Hydrochloride, 3 grams of Sodium Acetate and 4 milliliters of Resazurin (0.025%) in 1,000 milliliters of DI Water, and Combine the solutions.
[0434] Glucose is added to the combined solution at 60 or 120 grams per liter, and the solution is adjusted to a pH of about 5.5 with 5N sodium hydroxide. The batch medium then becomes anaerobic by autoclaving at 121°C for 20 minutes while sparging with the nitrogen that has passed through a 0.2 micron filter. Each batch fermentation is conducted in a sealed tank reactor and approximately 2 milliliters of the batch medium is used per gram of biocatalyst. In some of the reactors, n-butanol is injected to determine the effect of n-butanol on biocatalysts and fermentation. Fermentations are conducted at a temperature of about 37°C, and samples of the fermentation broth are taken periodically and analyzed by gas chromatography. Fermentations continue for 48 hours. Data are summarized in Table VI.TABLE VI
XII. ETHANOL
[0435] The biocatalyst of this invention is attractive for substrate conversion to ethanol. The maximum ethanol titer in fermentation broths that use yeast is typically about 15 to 18 percent, and the efficiency of converting substrates such as sugars and synthesis gas to ethanol in commercial processes that theoretically use yeast is typically lower. to about 95 percent. With other microorganisms that produce ethanol, such as cyanobacteria and Clostridia, their sensitivity to ethanol concentrations may be much greater than that of yeast. For example, 1.5 percent by volume of ethanol has been reported to cause a 50 percent growth reduction in Synechocystis sp. PCC 6803. Therefore, processes using these alternative microorganisms generate broths containing very dilute ethanol. U.S. patent 7,682,821 B2 discloses a closed photobioreactor that uses daily ambient temperature fluctuations as a means to reduce the cost of separating ethanol. Processes using the biocatalyst of this invention allow higher titers of ethanol to be produced, thereby reducing the costs of water/ethanol separation, and the conversion efficiency almost approaches theoretical efficiency due to phenotypic changes for the microorganism in biocatalysts of this invention.
[0436] In broad aspects, the processes to bioconvert the substrate to ethanol comprise: a. placing an aqueous medium in contact with a biocatalyst of this invention, said biocatalyst containing microorganisms capable of bioconverting said substrate to ethanol, wherein said aqueous medium is maintained under metabolic conditions that include the presence of nutrients for said microorganisms and contains said substrate; B. maintaining contact between the aqueous medium and the biocatalyst for a time sufficient to bioconvert at least a portion of said substrate to ethanol; and c. recovering ethanol from said aqueous medium.
[0437] The microorganism to be used will define the substrate. Substrates that have found application in ethanol production include carbon dioxide, sugars and synthesis gas. Microorganisms capable of producing ethanol include, but are not limited to, wild-type or recombinant bacteria and yeasts, for example, Clostridia such as C. ljungdahlii, Clostridium aceticum, and C. thermoaceticum, Acetogenium kivui, Acetobacterium woodii, Acetoanaerobium noterae, Butyribacterium methylotrophicum, Eubacterium limosum, Zymomonas mobilis, Zymomonas palmae, mesophilic yeasts such as Pichia stipitis, Pichia segobiensis, Candida shehatae, Candida tropicalis, Candida boidinii, Candida tenuis, Pachysolenulan, Candidal , Candida rugosa, Candica sonorensis, Issatchenkia terricola, Kloeckera apis, Pichia barkeri, Pichia cactophila, Pichia deserticola, Pichia norvegensis, Pichia membranefaciens, Pichia mexicana, Sacchrimyces cervisea and Torulaspora delbrueckii, and yeasts such as temophilica Candida, Candida, emberorum, Candida pintolopesii, Candida thermophila, Kluyvero myces marxianus, Kluyveromyces fragilis, Kazachstania telluris, Issatchenkia orientalis and Lachancea thermotolerans. The thermophilic bacteria include, among others, Clostridium thermocellum, Clostridium thermohydrosulphuricum, Clostridium thermosaccharolyticum, Thermoanaerobium brockii, acetoethylicus Thermobacteroides, Thermoanaerobacter ethanolicus, Clostridium thermoaceticum, Clostridium thermoautotrophicum, Acetogenium kivui, Desulfotomaculum nigrificans and Desulvovibrio thermophilus, Thermoanaerobacter tengcongensis, Bacillus stearothermophilus and Thermoanaerobacter mathranii. Genetically enhanced photoautotrophic cyanobacteria, algae and other photoautotrophic organisms have been adapted to bioconvert carbohydrates within the microorganism to ethanol. For example, genetically modified cyanobacteria that have constructs comprising DNA fragment encoding the enzymes pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) are described in U.S. patent 6.699,696. Cyanobacteria are photosynthetic bacteria that need light, inorganic elements, water and a source of carbon, usually carbon dioxide, to metabolize and develop. Ethanol production using genetically modified cyanobacteria has also been described in the PCT patent application published in WO 2007/084477. See also U.S. patent application 20120301937 for a listing of microorganisms that produce ethanol.
[0438] Bioconversion conditions often fall within the Typical Mesophilic Bioconversion Conditions and Typical Bioreactor Systems can be used. Continuous processes are especially preferred as the biocatalysts of this invention can provide high cell densities and thus, together with the improved bioconversion rate, provide high substrate conversion efficiencies with relatively short average residence times in the bioreactor, by example, often less than about 3 or 4 hours and sometimes less than about 30 minutes.
[0439] For photosynthetic processes, the combination of high cell concentrations per unit volume of liquid culture medium, the essential absence of residues from the microorganisms that thus provide a cleaner culture medium, and the phenotypic changes associated with The biocatalysts of this invention allow for a significant increase in ethanol that can be generated per unit time per unit surface area. Therefore, smaller footprints are required for photobioreactors, and closed processes such as disclosed in the U.S. patent 7,682,821 can generate even higher concentrations of ethanol in the condensate. Additionally, since in situ sterilization can be used, more reliable operations can occur as the population of any contaminating microorganisms can be controlled. The photobioreactor can contain a liquid culture medium with biocatalysts in it. The substrate, eg carbon dioxide, can be dissolved in the culture medium, or the biocatalyst can be brought into contact with the gaseous substrate and then the ethanol can be removed from the biocatalyst, for example, by evaporation or upon entering in contact with an extractant for ethanol such as water. EXAMPLE 232
[0440] A fluidized bed bioreactor is charged at about 75 volume percent of its capacity with the biocatalyst substantially as described in example 147. A continuous flow of water containing glucose at a concentration of 120 grams per liter or 250 grams per liter is supplied to the bioreactor at various rates to provide hydraulic dwell times of 4 or 10 hours. The bioreactor is kept at a temperature of about 37 °C. The effluent from the bioreactor is periodically analyzed for ethanol and glucose concentrations. At the 4-hour hydraulic retention time, sugar conversion produces about 95 to 97 percent theoretical ethanol production at each glucose concentration. At the 10-hour hydraulic retention time, sugar conversion produces about 98 to 99 percent theoretical ethanol production at each glucose concentration. XIII. ANAEROBIC DIGESTION
[0441] As discussed above, urban wastewater often undergoes aerobic bioconversion. Supplying oxygen to the bioreactor is a significant expense, even if air is used as the oxygen-containing gas for urban wastewater installation, and often makes up at least about 30 percent of the total costs. In addition, where tertiary treatment is required, an anaerobic bioconversion is used and thus the oxygen concentration in the water that is treated must be reduced. To meet regulatory requirements established in numerous jurisdictions, wastewater treatment must reduce the organic content as well as substantially reduce or eliminate pathogens.
[0442] Mesophilic anaerobic digestion has been proposed. While eliminating the cost of oxygen supply, such processes suffer from numerous disadvantages. Maintaining effective populations of microorganisms has proven difficult, especially since both acidogenesis and methanogenesis must be supported. The residence time is long, often in the range of 15 days, and the process often does not provide sufficient reduction in pathogens.
[0443] Thermophilic anaerobic digestion provides the advantage of a shorter residence time and a better ability to treat pathogens. See, for example, patent application published in U.S. 2013010539. The maintenance of the population of microorganisms still remains problematic, and the waste water must be brought up to and maintained at a temperature of at least 45°C for operation of the thermophilic microorganisms.
[0444] The biocatalysts of this invention provide improvements in anaerobic wastewater digestion in that not only can thermophilic microorganisms be targeted to the biocatalyst as opposed to conventional systems where microorganisms are often derived from the sludge, but also the biocatalyst can provide a high concentration of thermophilic microorganisms per unit volume of bioreactor. The bioconversion rate and thus the hydraulic residence time can be reduced. More importantly, since thermophilic anaerobic bioconversion is exothermic, the high concentration of microorganisms effectively serves as a heat source to obtain and maintain thermophilic bioconversion temperatures. Also, as thermophiles are found in the biocatalyst of this invention, the processes are useful even for the treatment of waste water that has a low organic content.
[0445] The processes for thermophilic anaerobic digestion of waste water containing organic compound comprises: a. placing under thermophilic conditions said waste water in contact with the biocatalyst of this invention containing thermophilic microorganisms suitable for the bioconversion of organic compounds into methane, preferably, said thermophilic conditions comprise a temperature of at least about 45 °C, say, between about 47°C and 65°C or 70°C, for a time sufficient to reduce organic compound concentrations, preferably to a BOD of less than about 10, preferably less than about 4 milligrams of oxygen per liter to supply treated water and biogas, b. separate biogas from waste water and c. separate the treated water from the biocatalyst.
[0446] Preferably, the microorganism used in the biocatalyst comprises a methanogen, especially one or more of the following microorganisms Methanosarcina acetivorans, Methanothermobacter thermautotrophicus, Methanobrevibacter smithii, Methanospirillum hungatei, Candidatus Brocadia anammoxidans, Kuenemonia sp., sp. , and Scalindua sp. The cell concentration in the biocatalyst is preferably at least about 100 or 200 grams per liter. Generally, bioconversion conditions include maintaining a pH in the range of about 6.5 to 9, say, about 7 and 8.5. Often, the oxygen concentration in the wastewater to be brought into contact with the biocatalyst is less than about 2 milligrams per liter. Any suitable bioreactor configuration can be used including, but not limited to, Typical Bioreactor Systems. Preferably, the bioreactor contains enough biocatalyst to provide at least about 100 grams of cells per liter of capacity. XIV. OTHER APPLICATIONS
[0447] The properties of the biocatalysts of this invention allow a wide range of specific applications. For example, the ability to target a high population of microorganisms with a stable population makes the biocatalysts of this invention useful for biological detection devices; coatings including, but not limited to, antifouling paint and coatings, such as for ship hulls and other surfaces immersed in surface water; and filters to remove unwanted components from gases and liquids. Biocatalysts can be used in biofuel cells.
[0448] The biocatalysts of this invention can be used to produce hydrogen or hydrogen equivalents using mesophilic or thermophilic, anaerobic or facultative anaerobic microorganisms. Hydrogen can be recovered or used in another chemical or metabolic process. In such a process, methane can be used to produce hydrogen and then the hydrogen used to reduce sulfate to sulfide using sulfate-reducing microorganisms. Since microorganisms are irreversibly retained in biocatalysts, cocultures, in the same biocatalyst or in different biocatalysts, can be maintained.
[0449] The biocatalysts of this invention may find application in the methanogenesis of carbonaceous substrates especially in methane. Microorganisms that have this bioactivity are typically syntrophs, and the biocatalyst enhances the stability of the syntrophic system.
[0450] The biocatalysts of this invention can be used to produce alkenes such as ethylene, propylene, butene, butadiene and styrene, for example, from carbohydrates such as sugars, synthesis gas and carbon dioxide. See, for example, patent application published in U.S. 20130122563.
[0451] The biocatalysts of this invention can be used for the treatment of various terrains, surfaces, urban wastewater and industrial water streams, as set out above. Additionally, beneficial applications include anaerobic digestion, removal of sulfate and sulfite anions, and a layered catalyst to drive aerobic wastewater treatment. APPENDIX A
[0452] Representative microorganisms include, without limitation, Acetobacter sp., Acetobacter aceti, Achromobacter, Acidiphilium, Acidovorax delafieldi P4-1, Acinetobacter sp. (A. calcoaceticus), Actinomadura, Actinoplanes, Actinomycetes, Aeropyrum pernix, Agrobacterium sp., Alcaligenes sp. (A. dentrificans), Alloiococcus otitis, Ancylobacter aquaticus, Ananas comosus (M), Arthrobacter sp., Arthrobacter sulfurous, Arthrobacter sp. (A. protophormiae), Aspergillus sp., Aspergillus niger, Aspergillus oryze, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus soybean, Aspergillus usaii, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amyloliquefaciens, Bacillus circulaus, Bacillus brevis, Bacillus brevis lentus, Bacillus licheniformis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Beijerinckia sp., Bifidobacterium, Brevibacterium sp. HL4, Brettanomyces sp., Brevibacillus brevis, Burkholderia cepacia, Campylobacter jejuni, Candida sp., Candida cylindracea, Candida rugosa, Carboxydothermus (Carboxydothermus hydrogenoformans), Carica papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium sp. , Citrobacter, Clostridium sp., Clostridium butyricum, Clostridium acetobutylicum, Clostridium kluyveri, Clostridium carboxidivorans, Clostridium thermocellum, Cornynebacterium sp. strain m15, Corynebacterium (glutamicum), Corynebacterium efficiens, Deinococcus radiophilus, Dekkera, Dekkera bruxellensis, Escherichia coli, Enterobacter sp., Enterococcus, Enterococcus faecium, Enterococcus gallinarium, Enterococcus gallinarium, Erbacwinia sp. Hansenula sp., Haloarcula, Humicola insolens, Humicola nsolens, Kitasatospora setae, Klebsiella sp., Klebsiella oxytoca, Klebsiella pneumonia, Kluyveromyces sp., Kluyveromyces fragilis, Kluyveromyces lactis, Kocuria sp. , Lactococcus lactis, Leuconostoc, Methylosinus trichosporum OB3b, Methylosporovibrio methanica 812, Methanothrix sp. Methanosarcina sp., Methanomonas sp., Methylocystis, Methanospirilium, Methanolobus siciliae, Methanogenium organophilum, Methanobacerium sp., Methanobacterium bryantii, Methanococcus sp., Methanomicrobium sp., Methanoplanus sp., Methanoplanus sp. sp., Methanopyrus sp., Methanocorpusculum sp., Methanosarcina, Methylococcus sp., Methylomonas sp., Methylosinus sp., Microbacterium imperiale, Micrococcus sp., Micrococcus lysodeikticus, Microlunatus, Moorella (e.g. Moorella (Clostridium), thermoacetica sp. (strain B), Morganella, Mucor javanicus, Mycobacterium sp. strain GP1, Myrothecium, Neptunomonas naphthovorans, Nitrobacter, Nitrosomonas (Nitrosomonas europea), Nitzchia sp., Nocardia sp., Pachysolen sp., Pantoea, Papaya carica, Pediococcus sp. , Penicillium roqueforti, Penicillum lilactinum, Penicillum multicolor, Phanerochaete chrysoporium, Pichia sp., Pichia stipitis, Paracoccus pantotrophus, Pleurotus ostreatus, Propionibacterium sp., Proteus, Pseudomonas (P. pavonaceae, Pseudomonas. of Pseudomonas PSI, P. cepacia G4, P. medocina KR, P. picketti PK01, P. vesicularis, P. paucimobilis, Pseudomonas sp.DLC-P11, P. mendocina, P. chichhori, strain IST 103), Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Ralstonia sp., Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus n. , Rhizopus oligosporus, Rhodococcus, (R. erythropolis, R. rhodochrous NCIMB 13064), Salmonella, Saccharomyces sp., Saccharomyces cerevisiae, Schizochytriu sp., Sclerotina libertina, Serratia sp., Shigella, Sphingobacterium multivorum, Sphingobium (Sphingbium chloroyphenolicum.), Sphingbium chloroyphenolicum. RW1). , Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon sp., Trichosporon penicillatum, Vibrio alginolyticus, Xanthomonas, Xanthobacter sp. (X autotrophicus GJ10, X flavus), Yeast, Yarrow lipolytica, Zygosaccharomyces rouxii, Zymomonas sp., Zymomonus mobilis, Geobacter sulfurreducens, Geobacter lovleyi, Geobacter metallireducens, Bacteroides succinogens, Euflavorus succinogens, Butyrivibrio bacterium fibrisolvens , Clostridium cellulosolvens, Clostridium cellulovorans, Clostridium thermocellum, Bacteroides cellulosolvens, and Acetivibrio cellulolyticus Gliricidia sp., Albizia sp., or Parthenium sp. Cupriavidus basilensis, Cupriavidus campinensis, Cupriavidus gilardi, Cupriavidus laharsis, Cupriavidus metallidurans, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus pinatubonensis, Cupriavidus respiraculi, Cupriavidus taiwanensis, Thidianthios, Thiidibacillus, spp. albertensis, Acidithiobacillus caldus, cuprithermicus Acidithiobacillus, Rhodopseudomonas palustris Rhodopseudomonas, Rhodobacter sphaeroides, capsulate Rhodopseudomonas, acidophila Rhodopseudomonas, Rhodopseudomonas viridis, Desulfotomaculum, Desulfotomaculum acetoxidans, Desulfotomaculum kuznetsovii, Desulfotomaculum nigrificans, Desulfotomaculum reducens, Desulfotomaculum carboxydivorans, Methanosarcina barkeri, Methanosarcina acetivorans, Moorella thermoacetica , Carboxydothermus hydrogenoformans, Rhodospirillum rubrum, Acetobacterium woodii, Butyribacterium methylotrophicu m, Clostridium autoethanogenum, Clostridium ljungdahlii, limosum Eubacterium, Oxobacter pfennigii, productus Peptostreptococcus, Rhodopseudomonas palustris P4 Rubrivivax gelatinosus, Citrobacter sp Y19, Methanosarcina acetivorans C2A, Methanosarcina barkeri, orientis Desulfosporosinus, desulfuricans Desulfovibrio, Desulfovibrio vulgaris, thermoautotrophica Moorella, Carboxydibrachium pacificus, Carboxydocella thermoautotrophica, Thermincola carboxydiphila, Thermolithobacter carboxydivorans, Thermosinus carboxydivorans, Methanothermobacter thermoautotrophicus, Desulfotomaculum carboxydivorans, Desulfotomaculum kuznetsovii, Desulfotomaculum nigricans, Desulfotomaculum sub thermo. thermosyntrophicum, Syntrophobacter fumaroxidans, Clostridium acidurici, Desulfovibrio africanus, C. pasteurianum, C. pasteurianum DSM 525, Paenibacillus polymyxa, Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achanthidium, Amphinory, Actinas, Actinas, Actinas Amphiprora, Amphithrix, Amphora, Anaaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesbianus, Apatococcus Bambusin, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleris, Carhisoneria, Cattle, Capdis, Cattles Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblelamy, Chlamydosa Chlamydoblepharis, Chlamydoblepharis, Chlamydoblepharis Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysolepidomonas, Chrysolepidomonas , Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmioneis, Crytagen, Crateriportula, Cryptophyta, Crateriportula, Cryptophyta , Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Dercodicarma, Destocula, Despella, Cymbellonitzschia, Derco Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymocystis, Didymogenes, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymocystis, Didymococcus, DicothrixDistrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Eustropia, Eurastrum, Eurastrum Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragillaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Glodecyste, Gloeococcus Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gongrosira, Gongrosira, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Haniuma Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Ha slea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotil, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalogonium, Hydrodoxy, Hydrodicum,leru, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagerheimia, Lampella, Lagerheimia Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloya, Melosyre, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Microsterias, Microchaete, Microcoleus Micron, Microncystis Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mou geotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitellopsis, Nitzstopchia, Odulae Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peranema Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomites, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Pclaeura, Pilectole Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophor a, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosidium, Pymosolidium, Pmosidium, P. Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Stiethioneisrix lla, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerphaenellopsis, Sphaerphaellocysta, Sphaerphaerello Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stikesylsium, Sticlococcus, Sticlococcus, Sticholsyls Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tespora, Tetras, Tetras, trumcham T horea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uvaski, Vacuolaria, Westvulina, Volvoxu Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, Zygonium, Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, Thermomicrobial Thiocystis, Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, Roseospira, Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrospira sp., Nitrospira sp., Nitrospira sp., Nitrospira sp. Nitrosospira sp., Nitrosolobus sp., Nitrosovibrio sp., Thiovulum sp., Thiobacillus sp., Th iomicrospira sp., Thiosphaera sp., Thermothrix sp., Hydrogenobacter sp., Siderococcus sp., Aquaspirillum sp. Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoproteus sp. ., Pyrodictium sp., Sulfolobus sp., Acidianus sp., Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp., oleaginous yeast, Idopsis thal Panicum virgatum, Miscandesse Miscanthus giganteus, Zea mays (plants), Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae), Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green sulphurous bacteria), Chloroflexus auranticusl, Chromatium tepidum and Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter paluspurate sulphurous, and non-sulfur capsulatus Rhodobactemonas.

权利要求:
Claims (19)
[0001]
1. BIOCATALYST, characterized in that it comprises: a solid structure of hydrated hydrophilic polymer that defines an interior structure that has a plurality of interconnected larger cavities that have a smaller dimension of between 5 and 100 microns and a HEV between 1000 and 200,000 and a population of microorganisms, substantially irreversibly retained in the interior structure, said population of microorganisms being at a concentration of between 60 and 750 grams per liter based on the volume defined by the exterior of the solid structure when fully hydrated, in which microorganisms maintain their population substantially stable .
[0002]
2. BIOCATALIZER according to claim 1, characterized in that the HEV is at least 20,000 and the concentration of microorganisms within the solid structure is at least 100 grams per liter based on the volume defined by the outside of the solid structure.
[0003]
3. BIOCATALIZER according to claim 2, characterized in that the solid structure defines an outer skin that has pores of an average diameter between 1 and 10 microns and the pores comprise 1 to 30% of the surface area of the outer skin.
[0004]
4. BIOCATALYST according to claim 1, characterized in that the larger cavities are quiescent.
[0005]
5. BIOCATALYST according to claim 1, characterized in that it contains an exo-network of said microorganisms.
[0006]
6. BIOCATALYST according to claim 1, characterized in that the biocatalyst additionally comprises polysaccharide.
[0007]
7. BIOCATALYST according to claim 1, characterized in that the biocatalyst further comprises a solid sorbent.
[0008]
8. BIOCATALYST according to claim 1, characterized in that said microorganism population is a photosynthetic microorganism and the biocatalyst further comprises a phosphorescent material.
[0009]
9. BIOCATALYST according to claim 1, characterized in that the biocatalyst additionally comprises isolated enzyme.
[0010]
10. BIOCATALYST according to claim 1, characterized in that the microorganism population exhibits metabolic alteration compared to planktonic metabolism, under substantially the same metabolic conditions, or increased resistance to toxins, or both.
[0011]
11. METHOD FOR PRODUCING A BIOCATALYST, as defined in claim 1, characterized in that it comprises: a. forming a liquid dispersion of solubilized precursor for hydrophilic polymer and microorganisms for said biocatalyst wherein the concentration of microorganisms in the liquid dispersion is between 60 and 750 grams per liter; B. subjecting said dispersion to solidification conditions to form a solid structure of the hydrophilic polymer wherein the solid structure has an interior structure that has a plurality of interconnected larger cavities containing said microorganisms, said larger cavities having a smaller dimension of between 5 and 100 microns and where the solid structure has a HEV of between 1000 and 200,000, said solidification conditions do not unduly adversely affect the population of said microorganisms; and c. maintain the solid structure containing microorganisms under conditions that do not adversely affect the population of said microorganisms within the solid structure for a time sufficient to allow the microorganisms to undergo phenotypic changes to maintain their population substantially stable and become so substantially irreversible retained within the solid structure.
[0012]
12. METABOLIC PROCESS, characterized in that it comprises subjecting the biocatalyst, as defined in claim 1, to metabolic conditions that include the presence of substrate to bioconvert said substrate into a bioproduct.
[0013]
13. Process according to claim 12, characterized in that the substrate is carbohydrate and the bioproduct is ethanol.
[0014]
Process according to claim 12, characterized in that a toxin is present and the biocatalyst exhibits increased tolerance to the toxin.
[0015]
15. PROCESS FOR METABOLIZING dissolved ORGANIC CARBON and ammonium cation in a wastewater stream, characterized by comprising: a. continuously passing said wastewater stream to a bioreactor containing biocatalyst, as defined in claim 1, which has substantially irreversibly retained therein microorganisms to metabolize dissolved organic carbon to carbon dioxide and ammonium cation to nitrate or nitrite anion ; B. contacting said bioreactor, said wastewater stream with said biocatalyst in the presence of oxygen for a time sufficient to provide an oxidized effluent that contains less than 5 by mass of ammonium cation and that has a biochemical oxygen demand (BOD) of less than 10 milligrams per liter, where substantially no solids pass from the biocatalyst to the oxidized effluent.
[0016]
16. PROCESS FOR THE BIOLOGICAL REDUCTION OF water-soluble PHOSPHATE, characterized in that it comprises contacting said water in a bioreactor with a biocatalyst, as defined in claim 1, having substantially irreversibly retained therein, phosphate accumulation microorganisms under conditions of accumulation of phosphate for a time sufficient to reduce the concentration of phosphate in said water.
[0017]
17. PROCESS TO TREAT WATER containing a soluble compound of metal or semimetal, characterized in that it comprises: (a) continuously introducing said water into a reaction zone containing a biocatalyst, as defined in claim 1, containing a microorganism to reduce metabolically said soluble compound; (b) contacting water with said biocatalyst for a time sufficient to reduce the concentration of said water-soluble compound; (c) maintaining said biocatalyst under metabolic conditions sufficient to metabolically reduce the oxidation state of the metal or semimetal to form semimetal or elemental metal or precipitated compound thereof; and (d) withdrawing water having a reduced concentration of said soluble compound from the bioreaction zone.
[0018]
18. PROCESS FOR BIOCONVERSION OF SUBSTRATE contained in a gas phase to a bioproduct, characterized in that it comprises: a. continuously contacting the gas phase with the biocatalyst as defined in claim 1, said contact being at a temperature suitable for metabolic bioconversion and for a time sufficient to effect said bioconversion of a portion of the substrate to a bioproduct; B. cycling a portion of the biocatalyst from step (a) to an immersion step in an aqueous medium for a time sufficient to substantially hydrate the biocatalyst and, wherein the aqueous medium, to one of said immersion step, which comprises nutrients for the microorganisms and said immersion is for a time sufficient to supply nutrients in said biocatalyst; ç. separating the biocatalyst and aqueous medium from an immersion step; and d. use a portion of the separate biocatalyst for step (a).
[0019]
19. Process according to claim 12, characterized in that the substrate is nitrate anion, or perchlorate anion, or both.

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公开号 | 公开日

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US10752528B2|2020-08-25|

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US9212358B2|2015-12-15|

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JP6298458B2|2018-03-20|

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法律状态:
2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-02-23| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/06/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261689925P| true| 2012-06-15|2012-06-15|

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US201261689945P| true| 2012-06-15|2012-06-15|

---

US201261689953P| true| 2012-06-15|2012-06-15|

---

US201261689921P| true| 2012-06-15|2012-06-15|

---

US201261689943P| true| 2012-06-15|2012-06-15|

---

US201261689939P| true| 2012-06-15|2012-06-15|

---

US201261689929P| true| 2012-06-15|2012-06-15|

---

US201261689940P| true| 2012-06-15|2012-06-15|

---

US201261689922P| true| 2012-06-15|2012-06-15|

---

US201261689930P| true| 2012-06-15|2012-06-15|

---

US201261689933P| true| 2012-06-15|2012-06-15|

---

US201261689924P| true| 2012-06-15|2012-06-15|

---

US201261689935P| true| 2012-06-15|2012-06-15|

---

US201261689932P| true| 2012-06-15|2012-06-15|

---

US201261689923P| true| 2012-06-15|2012-06-15|

---

US61/689,929|2012-06-15|

---

US61/689,924|2012-06-15|

---

US201361849725P| true| 2013-02-01|2013-02-01|

---

US61/849,725|2013-02-01|

---

US201361850631P| true| 2013-02-20|2013-02-20|

---

US201361851467P| true| 2013-03-08|2013-03-08|

---

US61/851,467|2013-03-08|

---

US201361852451P| true| 2013-03-15|2013-03-15|

---

US61/852,451|2013-03-15|

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PCT/US2013/046037|WO2013188858A2|2012-06-15|2013-06-14|Novel biocatalyst compositions and processes for use|

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