• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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  • 优先权
    • 专利摘要:
    The present invention comprises a bacterial strain designated as Bacillus aryabhattai DC100 (CECT 9226), for the elimination of colored compounds and organic matter in wastewater and/or effluents. The invention also relates to a process comprising the following steps: production of the inoculum of the strain Bacillus aryabhattai DC100 (CECT 9226), characterization and, if necessary, supplementation of the waste water and/or effluent to be treated, and finally the treatment of the same by the bacterial strain previously obtained. This procedure allows the complete decolorization of colored compounds preferably type triphenylmethane and indigotine, as well as reductions of dissolved organic matter or in suspension present in waters and/or effluents from industries that carry out some staining or coloring process, such as textile, paper, cosmetic, pharmaceutical and/or food. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2674338A1
    申请号:ES201601101
    申请日:2016-12-28
    公开日:2018-06-28
    发明作者:Alicia PÉREZ PAZ；José Manuel DOMÍNGUEZ GONZÁLEZ
    申请人:Universidade de Vigo；
    IPC主号:
    专利说明:

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    BACTERIAN BACILLUS CEPA AND PROCEDURE FOR THE ELIMINATION OF COLORED COMPOUNDS AND ORGANIC MATTER IN WASTEWATER
    SECTOR OF THE TECHNIQUE
    The present invention consists of a biological process for the elimination of colored compounds and organic matter present in wastewater and effluents from the activities of the textile, paper, cosmetic, pharmaceutical and / or food industry. This process is likely to be used by companies specialized in the treatment of water and residual effluents mentioned above.
    GLOSSARY OF TERMS AND ABBREVIATIONS
    The terms and abbreviations employed in the present invention have the following meaning.
    - Pigment: Biodegradable substance present in nature, which can be extracted from plants, insects and / or minerals, and which has the ability to color / dye materials.
    - Coloring: Synthetic colored compound non-biodegradable and highly toxic in solution, created artificially for staining materials.
    - Colored compound: In the present invention it refers to a substance that confers color, being able to be a pigment or dye.
    - Wastewater: Water from industrial activities, not suitable for human consumption. In the present invention, essentially those wastewater that has colored compounds and dissolved organic matter.
    - Effluents: In the present invention, liquid or semi-liquid waste from industries that carry out staining or coloring processes.
    - COD: Abbreviation for chemical oxygen demand. Parameter that indicates the amount of substances (dissolved or suspended organic matter) present in a liquid sample and that are susceptible to being oxidized. It allows to determine the degree of contamination and is expressed in milligrams of diatomic oxygen per liter (mgOi / L).
    - Species: Set of populations or bacterial strains, which have a high degree of morphological and phenotypic similarity, but which are clearly differentiable from other bacterial strains.
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    - Bacterial strain: A set of homogeneous cells from the reproduction of a single initial cell (selected and isolated).
    - Pure culture: Multiplication of a single bacterial strain.
    - Mixed culture: Multiplication of different bacterial strains, not necessarily of the same species.
    - Inocula: Amount of pure culture taken for the preferred embodiment of the procedure established in the present invention.
    BACKGROUND OF THE INVENTION
    The use of colored compounds for aesthetic and / or communicative purposes has been carried out since prehistoric times. Formerly pigments obtained from natural sources such as plants, insects and / or minerals were used, but their extraction and production was expensive and, over time, inefficient to supply all the demand. With the subsequent development of synthetic dyes, much more stable than natural ones, the reduction in production costs was notable. However, these economic advantages implied a series of negative aspects about the environment and human health.
    A dye, even in a very low concentration, is considered toxic or dangerous for several reasons. In the first place, its uncontrolled discharge into the environment can cause mortality of the existing fauna and flora, either by inhibition of photosynthesis in the case of autotrophic organisms, or by toxicity. Secondly, since anaerobic digestion is not easily biodegradable, it can generate highly toxic, carcinogenic and mutagenic aromatic amines. Third, there are numerous cases of human allergies and intolerances to these synthetic colored compounds. In addition to this problem, the wastewater from the industries that carry out some dyeing or coloring process, usually contain other types of substances, becoming much more complex and toxic effluents. Taking as an example a textile industry, in general its effluents are constituted by an amount of residual dye (10-200 mg / L) and auxiliary compounds such as salts, detergents and oils that help to fix them. Therefore, apart from being visually unsightly or undesirable, they are highly alkaline and saline, presenting suspended solids from the material to be dyed. This entails the need to
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    specific treatments before discharge, assuming an extra cost for companies by requiring additional technology or by hiring specialized external companies.
    Generally treatments are classified as chemical, physical and biological, giving priority to the use of the first 2 compared to the third. The disadvantages of physical and / or chemical treatments are that they involve a high economic investment, are usually specific for a certain type of compounds and also generate waste that is still necessary to treat. Biological treatments have the disadvantage of requiring more time for their performance, but they are much cleaner and cheaper procedures.
    Spanish regulations in line with European regulations set certain main parameters to be taken into account before allowing direct or indirect discharge (C. Hessel et al. 2007. Journal of Environmental Management. 83 171-180). Some of them are: biological oxygen demand (BOD), chemical oxygen demand (COD), suspended solids, salinity, color, the presence of oils and heavy metals. However, if the discharges are not made directly to the public channel, the concentration of each of them will depend on the purification capacity of the purification station and therefore the body that manages it. For example, in the wastewater treatment plants in Galicia, discharge limits of 1000 mg / L are established for the COD and for the color that is negligible in dilution 1/30 (DECREE 141/2012, of June 21, which approves the Framework Regulation of the Public Wastewater Sanitation and Purification Service of Galicia Official Gazette of Galicia, July 6, 2012. 129: 26924-26958).
    EXPLANATION OF THE INVENTION
    One aspect of the invention relates to a new bacterial strain belonging to the species Bacillus aryabhattai. This species has been discovered and deposited for the first time in the American and Japanese type culture collections in 2006. Isolated from cryotubes used for sampling upper areas of the atmosphere, the applications attributed so far have been in the field of heavy metal bioremediation; the production of biopolymers; or the production of asparaginase.
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    However, the bacterial strain Bacillus aryabhattai DC100 (CECT 9226) object of the invention has been isolated by depletion from contaminated cryovial containers of the species Streptomyces setonii contaminated. To do this, from a mixed culture, the different morphologies present were strewn, until pure culture plates were obtained, with the same morphology and Gram staining. The presence of the desired characteristics was then validated in some of the isolated bacterial strains and was identified by direct amplification by PCR of the 16S rRNA gene and partial sequencing thereof (with readings in both directions).
    This bacterial strain has been deposited in the Spanish Type Culture Collection (CECT), dated 11/23/2016, where it has been assigned the number 9226. The CECT is based in the research building of the University of Valencia, Stop Scientist University of Valencia Street Professor Agustín Escardino, 9; 46980 Paterna (Valencia).
    Another aspect of the invention relates to the method and use of the present bacterial strain Bacillus aryabhattai DC100 (CECT 9226), for the removal of colored compounds and organic matter in wastewater, which comprises the following steps:
    a) Production of the Bacillus aryabhattai DC100 inoculum (CECT 9226), which includes growth in suitable conditions until reaching the stationary phase, subsequent centrifugation, washing and resuspension with a buffer solution at a concentration between 2-5 g / l by weight dry.
    b) Characterization and supplement of the wastewater to be treated, which includes an analysis of the salt, nitrogen and carbon content of the effluent, and if necessary a subsequent supplement in order to reach at least 30-70% of nitrogen, between 1-10% carbon and between 10-30% of salts necessary for further development of the inoculum obtained in stage a) during the biological treatment.
    c) Biological treatment in the appropriate conditions of aeration, temperature and pH of the wastewater characterized and supplemented in section b) by the inoculum obtained in stage a)
    In a preferred embodiment, the production step of the Bacillus aryabhattai DC100 inoculum (CECT 9226) is performed in a medium comprising the following composition:
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    • 15-30 g / L casein peptone
    • 3-10 g / L of soy peptone
    • 3-10 g / L sodium chloride
    • 2-5 g / L glucose
    • 2-5 g / L hydrogen dipotassium phosphate
    The culture conditions are between 30-37 ° C and 100-150 rpm for 12-24h. Subsequently, centrifugation is performed at 6000 rpm for 10 minutes, and the subsequent washing step is carried out twice with phosphate buffered saline, until reaching a concentration around 2-5 g / L.
    In another preferred embodiment, the conditions of the biological treatment of the wastewater obtained in stage b) comprise incubating the inoculum obtained in stage a) at temperatures between 25-46 ° C, stirring between 100-400 rpm, and a pH between 6-12, for a time between 24-72 hours that will depend on the dye load to be degraded. The absorbance is monitored, and the COD is measured according to ISO 15705.
    With the procedure described here, some of the problems presented by wastewater in those industries that carry out staining or coloring processes are solved. In this way, the process developed here allows the complete decolorization of triphenylmethane and indigotine-type dyes and COD reductions that vary around 50% on effluents from the textile industry. But in addition, the process described here has several advantages. First of all and due to the extremophilic nature of the bacterial strain object of the invention, it is possible to treat the wastewater directly and without prior procedures such as neutralization of the effluent. Second, the low cost of the process object of the invention described herein. Third, depending on the composition of the wastewater to be treated, the brevity and simplicity of the treatment. Fourth, the diversity of compounds that it tolerates and that is capable of degrading the bacterial strain object of the invention.
    Another aspect of the invention relates to the method and use of the present bacterial strain Bacillus aryabhattai DC100 (CECT 9226), in the biological treatment of wastewater for the removal of colored compounds, more specifically of
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    triphenylmethane and indigotine type dyes.
    Finally, another aspect of the invention relates to the method and use of the present bacterial strain Bacillus aryabhattai DC100 (CECT 9226), in the biological treatment of wastewater for the reduction of organic matter (COD) present in them.
    All this entails highlighting the use of Bacillus aryabhattai DC100 (CECT 9226) in effluents from the textile, paper, cosmetic, pharmaceutical and / or food industry.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1.- Spectrophotometric scanning of the supernatant in the range of appearance of the triphenylmethane type dye used (Example 1). Continuous line: time Oh; dotted line: 24h time; dotted and striped line: 48h time and striped line: 72h time. Figure 2. - Discoloration process of a triphenylmethane type dye (Example 1). a) Time 0; b) Time 48h; c) Time 72h.
    Figure 3.- Spectrophotometric scanning of the supernatant in the range of appearance of the indigotine type dye used (Example 2). Continuous line: time Oh; striped line: 24h time; dotted line: time 48h.
    Figure 4.- Spectrophotometric scanning of the supernatant of the decolorization process of an effluent from a textile industry. Continuous line: time Oh; dotted and striped line: 24h time.
    Figure 5.- Percentages of COD reduction of the proposed examples. A. Line with a triangle-shaped marker corresponding to the bleaching process of triphenylmethane-type dyes with Bacillus aryabhattai DC100 (CECT 9226) (Example 1); B. Line with circle marker that corresponds to biological treatment of the effluent of a textile industry with Bacillus aryabhattai DC100 (CECT 9226) (Example 3); C. Line with corresponding square marker biological treatment of semi-liquid effluent from the textile industry using Bacillus aryabhattai DC100 (CECT 9226) (Example 4).
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    In the following, four possible examples of carrying out the biological procedure for the removal of colored compounds in wastewater are detailed. The first example concerns the use of Bacillus aryabhattai DC100 (CECT 9226) for the degradation of a triphenylmethane type dye; the second example refers to the discoloration of indigotine class dyes; the third example to a real application of Bacillus aryabhattai DC100 (CECT 9226) on a liquid effluent from the textile industry; and finally Example 4 refers to the biological treatment of a semi-liquid effluent from the textile industry with the bacterial strain object of the invention.
    A preferred embodiment of the invention would comprise obtaining the inoculum at the desired concentration. Subsequently, the characterization of the effluent to be treated and supplementation if necessary. Finally, the start-up of the biological treatment of the fading process.
    With the process described here, some of the problems presented by some industrial processes that consume high volumes of water are solved. Due to the lack of information regarding the composition of the wastewater generated by specific processes, as an example to explain the present invention, the average values of the different processes carried out by a company in the textile sector have been taken (table one). (Guide to best techniques available in Spain in the textile sector, 2004, Ministry of Environment).
    With the treatment described here the color degradation is complete for dyes of the triphenylmethane and indigotine type and partial in terms of the reduction of organic matter.
     Process  COD PH (mg / L) Color (mg Pt-Co-I)
     Fiber and Yarn Dyeing  10-12 500-1200 300-1000
     Dyeing and finishing of wool and cotton fabric  6-12 300-2800 500-3000
     Stamping and finishing  6-9 350-2300 Not quantified
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    Example 1.- Process of decolorization of a triphenylmethane type dye with Bacillus aryabhattai DC100 (CECT 9226).
    The process begins by reactivating a cryovial of Bacillus aryabhattai DC100 (CECT 9226), previously isolated in our laboratory and stored at -80 ° C in a 20% glycerol solution. For this, a culture medium is prepared with the nutritional requirements previously described (15-30 g / L of casein peptone; 3-10 g / L of soy peptone; 3-10 g / L of sodium chloride; 2-5 g / L of glucose and 2-5 g / L of dipotassium hydrogen phosphate) and it is left incubating a maximum of 24h, with a constant stirring between 100-150 rpm and a temperature of 30-37 ° C. Previously, its cell cycle, its optical density (D O Aeoo) and the production of biomass by dry weight will have been studied. For this, every 2 hours, 1.5 ml aliquots are taken and their absorbance in the spectrophotometer will be measured at a wavelength of 600 nm (D.O. 6oo). To determine the biomass by dry weight, 5 ml of the culture will be taken, filtered through a filter of pore size 0.20 pl and allowed to dry to constant weight at 105 ° C. When the culture is in a stationary phase, it will be centrifuged at 6000 rpm for 10 minutes and washed twice with phosphate buffered saline, this being the inoculum to be used in the biological treatment of the bleaching process.
    On the other hand a simulation of wastewater will be prepared. Firstly, a stock solution of 500 mg / L of a triphenylmethane-type dye will be available by filtration. A given volume of this solution will be added to a culture medium (previously autoclaved for 15 minutes at 121 ° C) containing between 30 and 70% nitrogen, between 1 and 10% carbon and between 10 and 30% salts. The pH will then be adjusted between 6-12 and inoculated with the Bacillus aryabhattai DC100 inoculum (CECT 9226) previously obtained. It will then be incubated at a temperature between 25 - 46 ° C and stirring between 100 - 400 rpm until complete discoloration. At regular intervals of time, 1.5 ml aliquots will be taken, the pH will be measured, the absorbance at the maximum length of! dye and COD according to ISO 15705. In addition, a spectrum scan will also be performed to confirm that no other type of compound is generated. As can be seen in Fig. 1, the decrease in the absorbance peak is notable, the percentage of discoloration being 99.163 ± 0.080%. In Fig. 2 we show the process of discoloration over time. As for the COD, it can be seen in Figure 5.A that the reduction is 42.06 ± 0.86%.
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    Example 2.- Process of decolorization of an indigotine type dye with Bacillus aryabhattai DC100 (CECT 9226).
    Initially we proceed to reactivate a cryovial of Bacillus aryabhattai DC100 (CECT 9226) isolated in our laboratory and stored at -80 ° C in a 20% glycerol solution. For this, a culture medium is prepared with the nutritional requirements previously described (15-30 g / L of casein peptone; 3-10 g / L of soy peptone; 3-10 g / L of sodium chloride; 2-5 g / L of glucose and 2-5 g / L of dipotassium hydrogen phosphate) and it is left incubating a maximum of 24h, with a constant stirring between 100-150 rpm and a temperature of 30-37 ° C. Previously, the cell cycle, optical density (D.O Aeoo) and biomass production by dry weight will have been studied. For this, every 2 hours, 1.5 ml aliquots are taken and their absorbance in the spectrophotometer will be measured at a wavelength of 600 nm (D.O. 6oo). To determine the biomass by dry weight, 5 ml of the culture will be taken, filtered through a filter of pore size 0.20 pl and allowed to dry to constant weight at 105 ° C. When the culture is in a stationary phase, it will be centrifuged at 6000 rpm for 10 minutes and washed twice with phosphate buffered saline. This will be the inoculum to be used in e! Biological treatment of the fading process.
    Then, the simulation of wastewater will be prepared, and for this a stock solution of 500 mg / L of an indigotine type dye sterilized by filtration will be available. A given volume of this solution will be added to a culture medium (previously autoclaved for 15 minutes at 121 ° C) containing between 30 and 70% nitrogen, between 1 and 10% carbon and between 10 and 30% salts. The pH will then be adjusted between 6-12 and inoculated with the Bacillus aryabhattai DC100 inoculum (CECT 9226) previously obtained. It will then be incubated at a temperature between 25 - 46 ° C and stirring between 100 - 400 rpm until complete discoloration. At regular intervals of time, 1.5 ml aliquots will be taken and the pH and absorbance will be measured at the maximum length of the dye. A spectrum scan will also be performed to confirm that no other type of compound is generated. As can be seen in Fig. 3, as in Example 1, the reduction is total, obtaining 99.546 ± 0.057% discoloration.
    Example 3. Process of decolorization of the effluent of a textile industry with Bacillus aryabhattai DC100 (CECT 9226).
    Initially we prepare a culture medium suitable for the growth of Bacillus
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    aryabhattai DC100 (CECT 9226), bacterial strain isolated in our laboratory and stored at -80 ° C in a 20% solution of glycerol. The composition is that described in the previous example: 15-30 g / L casein peptone; 3-10 g / L of soy peptone; 3-10 g / L sodium chloride; 2-5 g / L glucose and 2-5 g / L hydrogen dipotassium phosphate. The incubation time will be given by previous studies of its cell cycle and biomass production. To do this, 1.5 ml aliquots are taken every 2 hours and their absorbance in the spectrophotometer will be measured at a wavelength of 600 nm (D.O.6oo). As for the determination of biomass by dry weight, 5 ml of culture will be taken, filtered through a filter of pore size 0.20 pl and allowed to dry to constant weight at 105 ° C. When the inoculum is in a stationary phase, it will be centrifuged at 6000 rpm for 10 minutes and washed twice with phosphate buffered saline.
    Next, an effluent from a textile industry will be characterized and adapted by supplementation to the following parameters: between 30 and 70% nitrogen, between 1 and 10% carbon and around 10-30% of you go out. The pH will then be adjusted between 6-12 and inoculated with the Bacillus aryabhattai DC100 inoculum (CECT 9226) previously obtained. It will be incubated at a temperature between 25 - 46 ° C and stirring between 100 - 400 rpm. At regular intervals of time, 1.5 ml aliquots will be taken, the pH, the absorbance at the maximum length of the dye and the COD will be measured according to ISO 15705. A larger spectrum scan will also be performed to confirm that no one is generated. Other type of compound. As can be seen in Fig. 4, the fall in its absorption spectrum is total, reaching 100%. As for the COD, it can be seen in Figure 5.B that the reduction is 47.70 ± 0.78%.
    Example 4.- Treatment of a semi-liquid effluent from a textile industry with Bacillus aryabhattai DC100 (CECT 9226).
    In this example, it is based on semi-liquid effluents from a textile industry !, which, as in example 4, will be characterized and supplemented until a composition of between 30 and 70% nitrogen, 1 and 10% is obtained. carbon and about 10-30% salts. On the other hand, the inoculum of Bacillus aryabhattai DC100 (CECT 9226), bacterial strain isolated in our laboratory object of invention preserved at -80 ° C in a 20% glycerol solution, in a culture medium whose composition is: 15-30g / L casein peptone; 3-10 g / L of soy peptone; 3-10
    g / L sodium chloride; 2-5 g / L glucose and 2-5 g / L hydrogen dipotassium phosphate. Previously, their cell cycle and biomass production will have been studied. To do this, 1.5 ml aliquots are taken every 2 hours and their absorbance in the spectrophotometer will be measured at a wavelength of 600 nm (D or Á6oo). As for the determination of biomass by dry weight, 5ml of the culture will be taken, filtered through a filter of pore size 0.20 pl and allowed to dry until constant weight at 105 ° C. When the inoculum is in a stationary phase, it will be centrifuged at 6000 rpm for 10 minutes and washed twice with phosphate buffered saline. This culture will be used to inoculate the medium prepared with the semi-liquid effluents from a textile industry. The incubation will be carried out at a temperature between 25-46 ° C and stirring between 100-400 rpm. At regular intervals of time, aliquots will be taken to measure the COD according to ISO 15705. Figure 5.C shows that the reduction is 14.09 ± 0.71%.
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    权利要求:
    Claims (1)
    [1]
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    Bacillus aryabhattai DC100 bacterial strain deposited in the CECT with the CECT code 9226.
    Use of the bacterial strain Bacillus aryabhattai DC100 (CECT 9226), according to claim 1, in the biodegradation of dyes of the triphenylmethane or indigotine type.
    Process for the removal of colored compounds and organic matter in wastewater according to claims 1 and 2, comprising the following steps:
    a) Production of the Bacillus aryabhattai DC100 inoculum (CECT 9226) in a culture medium of composition: 15-30g / L of casein peptone; 3-10 g / L of soy peptone; 3-10 g / L sodium chloride; 2-5 g / L of glucose and 2-5 g / L of dipotassium hydrogen phosphate, until reaching the stationary phase, subsequent centrifugation and washing with a buffer solution and preparation of a concentration between 2-5 g / l in dry weight .
    b) Characterization and supplement of the wastewater and / or effluent to be treated that includes an analysis of the salt, nitrogen and carbon content, and subsequent supplement if necessary in order to reach at least 30-70% of nitrogen, between 1-10% carbon and 10-30% salts necessary for the development of the inoculum obtained in stage a) in the subsequent biological treatment.
    c) Biological treatment by inoculum obtained in stage a) of the wastewater characterized and supplemented in stage b).
    Method according to claim 3, characterized by the culture medium used for the production of the Bacillus aryabhattai DC100 inoculum (CECT 9226) according to step a) comprising the following composition:
    • 15-30 g / L casein peptone;
    • 3-10 g / L of soy peptone;
    • 3-10 g / L of sodium chloride;
    • 2-5 g / L glucose;
    • 2-5 g / L of hydrogen dipotassium phosphate;
    where the culture conditions are between 30 ° C-37 ° C and 100-150 rpm for 12-24h. Once the stationary phase is reached, it is centrifuged at 6000 rpm for 10 minutes, washed twice with phosphate buffered saline, and resuspended until
    reach a concentration around 2-5 g / L.
    Method according to claim 3, wherein the biological treatment according to step c) is characterized by incubating the inoculum obtained in step a. in the effluent characterized in step b) at temperatures in the range of 25-46 ° C, stirring between 100-400 rpm, and pH between 6-12, for a time between 24-72 hours that will depend on the dye load to degrade.
    Use of the Bacillus aryabhattai DC100 strain (CECT 9226) and procedure for the removal of colored compounds in wastewater and effluent.
    Use of the bacterial strain Bacillus aryabhattai DC100 (CECT 9226) and procedure for the reduction of the organic matter present in wastewater and effluent.
    Use of the bacterial strain Bacillus aryabhattai DC100 (CECT 9226) and procedure for the elimination of colored compounds and organic matter in wastewater and effluents from the textile, paper, cosmetic, pharmaceutical and / or food industry.
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    同族专利:
    公开号 | 公开日
    ES2674338B2|2018-11-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1994018312A1|1993-02-01|1994-08-18|The Chinese University Of Hong Kong|Microbial degradation of indigo and indigo carmine|
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