Procedure for the increase of lipase production in Halomonas cultures by chemical and biological ind

专利摘要:
The present invention relates to a process for increasing the production of lipases in cultures of microorganisms of the genus Halomonas by chemical and/or biological induction. The invention relates to a process of increased production of the enzyme lipase in an organism of the genus Halomonas by chemical induction comprising the design of the culture medium by the addition of the chemical agent, preparation of the Halomonas inoculum, growth of the pure culture of Halomonas. and production of lipase in the presence of the chemical agent. Or, by biological induction comprising preparation of the culture medium and Halomonas inoculum, growth of the culture and production of lipase in a mixed culture composed of Halomonas and the inducing microbial agent. (Machine-translation by Google Translate, not legally binding)
公开号:ES2677443A1
申请号:ES201700078
申请日:2017-02-01
公开日:2018-08-01
发明作者:Francisco Javier DEIVE HERVA；María Ángeles SANROMÁN BRAGA；Ana María RODRÍGUEZ RODRÍGUEZ；Esther GUTIERREZ ARNILLAS
申请人:Universidade de Vigo；
IPC主号:

专利说明:

Procedure for increasing lipase production in Halomonas crops
through chemical and biological induction.
5 SECTOR OF THE TECHNIQUE
The present invention is framed in the field of industrial microbiology, more specifically in the production of biomolecules from endophilic microorganisms. Because one of the biggest problems of enzymatic synthesis from this type of microorganisms lies in the low levels of activity achieved, 10 it is intended to increase, through chemical and / or biological induction, the low levels of lipase production of a recently isolated halophyla strain in southern Spain, Halomonas sp LM1C (Gutierrez-Arnillas et al., 2016, New sources of halophilic lipases: Isolation of bacteria from Spanish and Turkish saltworks, Biochem. Eng. J. 109,170-177), demonstrating its viability at a flask and bioreactor scale. The lipase produced by this procedure can be used in the food, textile, pharmaceutical, detergent and paper industry, in the production of biodiesel and cosmetics and in the treatment of wastewater (Daiha et al., 2015, Are Lipases Still Important Biocatalysts A Study of Scientific Publications and Patents for Technological Forecasting, PLoS ONE 10 (6)).
20 STATE OF THE TECHNIQUE
The report "Lipase Market by Source (Microbial Lipases, Animal Lipases), Application (Animal Feed, Dairy, Bakery, Confectionery, Others), & by Geography (North America, Europe, Asia-Pacific, Latin America, RoW) - Global Forecast to 2020 ", prepared by MarketsAndMarkets®, foresees that the lipase market will reach almost 600 million of 25 dollars in 2020, with a compound annual growth rate (CAGR, Compound annual growth rate) of 6.5% between 2015 and 2020. Lipases (EC 3.1.1.3) are catalysts with a high degree of specificity and speed of reaction, whose main function is the hydrolysis of triglycerides. In addition, in media with low water activity, they exert their catalytic function in esterification, transesterification and
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Intersterification The versatility of these enzymes allows their diversification in numerous industrial sectors, boosting their worldwide success. Industrial development promotes the demand for lipases by the detergent, food, textile, pharmaceutical, cosmetic, petrochemical industry, among others. Similarly, new emerging sectors, such as biofuels, are developing new more sustainable production techniques thanks to the use of lipases {Houde et al., 2004, Lipases and their industrial applications: An overview, Appl. Biochem Biotechnol Part A Enzym. Eng. Biotechnol. 118, 155-170).
Leading companies in the lipase market such as Novozymes® (Denmark), Koninklijke DSM N.V.®, (Netherlands), Chr. Hansen Holdings A / S® {Denmark), and £. I. Du Pont de Nemours and Company® (United States), orient their efforts to the search for new more competitive sources of production as well as new applications. Although there are lipases of animal and plant origin, it has been shown that microbial lipases have a greater interest due to advantages such as their great catalytic diversity, high yields, and the possibility of improving the competitiveness of production processes through genetic modification or the use of waste as substrates (Sangeetha et al., 2011, Bacterial lipases as potential industrial biocatalysts: An overview, Res. J. Microbiol., 1-24).
The need for new, more resistant enzymes, which maintain their ability to catalyze under extreme conditions usually found in industrial processes, has promoted the interest of finding new producing organisms among the extremity. Most of them are microorganisms that can be classified according to the type of extreme conditions in which they live. Thus, thermophilic or psychophilic organisms are those with an optimum growth temperature greater than 60 ° C or below 15 ° C, respectively. Similarly, organisms have been found surviving at pressures above 130 MPa (piezophils), while radiophilic organisms such as Deinococcus radiodurans support high levels of radiation (Rothschild and Mancinelli, 2001, Life in extreme environments, Nature 409,1092- 1101). Based on their survival in habitats with high (> 9) and low (near 0) pH levels, alkalophils and acidophilus are typical, respectively, and those organisms that are capable of growing in environments with
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Low water content are classified as xerophyls. Finally, halophilic organisms develop at high concentrations of salt (between 2 and 5 M NaCI) {Kamekura et al., 1998, Diversity of extremely halophilic bacteria, Extremophiles 2, 289-295).
As already mentioned, the main attraction of these microorganisms lies in their ability to synthesize enzymes that remain active when operating in the extreme conditions in which the microorganism is able to develop. In this sense, haloenzymes exert their catalytic action not only at high salt concentrations but also in the presence of organic solvents and high temperatures (Oren, 2010, Industrial and environmental applications of halophilic microorganisms, Environ. Technol. 31, 825-834) . Apart from these characteristics, the phylogenetic diversity observed in halophiles, which induces from archaeas, bacteria and fungi to protists, protozoa and algae, is another attraction to ensure the existence of enzymes with characteristics suitable for a given application.
One of the advantages of lipase microbial production is that the culture medium and operational conditions can be optimized to maximize enzymatic biosynthesis. However, in most cases production levels are low and the use of inducing agents that generate increases in enzymatic production in microbial cultures is necessary (Benjamin and Pandey, 1996, Optimization of liquid media for lipase production by Candida rugosa, Bior.Technol., 55, 167-170). The induction agent is chosen based on the enzyme of interest. Most of the substances used as inducers in microbial cultures are chemical or of an organic nature, in any case, it is an increase in production costs. In the case of lipases, several studies show good results of induction with compounds of a lipidic nature.
The inducing agents can be differentiated depending on the mechanism of action. In most cases the inductor used has a direct effect on the catalytic activity. So, the inducers widely used in the production of lipases are fatty acids, triglycerides and some esters. In the case of fatty acids, there is a relationship between chain size and enzymatic production. Increased lipolytic activity is observed with increasing chain length,
It may be related to the solubility of fatty acids in water and the phenomenon of interfacial activation. Also, it has been observed that fatty acids induce higher levels of production compared to other compounds of a lipidic nature (Obradors et al., 1993, Effects of different fatty acids in lipase 5 production by Candida rugosa, Biotechnol. Letters, 15, 357 -360). Triglycerides such as olive, sunflower, palm, coconut, corn, etc., exhibit good results in crops for lipase production {Gulati R, Saxena RK, Gupta et al., 1999, Parametric optimization of Aspergillus terreus lipase production and its potential in ester synthesis, Proc. Biochem., 35, 459-464). On the other hand, other compounds that can contribute to increase extracellular lipolytic activity due to their ability to increase the permeability of the cell membrane and facilitate the release of enzymes to the environment, are surfactants (Dominguez et al., 2003, Effect of lipids and surfactants on extracellular lipase production by Yarrowia lipolytica, J. Chem. Technol. and Biotechnol., 78,1166-1170).
15 This type of induction by means of a chemical agent is widely used and in many cases the increase in costs is balanced with the results obtained from enzymatic production. However, there is another method of induction that can substitute or coexist with chemical induction. Biological induction has been extensively studied in the production of lacquers and allows an increase in the levels of production of lacquers by mixed culture (Higher Council for Scientific Research. Method for increasing the production of laccase in Chorioolopsis by means of a biological inducer. Spanish patent 2 365 460,). However, there is no reference about biological induction processes in microbial cultures for lipase production. In fact, the absence of studies focusing on field 25 of halophilic lipases has so far prevented its large-scale marketing. For this reason, the investigation of agents that induce lipolytic activity and the implementation at bioreactor scale of cultures of production of halophilic lipase is crucial to contribute to the development of new catalytic processes with less environmental impact than conventional methods.
In one aspect of the present invention it refers to a process of lipase production by the bacterium of the genus Halomonas by chemical induction comprises the following steps:
5 a) design of the culture medium by adding the chemical agent;
b) preparation of Halomonas inoculum;
c) growth of a pure Halomonas culture and lipase production in the presence of the chemical agent.
The base composition of the culture medium is (modified CECT 17):
10 7.5 g / L casein peptone, 10.0 g / L yeast extract, 3.0 g / L citrate
trisodium, 2.0 g / L of KCI, 20g / L of MgSCV7H20, 0.05 g / L of FeS04-7H20, 0.25 mg / L of MnS04-4H20 and 150.0 g / L NaCI.
It should be noted that the base composition of the culture medium may undergo certain modifications depending on the type of induction chosen, that is, depending on the type of induction, chemical or biological, it is possible to opt for the use of other base compositions of the culture medium other than the one listed in the previous paragraph.
In a particular embodiment of the invention, the chemical induction will be carried out by means of a chemical agent, which is present in a concentration between 0.5-2 g of inductor per liter of dissolution of culture medium. The chemical agents used can be:
20 a) fatty acids of formula I, such as pentadecanoic acid corresponding to the acid with n = 13.
image 1
b) Glycerol or variants such as triglycerides with formula II can also be used. The composition of triglycerides in terms of saturated (x), monounsaturated (y) and polyunsaturated (z) acids implies more or less inductive effect on the
Lipase production. For example, sunflower oil with a higher percentage of saturated than unsaturated acids exhibits a high degree of induction.
image2
In view of the hydrophobic nature of most of these lipid substances, a surfactant is used to increase the bioavailability of the inducer in the medium. The surfactants selected and of which they are also used as inducing agents individually are the non-ionic surfactants of formula (III), where n is an integer that is selected from 6, 7, 8, 9,10,11, 12, which contain the polyoxyethylene 4- (tert-octyl) -phenyl ether structure. Some of them are known by their commercial name, for example the ether with n = 9 or 10 is commercially known as Triton® X-100, Nonidet® P-40 or IGEPAL®.
(Ill)
image3
c) 0, non-ionic surfactants of formula (IV) commercially known as polysorbate 80 6 Tween® 80, or surfactants of formula (V), commercially known as polysorbate 20 or Tween® 20
image4
image5
During the lipase production process by chemical induction, the selected chemical agent is added to the modified CECT17 medium and sterilization is carried out at 120 ° C for 20 minutes.
In another aspect of the invention, biological induction comprises the following steps:
a) preparation of the culture medium;
b) preparation of the inoculum of Halomonas;
c) growth of a culture and lipase production in a mixed culture composed of Halomonas and the inducing microbial agent;
10 where the mixed culture of Halomonas and inducing agent is prepared in stage b) of preparation of the inoculum or in stage c) of growth of the culture where the inducing microorganism is added after a time of growth of the Halomonas microorganism.
In a preferred embodiment, for the biological induction, species of microorganism belonging to the genera Acinetobacter, Actinopolyspora, Alteromonas, Arhodomonas, Bacillus, Chromohalobacter, Dichotomicrobium, Dunaliella, Exiguobacterium, Flavobacterium, Haloarcula, Halobacculus, Halobacculus, Halobacculus, Halobacculus Haloferax, Halomonas, Halorhabdus, Halorubum, Haloterrigena, Halothermothrix, Halovibrio, Marinobacter, Marinococcus, Natrialba, 20 Natronobacterium, Natronococcus, Natronomonas, Nesterenkonia, Nocardiopsis,
Ocenoabacillus, Pelagibacterium, Pseudalteromonas, Pseudomonas, Salinicoccus, Salinivibrio, Spirochaeta, Staphylococcus, Tetragenococcu, Vibrio or other microorganism capable of growing in high concentrations of salt: 10-15% NaCI.
During the lipase production process by biological induction, the modified CECT17 medium is sterilized at 120 ° C for 20 minutes.
In a more preferred embodiment, the inoculum of microorganisms of the genus Halomonas (2-5% v / v) is added to the culture medium selected, sterilized and tempered. In the case of biological induction, a mixed culture can be made by inoculating active cells of the genus Halomonas and the inducing microorganism with a concentration 2-5% v / v, or inoculum of the inducing organism (25% v / v) after a time of growth of the organism of the genus Halomonas.
The growth of the Halomonas genus cell culture can be carried out in 50-2,000 mL Erlenmeyer flasks, which will be covered with a cellulose cap that allows passive aeration. The culture carried out in this type of containers will be carried out in shakers that allow to control the temperature (10-40 ° C) and the agitation (50200rpm). Another type of vessel for the cultivation of Halomonas can be a bioreactor with different configurations: air-lift type or tank type with mechanical agitation. In both cases, the working volume can be 1-10 L and be connected to a control unit that allows setting the stirring speed (50-700 rpm), aeration (0.25-1 15 wm) and the operating temperature (10-40 ° C).
To prevent foaming, a defoamer is added which can be of the emulsion type of an active silicone polymer and a non-ionic emulsifier. The addition of said antifoam can be carried out automatically by means of a foam detection probe inside the culture vessels connected to a controller 20 that allows to regulate the pumping cycles of the antifoam to the reaction vessel.
During growth of the culture and production of lipase in the presence of the chemical or biological agent, the control of biomass and lipolytic activity comprises:
a) measure biomass and lipolytic activity by sampling the culture vessels
25 b) if the lipolytic activity in the culture vessel is around the
Optimal activity, the biological reaction is stopped and the biomass is separated from the culture medium by centrifugation and the supernatant solution is stored at 20 ° C, since its production is extracellular.
The tool used to determine biomass and lipohthic activity is ultraviolet spectrophotometry, considering both the values of cell concentration per calibration of dry weight (turbidimetry at 600 nm) and those of enzymatic production. In the latter case, the hydrolysis of p-nitrophenyl laurate (2.5 mM) is monitored to obtain p-nitrophenol, following the process detailed previously (Fucinos et a!, 2005, Production of thermostable lipolytic activity by Thermus species, Biotechnol. Prog. 21, 1198-1205) and considering a unit of activity as the amount of enzyme that produces 1 pmol of p-nitrophenol per minute.
The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPTION OF THE FIGURES
Figure 1. Monitoring of lipohic activity (U / L) Halomonas sp. LM1C in flask culture by biological induction of Staphylococcus equorum AMC7. The lipogenic activity of a pure culture of Halomonas sp. LM1C as a reference. Error bars express the standard deviation.
Figure 2. Monitoring of the lipoetic activity (U / L) and biomass (g / L) of Halomonas sp. LM1C in culture in agitated tank bioreactor by chemical induction by Triton X-100®. Error bars express the standard deviation. The lines 20 represent the adjustment of the data to a logistic model.
EXAMPLES OF AN EMBODIMENT OF THE INVENTION Example 1
Lipase production by Halomonas sp. LM1C by biological induction by 25 Staphylococcus equorum AMC7 in 250 mL Erlenmeyer flask
First, a culture of Halomonas sp. LMIC and Staphylococcus equorum AMC7 of 50 mL with modified CECT17 medium (pH 6.9) at 14 ° C and 150 rpm for 24
hours, which will serve as inoculum for the flask (3% v / v). Next, 50 mL of modified CECT17 medium (pH 6.9) is sterilized (120 ° C for 20 minutes) together with the 250 mL Erlenmeyer flask. The flask is filled with the medium maintaining sterile conditions and inoculated in a proportion of 3% v / v. A shaker (Thermo 5 Fischer Scientific® MAxQ 8000) is used to regulate the operational parameters at a temperature of 14 ° C and 150 rpm of agitation.
After 144 hours and verified the maximum production levels around 3,000 U / L (Figure 1), the biological process is stopped to proceed to the elimination of the cells of the culture medium by centrifugation at 10,000 rpm, and keeping the 10 cell-free culture broth at -20 "C.
Example 2
Lipase production by Halomonas sp. LM1C by chemical induction by pentadecanoic acid in 250 mL Erlenmeyer flask
First, a culture of Halomonas sp. 50 mL LM1C with modified CECT17 medium (pH 6.9) at 21.6 ° C and 150 rpm for 24 hours, which will serve as an inoculum for the flask (3% v / v). Then, 50 mL of modified CECT17 medium and 10 mg / L of pentadecanoic acid (pH 6.9} are sterilized (120 ° C for 20 minutes) together with the 250 mL Erlenmeyer flask. The flask is filled with the medium keeping the sterility conditions and inoculated in a proportion of 3% v / v. A shaker 20 (Thermo Fischer Scientific® MAxQ 8000) is used to regulate the operational parameters at 21.6 ° C temperature and 150 rpm of agitation.
After 144 hours and verified the maximum production levels around 700 U / L (Table 1), the biological process is stopped to proceed to the elimination of the cells of the culture medium by centrifugation at 10,000 rpm, and keeping the broth 25 cell-free culture at -20 ° C.
Table 1
 Biomass (g / L) Activity (U / L)
 Cultivation without inductor (CONTROL)  2.7 ± 0.3 120.7 ± 15.8
 Pentadecanoic acid  3.0 ± 0.08 690.5 ± 84.2
Example 3
Production of lipase by Halomonas sp. LM1C by chemical induction by oil of 5 sunflower in Erlenmeyer flask of 250 mL
In the first place a culture of Halomonas sp. 50 mL LM1C with modified CECT17 medium (pH 6.9) at 21.6 ° C and 150 rpm for 24 hours, which will serve as inoculum for the flask (3% v / v). Next, 50 mL of modified CECT17 medium and 1 g / L of sunflower oil is sterilized (120 ° C for 20 minutes) which is homogenized by agitation with Ultra Turrax® after adding 1 g / L of Tween 80® ( pH 6.9), together with the 250 mL Erlenmeyer flask. The flask is filled with the medium maintaining sterile conditions and inoculated in a proportion of 3% v / v. A shaker (Thermo Fischer Scientific® MAxQ 8000) is used to regulate the operational parameters at a temperature of 21.6 ° C and 150 rpm of agitation.
After 144 hours have elapsed and the maximum production levels around 1,400 U / L have been verified (Table 1), the biological process is stopped to proceed to the elimination of the cells from the culture medium by centrifugation at 10,000 rpm, and keeping the cell free culture broth at -20 ° C.
Table 2
 Biomass (g / L) Activity (U / L)
 Cultivation without inductor (CONTROL)  2.8 ± 0.4 212.2 ± 1.6
 Sunflower oil  2.8 ± 0.02 1.381184
Example 4
Production of lipase by Halomonas sp. LM1C by chemical induction in agitated tank bioreactor
First, a culture of Halomonas sp. 50 mL LM1C with modified CECT17 medium1 (pH 6.9) at 21.6 ° C and 150 rpm for 24 hours, which will serve as an inoculum for the bioreactor (3% v / v). Subsequently, 1.5 L of modified CECT17 medium containing 1 g / L of Triton X-100® (pH 6.9) 5 is sterilized (120 ° C for 20 minutes) together with the stirred tank bioreactor equipped with a dual type impeller Rushton The bioreactor is filled with the medium while maintaining sterility conditions. A control unit (Biostat B, Braun Melsungen, Germany) is used to regulate operational parameters. It is important to connect the foam probe to avoid the negative effects caused by the formation of foam in the middle before setting the 10 operational conditions at 300 rpm of mechanical agitation and 0.3 vvm of aeration. This probe is connected to a tank containing Sigma Aldrich® SE-15 antifoam. Once the temperature of the bioreactor is set at 21.6 ° C, the inoculation is carried out with 50 mL of viable cells (24 hours) of Halomonas sp. LM1C.
After 196 hours and verified the maximum production levels around 15,000 U / L (Figure 2), the biological process is stopped to proceed to the elimination of
the cells of the culture medium by centrifugation at 10,000 rpm, and keeping the cell-free culture broth at -20 ° C.

权利要求:
Claims (12)
[1]
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1. Procedure for increasing lipase production in Halomonas cultures by chemical induction comprising the following stages:
a) design of the culture medium by the addition of the chemical agent;
b) preparation of Halomonas inoculum;
c) growth of a pure Halomonas culture and lipase production in the presence of the chemical agent.
[2]
2. Procedure for increasing lipase production in Halomonas cultures by biological induction comprising the following stages:
a) preparation of the culture medium;
b) preparation of Halomonas inoculum;
c) growth of the lipase culture and production in a mixed culture composed of Halomonas and the inducing microbial agent;
where the mixed culture of Halomonas and inducing microbial agent is prepared in stage b) of inoculum preparation or in stage c) of crop growth where the inducing microorganism is added after a time of growth of the Halomonas microorganism.
[3]
3. The method according to claim 1, characterized in that the chemical agent is a molecule selected from the following formulas:
a) a fatty acid of formula I
OR
OH
(I)
where:
n is an integer number of carbon atoms;
image 1
b} glycerol or its variants such as triglycerides with formula II
image2
where:
x the number of saturated acids 5 and the number of monounsaturated acids z the number of polyunsaturated acids;
c) non-ionic surfactants of formula III, formula IV or V, or compounds that increase the bioavailability of the inductor
image3
5
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25
where:
n is an integer that is selected from 6 to 12, which contain the polyoxyethylene 4- (tert-octyl) -phenyl ether structure.
[4]
4. Method according to claim 1, characterized in that the chemical agent is added in step a) of design of the culture medium in a concentration between 0.5-2 gr.
[5]
5. Method according to claim 2, characterized by the biological inductor is a
microorganisms selected from the following genera: Acinetobacter, Actinopolyspora, Alteromonas, Arhodomonas, Bacillus, Chromohalobacter,
Dichotomicrobium, Dunaliella, Exiguobacterium, Flavobacterium, Haloarcula,
Halobacillus, Halobacterium, Halobaculum, Halococcus, Haloferax, Halomonas, Halorhabdus, Halorubum, Haloterrigena, Halothermothrix, Halovibrio, Marinobacter, Marinococcus, Natrialba, Natronobacterium, Natronococcus, Natronomonas, Nesterenkonia, Nocardiopsis, Ocenoabacillus, Pelagibacterium, Pseudalteromonas, Pseudomonas, Salinicoccus, Salinivibrio, Spirochaeta, Staphylococcus, Tetragenococcus and / or Vibrio.
[6]
Method according to claims 1 to 2 and 5, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent is inoculated a pure or mixed culture in a proportion comprised between 2 and 5% v / v.
[7]
Method according to claims 1 to 6, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent is carried out in 50-2000 mL Erlernmeyer flask vessels.
[8]
Method according to claims 1 to 7, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent is carried out in bioreactor vessels with air-lift configuration or tank type with mechanical agitation, with working volume in the range of 1-10 L.
[9]
9. The method according to claims 7 to 8, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent utilizes crop agitation speeds between 50-700 rpm.
[10]
10. Method according to claims 1 to 9, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent employs levels of aeration of the culture in the range 0.25-1 wm.
[11]
11. Method according to claims 1 to 10, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent is
operates at cultivation temperatures between 10 and 40 ° C.
[12]
12. Method according to claims 1 to 11, characterized in that step c) of growth of the Halomonas culture in the presence of the chemical or biological agent is carried out in the presence of a defoamer in a range 0.01-1% (v / v) .
Method according to claims 1 to 12, characterized in that step c) of
Growth of the Halomonas culture in the presence of the chemical or biological agent comprises:
a) determination of biomass and lipolytic activity by sampling the culture
15 b) when the lipolytic activity in the culture is around the optimal activity, depending on the inductor used, the biological reaction is stopped and the biomass is separated from the culture medium by centrifugation and preservation of the supernatant solution at 20 ° C
Lipolytic activity (U / L)
3000
2500 -
2000 -
1500 -
1000 -
500
0 ■
image4
144
Figure 1
3500
2625
1750
875
image5
Time (h)
Figure 2
Biomass (g / L)

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ES2677443B2|2019-02-27|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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EP1475431A1|2003-05-07|2004-11-10|Cognis Deutschland GmbH & Co. KG|Method for the production of lipase|CN109385388A|2018-12-29|2019-02-26|中蓝连海设计研究院有限公司|Thermophilic salt denitrifying bacterium YL5-2 and its application|

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CN109439602A|2018-12-29|2019-03-08|中蓝连海设计研究院有限公司|Halophilic vibrio YL5-2 and its microbial inoculum are degraded the application in conversion pollutant under high salt conditions|

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CN111377544A|2018-12-29|2020-07-07|中蓝连海设计研究院有限公司|High-salinity wastewater treatment process utilizing halophilic bacteria YL5-2|

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ES201700078A|ES2677443B2|2017-02-01|2017-02-01|Procedure for the increase of lipase production in Halomonas cultures by chemical and biological induction|ES201700078A| ES2677443B2|2017-02-01|2017-02-01|Procedure for the increase of lipase production in Halomonas cultures by chemical and biological induction|