CN106755144B - Method for preliminary optimization of xylose-converted PHA artificial dual-bacterium system - Google Patents
Method for preliminary optimization of xylose-converted PHA artificial dual-bacterium system Download PDFInfo
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Abstract
The invention relates to a method for primarily optimizing a PHA artificial double-bacterium system by xylose conversion. Aiming at an artificial double-bacterium system of pseudomonas and saccharomyces cerevisiae, the PHA is produced by using a cheap carbon source xylose through an inoculation optimization method and a culture optimization method. Culturing the saccharomyces cerevisiae in a culture medium, measuring a growth curve of the saccharomyces cerevisiae, and determining a logarithmic phase; determining the appropriate inoculation time of the double bacteria; and (4) determining the appropriate inoculation ratio of the double bacteria. Finally, the optimal culture conditions can be obtained by comparing the data of the cell dry weight and the PHA yield of each experimental group. Finally, it was confirmed that the production of PHA was maximized under the conditions that the pH was 7, the sugar concentration was 15g/L, and the inorganic salt content was sufficient. By comparing the yields of PHA, the optimal seeding conditions were finally determined to be: and (3) saccharomyces cerevisiae: inoculating pseudomonas at the ratio of 1:2, and inoculating pseudomonas 12h later.
Description
Technical Field
The invention relates to a method for primarily optimizing a PHA artificial double-bacterium system by xylose conversion.
Background
Polyhydroxyalkanoate (PHA) is an environment-friendly polymer bioplastic, can perfectly replace the traditional petrochemical products, and is widely applied to the fields of medicine packaging, plastic manufacturing, polymer materials and the like due to good biodegradability and biocompatibility. From 2011 to 2020, the global demand for PHA will increase from 3 to over 12 million tons [1,2 ]. However, limited to cost and technical problems, it is difficult to realize large-area industrial production of PHA with the same grade as conventional plastics. According to research of Qinghua university, the substrate consumption accounts for 50% of PHA cost, and the energy consumption accounts for 30%. To reduce substrate costs, people look at the transition from expensive substrates such as glucose to non-grain raw materials such as xylose, which accounts for 18-30% of the natural lignocellulose content. Since 1990, researches such as screening and process optimization of PHA-producing strains by using xylose have been widely carried out, but major breakthroughs have not been made all the time, because PHA is mainly produced by fermentation of traditional single microbial strains, but the xylose conversion efficiency, PHA yield and the like are lower than those of PHA produced by using glucose as a substrate. The limitation of the metabolic capability of single bacteria of the microorganism is the bottleneck of the high efficiency of converting PHA from xylose.
In nature, a mixed strain system that functions in interdependence can solve problems such as metabolic burden well [3 ]. Compared with single bacterium, the microorganism mixed bacterium system has three advantages [4], and is combined with the analysis of a xylose-produced PHA system: tasks such as xylose utilization, PHA synthesis and the like are distributed to different strains to be executed, so that the problem of overweight metabolic load of a single bacterium can be avoided; the intracellular accumulation of PHA is formed under the severe environment, and the mixed bacteria system can maintain the relative stability of the whole system through the modes of metabolic substance exchange, energy level balance and the like among different bacterial strains under the condition of environmental fluctuation; the transformation space of PHA produced by single bacterium using xylose is small due to the limitation of the genome size, and the mixed bacterium system has various cells, and the functions of the system can be expanded by adding new bacteria or introducing new genes. The microbial mixed system is utilized to realize the efficient conversion of xylose into PHA, and the technical bottleneck of PHA production by xylose can be broken. However, the formation of natural mixed bacteria system is often aimed at survival, and it is difficult to achieve specific production target according to human expectation. In addition, as the number of cell types in the system increases, intercellular communication is also hindered, and the efficiency of the system is affected. The mismatching of the growth speed, the metabolic capability and the like of different cells also brings difficulty to mixed fermentation. Therefore, the explanation of the design and construction principle of the mixed bacteria system and the optimization of the mixed bacteria system are very significant.
[1]Lara D,Michael C,Achim R,et al.Market developments of andopportunities for biobased products and chemicals.Nova-Institute for Ecologyand Innovation Reprot,2013,52202
[2]Sudesh K,Bhubalan K,Chuah J A,et al.Synthesisofpolyhydroxyalkanoate from palm oil and some new applications.AppliedMicrobiology and Biotechnology,2011,89(5):1373-1386[4]S.Y.Lee.Poly(3-Hydroxybutyrate)Production from Xylose by Recombinant EscherichiaColi.bioprocess engineering,1998,18(5):397-399
[3]Hao Song,Mingzhu Ding,Xiaoqiang Jia,et al.Synthetic microbialconsortia:from systematic analysis to construction and applications.ChemicalSociety Reviews,2014,43(20):6954-6981
[4]Brenner K,Lingchong You,Frances H.Arnold,Engineering microbialconsortia:a new frontier in synthetic biology.Trends in Biotechnology,2008,26(9):483-489
Disclosure of Invention
In view of the above, the invention provides a method for primarily optimizing a xylose-converted PHA artificial dual-bacteria system, and the specific technology is as follows:
a method for primary optimization of an artificial double-bacterium system for converting PHA through xylose is characterized in that aiming at the artificial double-bacterium system of pseudomonas and Saccharomyces cerevisiae, the PHA is produced by using a cheap carbon source xylose by an inoculation optimization method and a culture optimization method, the Saccharomyces cerevisiae is a gift offered by Yuan Ying professor of Tianjin university, and a starting strain of the Saccharomyces cerevisiae L2612 is; the PHA-accumulating Pseudomonas aeruginosa SA1/TJU-J-05 was deposited in the China general microbiological culture Collection center at 18/2/2016.
The technical scheme of the invention is as follows:
a method for primarily optimizing a xylose conversion PHA (polyhydroxyalkanoate) artificial dual-bacterium system is characterized in that aiming at the artificial dual-bacterium system of pseudomonas and saccharomyces cerevisiae, the PHA is produced by using a cheap carbon source xylose through an inoculation optimization method and a culture optimization method. The method comprises the following specific steps:
1) culturing the saccharomyces cerevisiae in a culture medium, measuring a growth curve of the saccharomyces cerevisiae, and determining a logarithmic phase;
2) determining the appropriate inoculation time of the double bacteria;
3) and (4) determining the appropriate inoculation ratio of the double bacteria.
The culture medium used was: YPD medium and xylose medium.
Finally, the optimal inoculation conditions are determined as follows: the inoculation ratio of the pseudomonas is 1:2, and the pseudomonas is inoculated after the saccharomyces cerevisiae is independently cultured for 12 hours.
Preferably, the maximum PHA production is achieved at a pH of 7, a sugar concentration of 15g/L and a sufficient inorganic salt content.
The concrete description is as follows:
the inoculation optimization method mainly comprises the steps of optimally selecting the inoculation time and the inoculation proportion, namely culturing the saccharomyces cerevisiae, measuring the growth curve of the saccharomyces cerevisiae, carrying out double-bacterium co-culture through a rational design experiment, and obtaining the optimal inoculation condition to realize the inoculation optimization, wherein the used culture medium is as follows:
YPD medium: the yeast activating agent is used for activating yeast and comprises the following components: peptone 20, glucose 20, yeast extract 10. The medium was prepared with distilled water and sterilized in a sterilizer at 115 ℃ for 30 minutes. Wherein the glucose is sterilized separately, and all components are mixed uniformly in a sterile environment after sterilization. To the solid medium, 2% agar was added, and the rest conditions were the same.
Xylose culture medium: it can be used for co-culture of yeast and Pseudomonas and PHA accumulation. Wherein the carbon source is 1g of xylose, no glucose is contained, and inorganic salt components, sulfate, phosphate and trace elements thereof are added, and the concrete composition is as follows:
sulfate (200 ml): 10g of ammonium sulfate and 2g of anhydrous magnesium sulfate, diluting the mixture to 200mL by using distilled water, and sterilizing the mixture for 20min at 121 ℃;
phosphate (200 ml): 96.5g of disodium hydrogen phosphate dodecahydrate and 15g of dipotassium hydrogen phosphate, fixing the volume to 200ml by using distilled water, and sterilizing for 20min at 121 ℃;
trace elements (200 ml): 10mL of 50g/L ferric ammonium citrate solution, 10mL of 29.8g/L calcium chloride hexahydrate, 4.17mL of 12mol/L concentrated hydrochloric acid, 1mg of zinc sulfate heptahydrate, 0.3mg of manganese chloride tetrahydrate, 3mg of boric acid, 2mg of cobalt chloride hexahydrate, 0.064mg of copper sulfate, 0.2mg of nickel chloride and 0.3mg of sodium molybdate dihydrate are subjected to constant volume to 200mL by using distilled water and sterilized at 121 ℃ for 20 min.
The specific method comprises the following steps:
under sterile conditions, as initial OD600Inoculating yeast seed liquid into 100mL of xylose culture medium at an inoculation ratio of about 0.1, performing shake culture, sampling every 6h, storing sample bacterial liquid in a 2mL centrifuge tube, and measuring the OD600 value of the bacterial liquid under 600nm by using an ultraviolet spectrophotometer. Three samples are taken in parallel every time, and the growth curve of the saccharomyces cerevisiae is drawn according to the obtained result.
The time for the yeast to enter logarithmic phase under the condition is determined according to the growth curve of the yeast (hereinafter referred to as S bacteria) under the xylose culture medium determined before. In order to determine whether the yeast and the pseudomonas (hereinafter referred to as P bacteria) have symbiotic effect and whether PHA is accumulated or not in the environment of the xylose culture medium, a plurality of groups of mixed bacteria combinations are designed to achieve the purpose of producing PHA by using xylose by the mixed bacteria, and the specific design scheme is shown in Table 1:
TABLE 1 combination of mixed bacteria
After the culture is finished, the bacterial liquid of six groups of experiments is taken to measure the OD600And (4) performing comparative analysis, extracting dried bacteria from the bacteria liquid, performing esterification treatment, detecting the content of PHA by using gas chromatography, and comparing six groups of experimental results. The PHA content determination method adopts an internal standard method for determination, firstly, a sample and an internal standard are subjected to methyl esterification, and then the content of the sample and the internal standard is detected by gas chromatography, and the specific operation steps are as follows:
taking 1mL of shake flask bacterial liquid in a clean centrifugal tube, and measuring the final OD value. And (3) putting 40mL of bacterial liquid into a 50mL centrifuge tube, carrying out refrigerated centrifugation at 9000rpm for 10min, removing supernatant, adding distilled water to wash the thalli, shaking for resuspension, centrifuging again, repeating twice, putting into a freeze dryer, carrying out freeze drying for 24h at-40 ℃, weighing and recording the rest thalli after freeze drying.
500mL of an esterification solution was prepared from 0.5g of benzoic acid, 15mL of concentrated sulfuric acid, and 485mL of methanol, and about 50mg of the lyophilized cell sample, 2mL each of chloroform and the esterification solution were added to an esterification tube, which was then sealed with a lid, heated in a water bath at 100 ℃ and reacted for 4 hours. And (3) when the sample is cooled to room temperature, adding a proper amount of distilled water and shaking, standing for a moment to wait for layering, and then taking a lower-layer organic phase to analyze in a gas chromatography. Meanwhile, 20mg of PHB standard substance is weighed, and esterification reaction and detection are carried out according to the same method.
The gas chromatography procedure was as follows: column temperature 80 ℃, injector temperature 240 ℃, detector temperature 250 ℃, split ratio 1: 10. temperature rising conditions are as follows: the temperature is maintained at 80 ℃ for 90s, the temperature is increased to 140 ℃ at the speed of 30 ℃/min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, and the temperature is maintained at 250 ℃ for 2 min. The sample size was 2. mu.L.
The PHA content in the sample is calculated according to the peak area ratio of the standard and the internal standard in the chromatogram.
Finally, through the comparative analysis of the measured results of the growth of the bacteria and the PHA yield of different experimental groups, the optimal inoculation condition of the system can be determined.
The culture optimization method mainly obtains the optimal culture conditions and limiting factors through rational design of PHA (polyhydroxyalkanoate) production experiments of pseudomonas and comprises the following specific steps:
different experimental groups are designed by taking sugar concentration, pH and inorganic salt components as influencing factors, and the PHA accumulation of the pseudomonas under different conditions is tested. Wherein, the variables of pH are 6, 7 and 8, the variables of sugar concentration are 10g/L, 15g/L and 20g/L, the variables of inorganic salt components are missing sulfate components (ammonium salt and magnesium ions are abbreviated as component 1 in Table 2), missing phosphate components (component 2 in Table 2 is mainly a phosphorus source) and all additions, and the trace metal ions in Table 2 are abbreviated as component 3, and the detailed composition is referred to the content of the invention. The specific experimental protocol is shown in table 2:
TABLE 2 different fermentation condition optimizations
Wherein, in each three groups of experiments, three groups of variables with different sugar concentrations exist, and meanwhile, different combinations of inorganic salt components are used for testing the effects of nitrogen limitation and phosphorus limitation.
After the shake flask fermentation is finished, 1mL of bacterial liquid is taken, the OD600 value of 9 groups of bacterial liquid is measured, 40mL of fermentation liquid is taken and placed in a 50mL centrifuge tube for centrifugation, the supernatant is discarded after the centrifugation, the supernatant is placed in a refrigerated centrifuge for drying for 24h, and the dry weight of the thalli is weighed. The accumulated amount of PHA was determined using gas chromatography.
Finally, the optimal culture conditions can be obtained by comparing the data of the cell dry weight and the PHA yield of each experimental group.
Drawings
FIG. 1: a yeast growth curve diagram;
in the xylose culture medium, the yeast enters a logarithmic phase approximately 12 hours after inoculation, and the amount of the yeast is not obviously increased about 60 hours, and finally the maximum value of OD600 reaches about 7.5.
FIG. 2: PHA production for 6 groups of samples;
after mixed bacteria co-culture is finished, extracting dry bacteria from the bacteria liquid, performing esterification treatment, and detecting the PHA content by using gas chromatography.
Detailed Description
The invention is described below by taking the optimization of a dual-bacteria system composed of Pseudomonas aeruginosa SA1/TJU-J-05 and Saccharomyces cerevisiae L2612 as an example, and the specific steps are as follows:
1. method of optimizing conditions
Different experimental groups are designed by taking sugar concentration, pH and inorganic salt components as influencing factors, and the PHA accumulation of the pseudomonas under different conditions is tested. Wherein, the variables of pH are 6, 7 and 8, the variables of sugar concentration are 10g/L, 15g/L and 20g/L, the variables of inorganic salt components are missing sulfate components (ammonium salt and magnesium ions are abbreviated as component 1 in the table 3), missing phosphate components (component 2 in the table 3 is mainly a phosphorus source) and all additions, and the trace metal ions in the table 3 are abbreviated as component 3, and the detailed composition is referred to the content of the invention. The specific experimental protocol is shown in table 3:
TABLE 3 different fermentation condition optimizations
Wherein, in each three groups of experiments, three groups of variables with different sugar concentrations exist, and meanwhile, different combinations of inorganic salt components are used for testing the effects of nitrogen limitation and phosphorus limitation.
After the shake flask fermentation is finished, 1mL of bacterial liquid is taken, the OD600 value of 12 groups of bacterial liquid is measured, 40mL of fermentation liquid is taken and placed in a 50mL centrifuge tube for centrifugation, the supernatant is discarded after the centrifugation, the supernatant is placed in a refrigerated centrifuge for drying for 24h, and the dry weight of the thalli is weighed. The accumulated amount of PHA was determined using gas chromatography.
The method for measuring the PHA content in the experiment adopts an internal standard method, firstly, a sample and an internal standard methyl are esterified, and then the content of the sample and the internal standard methyl is detected by using gas chromatography, and the specific operation steps are as follows:
taking 1mL of shake flask bacterial liquid in a clean centrifugal tube, and measuring the final OD value. And (3) putting 40mL of bacterial liquid into a 50mL centrifuge tube, carrying out refrigerated centrifugation at 9000rpm for 10min, removing supernatant, adding distilled water to wash the thalli, shaking for resuspension, centrifuging again, repeating twice, putting into a freeze dryer, carrying out freeze drying for 24h at-40 ℃, weighing and recording the rest thalli after freeze drying.
500mL of an esterification solution was prepared from 0.5g of benzoic acid, 15mL of concentrated sulfuric acid, and 485mL of methanol, and about 50mg of the lyophilized cell sample, 2mL each of chloroform and the esterification solution were added to an esterification tube, which was then sealed with a lid, heated in a water bath at 100 ℃ and reacted for 4 hours. And (3) when the sample is cooled to room temperature, adding a proper amount of distilled water and shaking, standing for a moment to wait for layering, and then taking a lower-layer organic phase to analyze in a gas chromatography. Meanwhile, 20mg of PHB standard substance is weighed, and esterification reaction and detection are carried out according to the same method.
The gas chromatography procedure was as follows: column temperature 80 ℃, injector temperature 240 ℃, detector temperature 250 ℃, split ratio 1: 10. temperature rising conditions are as follows: the temperature is maintained at 80 ℃ for 90s, the temperature is increased to 140 ℃ at the speed of 30 ℃/min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, and the temperature is maintained at 250 ℃ for 2 min. The sample size was 2. mu.L.
The PHA content in the sample is calculated according to the peak area ratio of the standard and the internal standard in the chromatogram.
Finally, it was confirmed that the production of PHA was maximized under the conditions that the pH was 7, the sugar concentration was 15g/L, and the inorganic salt content was sufficient.
2. Method of vaccination optimisation
(1) Growth curve determination of saccharomyces cerevisiae
Under aseptic conditions, inoculating yeast seed liquid into 100mL of xylose culture medium according to an inoculation ratio of initial OD600 of about 0.1, sampling every 6h, storing the sample bacterial liquid in a 2mL centrifuge tube, and measuring the OD600 value of the bacterial liquid under 600nm by using an ultraviolet spectrophotometer. Three replicates were made for each sampling.
(2) Mixed culture of pseudomonas and saccharomyces cerevisiae
The approximate time for the yeast to enter log phase under these conditions was determined from the previously determined growth curve of the yeast (hereinafter referred to as S) in xylose medium (as shown in FIG. 1). In order to determine the optimal inoculation time and the optimal inoculation ratio, a plurality of groups of mixed bacteria combinations are designed so as to achieve the purpose of producing PHA by using xylose by the mixed bacteria, and the specific design scheme is shown in Table 2:
TABLE 4 combination of mixed bacteria
The general operation flow is as follows: under the aseptic condition, inoculating the yeast seed liquid in the logarithmic phase into a xylose culture medium, and adding the pseudomonas liquid at the same time or after the yeast enters the logarithmic phase. The ratio of mixed bacteria is in accordance with the initial OD600Determine, and maintain Total initial OD of all experimental groups600About 0.1.
By comparing the production of PHA (as shown in figure 2), the optimal seeding conditions were finally determined as: and (3) saccharomyces cerevisiae: inoculating pseudomonas at the inoculation ratio of 1:2 (S: P), and inoculating pseudomonas 12h later.
(3) PHA (polyhydroxyalkanoate) production experiment by mixed bacteria co-culture
After the mixed bacteria co-culture is finished, the bacteria liquid is taken to extract dry bacteria and then is subjected to esterification treatment, the PHA content of the bacteria liquid is detected by using gas chromatography, and the detailed operation steps are the same as the PHA content determination method.
The primary optimization of the yeast and pseudomonas system is realized through an inoculation optimization and condition optimization method, so that the two bacteria can be well symbiotic under a xylose culture medium, the mixed bacteria can utilize xylose to produce PHA, and a reference and a foundation are provided for the further optimization of the system.
Claims (5)
1. A method for primarily optimizing a xylose-converted PHA (polyhydroxyalkanoate) artificial dual-bacterium system is characterized in that aiming at the artificial dual-bacterium system of pseudomonas and saccharomyces cerevisiae, the PHA is produced by using a cheap carbon source xylose through an inoculation optimization method and a culture optimization method; the saccharomyces cerevisiae isSaccharomyces cerevisiaeL2612; pseudomonas of PHAPseudomonas aeruginosaSA1/TJU-J-05。
2. The method of claim 1, characterized by the steps of:
1) culturing the saccharomyces cerevisiae in a culture medium, measuring a growth curve of the saccharomyces cerevisiae, and determining a logarithmic phase;
2) determining the appropriate inoculation time of the double bacteria;
3) and (4) determining the appropriate inoculation ratio of the double bacteria.
3. The method as claimed in claim 2, wherein the culture medium used is: YPD medium and xylose medium.
4. The method according to claim 1, characterized in that the optimal inoculation conditions are finally determined as Saccharomyces cerevisiae: the inoculation ratio of the pseudomonas is 1:2, and the pseudomonas is inoculated after the saccharomyces cerevisiae is independently cultured for 12 hours.
5. The method as set forth in claim 1, wherein the maximum amount of PHA is produced under conditions of pH 7, sugar concentration of 15g/L and sufficient inorganic salt content.
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| CN1495215A (en) * | 2002-04-26 | 2004-05-12 | 佳能株式会社 | Method for preparing polyhydroxyalkanoate by utilizing paraffin in whose molecule the residue containing aromatic ring is possessed |
| WO2014144293A2 (en) * | 2013-03-15 | 2014-09-18 | Genomatica, Inc. | Development and use of microbes for impurity reduction in biomass hydrolysates and fermentation broths |
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| CN1495215A (en) * | 2002-04-26 | 2004-05-12 | 佳能株式会社 | Method for preparing polyhydroxyalkanoate by utilizing paraffin in whose molecule the residue containing aromatic ring is possessed |
| WO2014144293A2 (en) * | 2013-03-15 | 2014-09-18 | Genomatica, Inc. | Development and use of microbes for impurity reduction in biomass hydrolysates and fermentation broths |
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