Method for constructing PHA synthetic flora

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for constructing a PHA synthetic flora. The method for constructing the PHA synthetic flora provided by the invention comprises the following steps: enriching the PHA synthetic flora from activated sludge by respectively adopting culture media containing different single short-chain fatty acids or salts thereof, and then mixing the PHA synthetic flora enriched by the culture media containing different single short-chain fatty acids or salts thereof. According to the method, the PHA synthesis flora with relatively high utilization capability on different short-chain fatty acids can be obtained, flora corresponding to various short-chain fatty acids can be flexibly compounded according to the composition of the short-chain fatty acids in an actual substrate, and the utilization rate of the anaerobic fermentation liquid of the organic waste and the PHA synthesis efficiency can be remarkably improved. In addition, the construction method of the PHA synthesis flora can also significantly improve the enrichment efficiency of the PHA synthesis flora, is beneficial to obtaining the flora with stronger PHA synthesis ability, and improves the synthesis efficiency and yield of the PHA.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a method for constructing a PHA-synthesizing microbial community. Background Art

[0002] In human production and life, petroleum-based traditional plastics are a commonly used material, and their extensive application has generated a large amount of plastic waste. However, currently, there is still a lack of effective plastic waste treatment methods globally, and most plastic wastes are discarded or landfilled into natural systems. Due to the non-degradability of petroleum-based plastics, plastic waste in the natural environment seriously threatens the survival of wild animals and accumulates in the food chain in the form of "microplastics", thus directly threatening human health. Therefore, it is urgent to develop "biodegradable plastics" that can be biodegraded. Polyhydroxyalkanoates (PHA) have characteristics such as good biodegradability, biocompatibility, and complete biosynthesis, and are regarded as the most potential substitutes for petroleum-based plastics. Currently, PHA is mainly synthesized by pure bacteria fermentation, but due to the need for refined substrates and a strictly sterile environment, PHA still cannot be produced and applied on a large scale. To reduce costs, a mixed bacteria fermentation process for synthesizing PHA using various organic wastes (such as straw, kitchen waste, etc.) as substrates has been developed. This process converts organic wastes into short-chain fatty acids through anaerobic fermentation, enriches a PHA-synthesizing microbial community from the natural environment (such as activated sludge) using short-chain fatty acids, and then uses the enriched microbial community to synthesize PHA with an anaerobic fermentation broth rich in short-chain fatty acids as the substrate. However, since the microbial community enriched using a single short-chain fatty acid or a mixed acid of different short-chain fatty acids cannot efficiently utilize the actual anaerobic fermentation broth composed of different short-chain fatty acids, the PHA synthesis efficiency is relatively low. Therefore, constructing a microbial community that can more efficiently and flexibly utilize mixed acids composed of different short-chain fatty acids for PHA synthesis is very important for the mixed bacteria fermentation process of PHA. Summary of the Invention

[0003] The present invention provides a method for constructing a PHA-synthesizing microbial community.

[0004] In the research process of enriching PHA - synthesizing bacteria from activated sludge using short - chain fatty acids, it was found that when using a single short - chain fatty acid or a mixture of different short - chain fatty acids (mixed acid) to enrich PHA - synthesizing bacteria, it is difficult to obtain a bacterial community that can efficiently utilize various short - chain fatty acids in the anaerobic fermentation broth of organic waste to synthesize PHA, which limits the synthesis efficiency of PHA. For example, even when using a mixed acid composed of acetic acid, propionic acid, and butyric acid to enrich PHA - synthesizing bacteria, the obtained PHA - synthesizing bacteria are still difficult to ensure high utilization efficiency for acetic acid, propionic acid, and butyric acid. To solve the above problems, the present invention attempts to enrich bacterial communities using different single short - chain fatty acids (one of acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid) respectively, obtain bacterial communities that can efficiently utilize each single short - chain fatty acid for PHA synthesis, and then mix these bacterial communities to obtain a PHA - synthesizing bacterial community that can simultaneously and efficiently utilize different short - chain fatty acids.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a PHA - synthesizing bacterial community, the method comprising: respectively enriching PHA - synthesizing bacteria from activated sludge using culture media containing different single short - chain fatty acids or their salts, and then mixing the PHA - synthesizing bacteria enriched by the culture media containing different single short - chain fatty acids or their salts.

[0007] In the present invention, the method for respectively enriching PHA - synthesizing bacteria from activated sludge using culture media containing different single short - chain fatty acids or their salts is specifically as follows: respectively enriching PHA - synthesizing bacteria from activated sludge using culture media containing acetic acid or its salt, propionic acid or its salt, butyric acid or its salt, valeric acid or its salt, and lactic acid or its salt. The culture medium contains only one short - chain fatty acid. After enrichment using the culture medium containing one short - chain fatty acid or its salt, the short - chain fatty acid - enriched bacterial community is obtained. Mixing the enriched bacterial communities corresponding to different short - chain fatty acids gives the PHA - synthesizing bacterial community.

[0008] In some embodiments of the present invention, the method for constructing a PHA - synthesizing bacterial community comprises: enriching PHA - synthesizing bacteria from activated sludge using a culture medium containing acetic acid or its salt to obtain an acetic - acid - enriched bacterial community; enriching PHA - synthesizing bacteria from activated sludge using a culture medium containing propionic acid or its salt to obtain a propionic - acid - enriched bacterial community; enriching PHA - synthesizing bacteria from activated sludge using a culture medium containing butyric acid or its salt to obtain a butyric - acid - enriched bacterial community; enriching PHA - synthesizing bacteria from activated sludge using a culture medium containing valeric acid or its salt to obtain a valeric - acid - enriched bacterial community; enriching PHA - synthesizing bacteria from activated sludge using a culture medium containing lactic acid or its salt to obtain a lactic - acid - enriched bacterial community; and then mixing at least two of the acetic - acid - enriched bacterial community, the propionic - acid - enriched bacterial community, the butyric - acid - enriched bacterial community, the valeric - acid - enriched bacterial community, and the lactic - acid - enriched bacterial community.

[0009] The above-mentioned acetic acid-enriched flora, propionic acid-enriched flora, butyric acid-enriched flora, valeric acid-enriched flora, and lactic acid-enriched flora can be provided by the bacterial liquid obtained after the enrichment is completed.

[0010] In some embodiments of the present invention, the acetic acid-enriched flora, the propionic acid-enriched flora, the butyric acid-enriched flora, the valeric acid-enriched flora, and the lactic acid-enriched flora are mixed according to a mass ratio of (15-40):(15-40):(15-40):(15-40):(15-40).

[0011] In some embodiments of the present invention, the acetic acid-enriched flora, the propionic acid-enriched flora, the butyric acid-enriched flora, the valeric acid-enriched flora, and the lactic acid-enriched flora are mixed according to a mass ratio of (8.3-25):(12.5-37.5):(8.3-25):(12.5-37.5):(8.3-25).

[0012] In some embodiments of the present invention, the propionic acid-enriched flora and the valeric acid-enriched flora are mixed according to a mass ratio of (45-55):(45-55).

[0013] In some embodiments of the present invention, the acetic acid-enriched flora, the butyric acid-enriched flora, and the lactic acid-enriched flora are mixed according to a mass ratio of (30-35):(30-35):(30-35).

[0014] In the above method, the culture medium comprises the following components: short-chain fatty acids or their salts, amino acids, macroelements, and microelements.

[0015] Through a large number of studies on the genomes of various microorganisms in the flora contained in activated sludge, the present invention has found that the species with the ability to synthesize PHA are often auxotrophic for certain amino acids, while many microorganisms without the ability to synthesize PHA have the ability to synthesize these amino acids. Therefore, it is possible that these non-PHA-synthesizing bacteria act as amino acid providers in the community, making it difficult to remove them through a simple enrichment strategy, resulting in a large amount of short-chain fatty acids being utilized and wasted by these non-PHA-synthesizing bacteria. Based on the above findings, the present invention attempts to supplement trace amino acids in the enrichment medium and finds that this method can reduce the dominance of these non-PHA-synthesizing bacteria, thereby enhancing the enrichment and screening intensity of PHA-synthesizing bacteria, improving the enrichment and screening efficiency of PHA-synthesizing flora, and being more conducive to obtaining flora that can efficiently utilize short-chain fatty acids to synthesize PHA.

[0016] Preferably, the culture medium comprises the following components: 1-5 g / L of short-chain fatty acids or their salts, 1-5 mg / L of amino acids, 0.5-1.2 g / L of macroelements, and 1-3 mg / L of microelements.

[0017] The amino acids described above are lysine, isoleucine, tyrosine, histidine, phenylalanine, and tryptophan.

[0018] Preferably, the amino acids comprise the following components: lysine 0.1 - 0.3 mg / L, isoleucine 0.2 - 0.5 mg / L, tyrosine 0.1 - 0.4 mg / L, histidine 0.3 - 0.6 mg / L, phenylalanine 0.3 - 0.5 mg / L, and tryptophan 0.2 - 0.4 mg / L.

[0019] The present invention discovers that adding the above amino acids can significantly improve the enrichment efficiency of the PHA - synthesizing microbial community, and is more conducive to obtaining a microbial community that can efficiently utilize short - chain fatty acids to synthesize PHA.

[0020] The short - chain fatty acids described above are one of acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid.

[0021] The salts of the short - chain fatty acids include but are not limited to sodium salts, potassium salts, etc.

[0022] The macronutrients include one or more of magnesium, calcium, phosphorus, and potassium.

[0023] The micronutrients include one or more of iron, boron, copper, iodine, manganese, molybdenum, zinc, and cobalt.

[0024] Among them, the macronutrients and micronutrients can be provided by adding inorganic salts. The present invention has no special limitation on the types of inorganic salts that provide the above - mentioned macronutrients and micronutrients. Those skilled in the art can understand that as long as the above - mentioned macronutrients and micronutrients can be provided, any inorganic salt can achieve the expected effect.

[0025] The culture medium described above further comprises a chelating agent and a reducing agent.

[0026] Preferably, the chelating agent is EDTA, and / or the reducing agent is thiourea.

[0027] In some embodiments of the present invention, the culture medium contains 1-5 g / L of short-chain fatty acids or their salts, 0.5-0.8 g / L of MgSO4·7H2O, 0.1-0.2 g / L of EDTA, 0.05-0.1 g / L of CaCl2·2H2O, 0.04-0.06 g / L of K2HPO4, 0.02-0.05 g / L of KH2PO4, 5-15 mg / L of thiourea, 0.1-0.3 mg / L of lysine, 0.2-0.5 mg / L of isoleucine, 0.1-0.4 mg / L of tyrosine, 0.3-0.6 mg / L of histidine, 0.3-0.5 mg / L of phenylalanine, 0.2-0.4 mg / L of tryptophan, 1-2 mg / L of FeCl3·6H2O, 0.1-0.2 mg / L of H3BO3, 0.02-0.04 mg / L of CuSO4·5H2O, 0.02-0.04 mg / L of KI, 0.1-0.2 mg / L of MnCl2·4H2O, 0.05-0.07 mg / L of Na2MoO4·2H2O, 0.1-0.2 mg / L of ZnSO4·7H2O, and 0.1-0.2 mg / L of CoCl2·6H2O, wherein the short-chain fatty acids are respectively one of acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid.

[0028] Preferably, the pH of the culture medium is 6.8-7.2.

[0029] It has been verified that by using the above culture medium to enrich the microbial community from activated sludge, a microbial community that can efficiently utilize short-chain fatty acids to synthesize PHA can be obtained, and the PHA yield of the obtained PHA-synthesizing microbial community is significantly increased.

[0030] The present invention uses the aerobic transient feeding method (ADF) to enrich the PHA-synthesizing microbial community. Through the optimization of the enrichment process, it is found that by setting a relatively long starvation aeration stage (without adding new carbon source, but continuing to aerate to supplement oxygen) after every 2-3 enrichment cycles, the enrichment intensity of the enrichment strategy for the PHA-synthesizing microbial community can be enhanced to obtain a microbial community with stronger PHA synthesis ability.

[0031] In the method for constructing the PHA-synthesizing microbial community described above, the enrichment uses the aerobic transient feeding method, and in each enrichment cycle, feeding, aeration, sedimentation, and drainage are carried out in sequence;

[0032] After every 2-3 enrichment cycles (preferably every 2 enrichment cycles), there is also an aeration stage without adding carbon source.

[0033] Preferably, the time of the aeration stage without adding carbon source is 620-680 min; and / or, the time of the aeration stage in each enrichment cycle is 620-680 min.

[0034] The feed during the enrichment cycle is the medium described above; the medium from which short-chain fatty acids and their salts have been removed is added during the aeration stage without additional carbon source supplementation.

[0035] Preferably, after 1.5 - 2.5 h of feeding, an ammonium salt is added, and the addition amount of the ammonium salt is 0.2 - 0.5 g / L. More preferably, it is 0.3 - 0.4 g / L.

[0036] Preferably, the time for each enrichment cycle is 11 - 13 h.

[0037] Preferably, the sedimentation time is 40 - 60 min, the feeding time is 5 - 15 min, and the drainage time is 5 - 15 min.

[0038] The method for constructing a PHA-synthesizing microbial community provided by the present invention is generally applicable to various aerobic treatment sludges. By optimizing the medium and the enrichment process, the enrichment efficiency of the PHA-synthesizing microbial community is significantly improved, and the PHA synthesis ability of the enriched PHA-synthesizing microbial community is enhanced; by mixing the microbial communities enriched from different single fatty acids, a mixed microbial community capable of efficiently utilizing mixed acids to synthesize PHA is obtained.

[0039] Preferably, the activated sludge is the aerobic treatment sludge of winery cellar bottom wastewater. Since the main components of winery cellar bottom wastewater are various fatty acids, the microbial resources that utilize various short-chain fatty acids in the cellar bottom aerobic treatment sludge are richer, which is more conducive to enriching a microbial community with better short-chain fatty acid utilization ability.

[0040] The present invention further provides a PHA-synthesizing microbial community constructed by the method for constructing a PHA-synthesizing microbial community described above.

[0041] The present invention provides the application of the above-mentioned PHA-synthesizing microbial community in PHA production or in constructing a PHA-synthesizing microbial community.

[0042] The present invention also provides a medium for enriching a PHA-synthesizing microbial community, and the medium contains the following components: short-chain fatty acids or their salts 1 - 5 g / L, amino acids 1 - 5 mg / L, macronutrients 0.5 - 1.2 g / L, and micronutrients 1 - 3 mg / L;

[0043] Among them, the amino acids are lysine, isoleucine, tyrosine, histidine, phenylalanine, and tryptophan.

[0044] Preferably, the amino acids contain the following components: lysine 0.1 - 0.3 mg / L, isoleucine 0.2 - 0.5 mg / L, tyrosine 0.1 - 0.4 mg / L, histidine 0.3 - 0.6 mg / L, phenylalanine 0.3 - 0.5 mg / L, and tryptophan 0.2 - 0.4 mg / L.

[0045] Preferably, the short-chain fatty acid is one of acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid.

[0046] The macronutrients include one or more of magnesium, calcium, phosphorus, and potassium.

[0047] The micronutrients include one or more of iron, boron, copper, iodine, manganese, molybdenum, zinc, and cobalt.

[0048] In some embodiments of the present invention, the culture medium contains 1-5 g / L of short-chain fatty acid or its salt, 0.5-0.8 g / L of MgSO4·7H2O, 0.1-0.2 g / L of EDTA, 0.05-0.1 g / L of CaCl2·2H2O, 0.04-0.06 g / L of K2HPO4, 0.02-0.05 g / L of KH2PO4, 5-15 mg / L of thiourea, 0.1-0.3 mg / L of lysine, 0.2-0.5 mg / L of isoleucine, 0.1-0.4 mg / L of tyrosine, 0.3-0.6 mg / L of histidine, 0.3-0.5 mg / L of phenylalanine, 0.2-0.4 mg / L of tryptophan, 1-2 mg / L of FeCl3·6H2O, 0.1-0.2 mg / L of H3BO3, 0.02-0.04 mg / L of CuSO4·5H2O, 0.02-0.04 mg / L of KI, 0.1-0.2 mg / L of MnCl2·4H2O, 0.05-0.07 mg / L of Na2MoO4·2H2O, 0.1-0.2 mg / L of ZnSO4·7H2O, and 0.1-0.2 mg / L of CoCl2·6H2O, wherein the short-chain fatty acid is one of acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid.

[0049] Preferably, the pH of the culture medium is 6.8-7.2.

[0050] It has been verified that by using the above culture medium to enrich the microbial community from activated sludge, a microbial community that can efficiently utilize short-chain fatty acids to synthesize PHA can be obtained, improving the enrichment efficiency of the PHA-synthesizing microbial community, which is beneficial to obtaining a PHA-synthesizing microbial community with stronger PHA synthesis ability.

[0051] The present invention provides the application of the above-mentioned culture medium in enriching the PHA-synthesizing microbial community.

[0052] Preferably, the application is to enrich the PHA-synthesizing microbial community from activated sludge.

[0053] In the above application, the enrichment adopts an aerobic instantaneous feeding method, and feeding, aeration, sedimentation, and drainage are carried out in sequence in each enrichment cycle;

[0054] After every 2 - 3 enrichment cycles (preferably every 2 enrichment cycles), there is also an aeration stage without adding a carbon source.

[0055] Preferably, the time of the aeration stage without adding a carbon source is 620 - 680 min; and / or, the time of the aeration stage of each enrichment cycle is 620 - 680 min.

[0056] The feed in the enrichment cycle is the above - mentioned culture medium; the culture medium from which short - chain fatty acids and their salts are removed can be added in the aeration stage without adding a carbon source.

[0057] Preferably, after feeding for 1.5 - 2.5 h, an ammonium salt is added, and the addition amount of the ammonium salt is 0.2 - 0.5 g / L. More preferably, it is 0.3 - 0.4 g / L.

[0058] Preferably, the time of each enrichment cycle is 11 - 13 h.

[0059] Preferably, the sedimentation time is 40 - 60 min, the feeding time is 5 - 15 min, and the drainage time is 5 - 15 min.

[0060] Preferably, the activated sludge is the aerobic treatment sludge of winery cellar bottom wastewater.

[0061] The beneficial effects of the present invention at least include: The method for constructing a PHA - synthesizing bacterial community of the present invention can obtain a PHA - synthesizing bacterial community with high utilization ability for different short - chain fatty acids, and can flexibly compound the bacterial communities corresponding to various short - chain fatty acids according to the composition of short - chain fatty acids in the actual substrate, which can significantly improve the utilization rate of anaerobic fermentation liquid of organic waste and the PHA synthesis efficiency. In addition, the method for constructing a PHA - synthesizing bacterial community of the present invention can also significantly improve the enrichment efficiency of the PHA - synthesizing bacterial community, which is beneficial to obtaining a bacterial community with stronger PHA - synthesizing ability and improving the PHA synthesis efficiency and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0063] Figure 1 It is a schematic diagram of the enrichment reactor used in Example 3 of the present invention.

[0064] Figure 2 It is a schematic diagram of the process of each enrichment cycle in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0065] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0066] Example 1

[0067] This example provides a medium for enriching PHA-synthesizing bacteria. The medium contains short-chain fatty acids or their salts and also contains the following components:

[0068] MgSO4·7H2O, 0.67 g / L; EDTA, 0.11 g / L; CaCl2·2H2O, 0.08 g / L; K2HPO4, 0.048 g / L; KH2PO4, 0.038 g / L; thiourea 10 mg / L; lysine 0.3 mg / L; isoleucine 0.26 mg / L; tyrosine 0.36 mg / L; histidine 0.31 mg / L; phenylalanine 0.33 mg / L; tryptophan 0.4 mg / L; trace element solution 1 ml / L;

[0069] Among them, the trace element solution contains the following components: FeCl3·6H2O, 1.5 g / L; H3BO3, 0.15 g / L; CuSO4·5H2O, 0.03 g / L; KI, 0.03 g / L; MnCl2·4H2O, 0.12 g / L; Na2MoO4·2H2O, 0.06 g / L; ZnSO4·7H2O, 0.12 g / L; CoCl2·6H2O, 0.15 g / L.

[0070] Among them, the short-chain fatty acid or its salt is sodium acetate 3.418 g / L (at this time, the medium is an acetate-enriched bacteria medium), or sodium propionate 2.668 g / L (at this time, the medium is a propionate-enriched bacteria medium), or sodium butyrate 2.29 g / L (at this time, the medium is a butyrate-enriched bacteria medium), or valeric acid 1.7 g / L (at this time, the medium is a valerate-enriched bacteria medium), or lactic acid 2.5 g / L (at this time, the medium is a lactate-enriched bacteria medium).

[0071] The pH of the above medium is 7.0 ± 0.1.

[0072] Example 2

[0073] This example provides a medium for enriching PHA-synthesizing bacteria. The medium contains short-chain fatty acids or their salts and also contains the following components:

[0074] MgSO4·7H2O, 0.67 g / L; EDTA, 0.11 g / L; CaCl2·2H2O, 0.08 g / L; K2HPO4, 0.048 g / L; KH2PO4, 0.038 g / L; thiourea 10 mg / L; lysine 0.1 mg / L; isoleucine 0.45 mg / L; tyrosine 0.15 mg / L; histidine 0.55 mg / L; phenylalanine 0.41 mg / L; tryptophan 0.21 mg / L; trace element solution 1 ml / L;

[0075] Among them, the trace element solution contains the following components: FeCl3·6H2O, 1.5 g / L; H3BO3, 0.15 g / L; CuSO4·5H2O, 0.03 g / L; KI, 0.03 g / L; MnCl2·4H2O, 0.12 g / L; Na2MoO4·2H2O, 0.06 g / L; ZnSO4·7H2O, 0.12 g / L; CoCl2·6H2O, 0.15 g / L.

[0076] Among them, the short-chain fatty acid or its salt is sodium acetate 3.418 g / L (in this case, the culture medium is an acetate-enriched bacterial community culture medium), or sodium propionate 2.668 g / L (in this case, the culture medium is a propionate-enriched bacterial community culture medium), or sodium butyrate 2.29 g / L (in this case, the culture medium is a butyrate-enriched bacterial community culture medium), or valeric acid 1.7 g / L (in this case, the culture medium is a valerate-enriched bacterial community culture medium), or lactic acid 2.5 g / L (in this case, the culture medium is a lactate-enriched bacterial community culture medium).

[0077] The pH of the above culture medium is 7.0 ± 0.1.

[0078] Example 3

[0079] This example provides a method for constructing a PHA-synthesizing bacterial community, which is to enrich acetate-enriched bacterial community, propionate-enriched bacterial community, butyrate-enriched bacterial community, valerate-enriched bacterial community, and lactate-enriched bacterial community from activated sludge by using the acetate-enriched bacterial community culture medium, propionate-enriched bacterial community culture medium, butyrate-enriched bacterial community culture medium, valerate-enriched bacterial community culture medium, and lactate-enriched bacterial community culture medium in Example 1 respectively, and mixing at least two of the above bacterial communities to obtain a PHA-synthesizing bacterial community; among them, the activated sludge is aerobic treatment sludge from winery cellar bottom wastewater.

[0080] The schematic diagram of the enrichment reactor used for enriching bacterial communities with various short-chain fatty acid-enriched bacterial community culture media is as Figure 1 shown, and the enrichment method is as follows:

[0081] The effective working volume of the reactor is 2 L. A peristaltic pump and an air pump are used to control the inlet and outlet of materials and the air supply respectively, and a timing relay is used to control the operation of the reactor under the ADF process. Each operation cycle of the reactor is 12 h (schematic diagram as shown in Figure 2 ). In each cycle, feeding (10 min), aeration (650 min), sedimentation (50 min), and drainage (10 min) are carried out in sequence. Among them, during the feeding process, the acetic acid-enriched flora medium, propionic acid-enriched flora medium, butyric acid-enriched flora medium, valeric acid-enriched flora medium, and lactic acid-enriched flora medium of Example 1 are supplemented respectively. After 2 h of feeding, 50 mL of ammonium chloride solution with a concentration of 6.05 g / L is added to the reactor. After every 2 cycles, a starvation aeration stage without carbon source supplementation is carried out, that is, the supplemented medium is the medium supplemented during the above feeding process minus short-chain fatty acids and their salts, and it operates for 650 min. After standing and draining, the medium containing the corresponding short-chain fatty acids is normally added for enrichment.

[0082] Example 4

[0083] This example provides a method for constructing a PHA-synthesizing flora, which is to enrich acetic acid-enriched flora, propionic acid-enriched flora, butyric acid-enriched flora, valeric acid-enriched flora, and lactic acid-enriched flora from activated sludge by using the acetic acid-enriched flora medium, propionic acid-enriched flora medium, butyric acid-enriched flora medium, valeric acid-enriched flora medium, and lactic acid-enriched flora medium of Example 2 respectively, and mix at least two of the above-mentioned floras to obtain a PHA-synthesizing flora; among them, the activated sludge is taken from the aerobic treatment sludge of winery cellar bottom wastewater.

[0084] The enrichment reactor and enrichment method used for flora enrichment with various short-chain fatty acid-enriched flora media are the same as those in Example 3.

[0085] Experimental Example 1

[0086] The acetic acid-enriched flora (A-MMC), propionic acid-enriched flora (P-MMC), butyric acid-enriched flora (B-MMC), valeric acid-enriched flora (V-MMC), and lactic acid-enriched flora (L-MMC) enriched by using different short-chain fatty acid-enriched flora media in Example 3 are respectively used to synthesize PHA with different single short-chain fatty acids to detect the ability of each single short-chain fatty acid-enriched flora to synthesize PHA with different short-chain fatty acids. The specific method is as follows:

[0087] 200 mL of bacterial liquid was taken from the enrichment reactors corresponding to various short-chain fatty acids and added to 5 glass containers of 400 mL, and then 200 mL of substrates (acetic acid-enriched bacterial community medium Medium1, propionic acid-enriched bacterial community medium Medium2, butyric acid-enriched bacterial community medium Medium3, valeric acid-enriched bacterial community medium Medium4, lactic acid-enriched bacterial community medium Medium5) were added respectively. Air was introduced into the containers (3 L / min) for reaction. After one hour, the air supply was stopped. After the reaction system was allowed to stand, 200 mL of the supernatant was discharged. After adding 200 mL of fresh medium, aeration reaction was carried out. After repeating the above operations 5 times, an appropriate amount of bacterial liquid was taken for centrifugation to obtain bacterial cells. After freeze-drying the bacterial cells, the PHA content in the bacterial cells was measured by GC-MS.

[0088] PHA is divided into two parts: poly-3-hydroxybutyrate (PHB) and poly-3-hydroxyvalerate (PHV), and their cell contents are calculated as follows:

[0089]

[0090]

[0091] PHA% = PHB% + PHV%

[0092] M represents the mass (mg) of the dried bacterial cell mass, M PHB represents the mass (mg) of PHB in the bacterial cells, M PHV represents the mass (mg) of PHV in the bacterial cells.

[0093] The experimental results are shown in Table 1.

[0094] Table 1 Synthesis of PHA by enriched bacterial communities using single short-chain fatty acids

[0095]

[0096]

[0097] The results showed that the enriched microbial communities all had a certain PHA synthesis ability. Among them, the acetate-enriched microbial community (A-MMC) accumulated PHA in the range of 24.8% - 61.5% in cells using different short-chain fatty acids. A-MMC could accumulate more PHA (61%) when using acetate (experimental group 1), while only 24.8% of PHA could be accumulated when using propionate as the substrate (experimental group 2). The propionate-enriched microbial community (P-MMC) synthesized PHA in the range of 18.3% - 39.9% in cells using different short-chain fatty acids. When P-MMC used acetate and propionate (experimental groups 6 and 7), it accumulated 38% and 39.9% of PHA in cell dry weight respectively. When using lactate (experimental group 10), the least amount of PHA (18.3%) was accumulated. The butyrate-enriched microbial community (B-MMC) accumulated PHA in cells in the range of 16.7% - 62.9% using different short-chain fatty acids. B-MMC could accumulate more PHA in cells when using butyrate and valerate (experimental groups 13 and 14), accounting for 60.5% and 62.9% of cell dry weight respectively. In contrast, less PHA was produced when using propionate, only accounting for 16.7% of cell dry weight (experimental group 12). The valerate-enriched microbial community (V-MMC) synthesized PHA in the range of 19.7% - 59.6% using different short-chain fatty acids. When V-MMC used butyrate and valerate (experimental groups 18 and 19), it could synthesize more PHA, accounting for 59.6% and 59.5% of cell dry weight respectively. In contrast, when V-MMC used propionate and lactate (experimental groups 17 and 20), only 19.7% and 25.5% of PHA could be accumulated. The lactate-enriched microbial community (L-MMC) synthesized PHA in the range of 31.4% - 59% in cells using different short-chain fatty acids. When L-MMC used lactate (experimental group 25), it could accumulate more PHA, accounting for 59% of cell dry weight. Similarly, less PHA could be accumulated when using propionate (experimental group 22), accounting for 31.4% of cell dry weight. This indicated that when the enriched microbial communities used short-chain fatty acids for PHA synthesis, they had obvious preferences for the short-chain fatty acids used in the enrichment process.

[0098] In addition, the proportions of intracellular PHB and PHV were uneven and had an obvious correlation with the types of short-chain fatty acids. When the enriched microbial communities used acetate, butyrate and lactate (i.e., Medium1, 3 and 5), the proportion of PHB in cells was much larger than that of PHV. While when the enriched microbial communities used propionate and valerate (i.e., Medium2 and 4), the proportion of PHV in cells was much larger than that of PHB.

[0099] Experimental Example 2

[0100] The enriched microbial communities of single short-chain fatty acids (A-MMC, P-MMC, B-MMC, V-MMC, and L-MMC) were artificially combined to obtain five types of mixed microbial communities (Atype-MMC, Ptype-MMC, Btype-MMC, Vtype-MMC, and Ltype-MMC). These mixed microbial communities were respectively used to synthesize PHA with mixed acid solutions of different short-chain fatty acid compositions as substrates. The specific method is as follows:

[0101] 1. Mixed microbial communities and mixed acid solution substrates

[0102] The enriched microbial communities of single short-chain fatty acids were mixed according to the mass ratio to construct mixed microbial communities. The specific ratios are shown in Table 2.

[0103] Table 2 Ratios of mixed microbial communities

[0104]

[0105] The mixed acid solution substrate was prepared by removing short-chain fatty acids or their salts from the culture medium of Example 1 and then adding acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid for proportioning. The specific ratios are shown in Table 3.

[0106] Table 3 Composition of short-chain fatty acids in the mixed acid solution substrate

[0107]

[0108]

[0109] 2. Synthesis of PHA by mixed microbial communities using mixed acids

[0110] The specific experimental operations and calculation methods were the same as those in the PHA synthesis process of Experimental Example 1. The experimental results are shown in Table 4.

[0111] Table 4 Synthesis of PHA by mixed microbial communities using mixed acids

[0112]

[0113]

[0114] The results showed that when the mixed microbial community dominated by acetic acid-enriched bacteria (Atype-MMC) used different mixed acids (Medium6-10) for PHA synthesis, the proportion of PHA accumulated accounted for 52.58%-71.8% of the cell dry weight. Atype-MMC could accumulate an average of 65% of the cell dry weight of PHA. Compared with the average accumulation of 47% of PHA by the single microbial community (A-MMC), the PHA accumulation increased by 38%. When the mixed microbial community dominated by propionic acid bacteria (Ptype-MMC) used mixed acids for PHA synthesis, the proportion of PHA accumulated accounted for 46.2%-59% of the cell dry weight. The average PHA accumulation of Ptype-MMC using mixed acids was 54.24%. Compared with the average accumulation of 32.18% of PHA by P-MMC, the PHA accumulation increased by 68%. When Btype-MMC used mixed acids for PHA synthesis, the PHA content accumulated in the cells was between 56% and 65.9%. The average PHA accumulation of Btype-MMC using mixed acids was 60.28%. Compared with the average accumulation of 43.68% of PHA by B-MMC, the PHA accumulation increased by 38%. When Vtype-MMC used mixed acids for PHA synthesis, the PHA content accumulated in the cells was between 56.6% and 74.6%. The average PHA accumulation of Btype-MMC using mixed acids was 64.34%. Compared with the average accumulation of 42.4% of PHA by B-MMC, the PHA accumulation increased by 51.7%. When Ltype-MMC used mixed acids for PHA synthesis, the PHA content accumulated in the cells was between 58.5% and 73.75%. The average PHA accumulation of Ltype-MMC using mixed acids was 63.05%. Compared with the average accumulation of 49.16% of PHA by B-MMC, the PHA accumulation increased by 28.4%. The above results indicate that the mixed microbial communities can all adapt to mixed acids with different short-chain fatty acids mixed in different proportions for efficient PHA synthesis and accumulation.

[0115] Experimental Example 3

[0116] The single short-chain fatty acid-enriched microbial communities were mixed according to the mass ratio to construct five types of mixed microbial communities (MMC1, MMC2, MMC3, MMC4, and MMC5), and the anaerobic fermentation broth of straw was used for PHA synthesis. The specific method is as follows:

[0117] 1. Mixed microbial communities and anaerobic fermentation broth

[0118] The single short-chain fatty acid-enriched microbial communities were mixed according to the mass ratio to construct mixed microbial communities. The specific ratios are shown in Table 5.

[0119] Table 5 Proportions of mixed microbial communities

[0120]

[0121] The short-chain fatty acids in the anaerobic fermentation broth of straw mainly consist of acetic acid (1070 mg / L), propionic acid (200 mg / L), and butyric acid (666 mg / L).

[0122] 2. Synthesis of PHA by mixed microbial communities using anaerobic fermentation broth

[0123] The specific experimental operations and calculation methods are the same as those in the PHA synthesis process of Experimental Example 1. The calculation method of carbon conversion rate (Y) is as follows:

[0124]

[0125] The experimental results are shown in Table 6.

[0126] Table 6 Production of PHA by mixed microbial communities using anaerobic fermentation broth of straw

[0127]

[0128] The results show that different mixed microbial communities can all use the anaerobic fermentation broth of straw for PHA synthesis and accumulation, and the proportion of PHA in the cell dry weight is between 41% and 48%. By calculating the carbon conversion rates of the consumed short-chain fatty acids and the accumulated PHA, it is known that the carbon conversion capabilities of these mixed microbial communities are all above 60%, indicating that these mixed microbial communities can efficiently convert short-chain fatty acids in straw into PHA.

[0129] The present invention uses the method for constructing PHA-synthesizing microbial communities in the above-mentioned examples, and uses the aerobic treatment sludge of cellar bottom wastewater from wineries with different sources as raw materials for the enrichment of PHA-synthesizing bacteria and the construction of mixed microbial communities. Through a large number of experimental verifications, the constructed mixed microbial communities can all efficiently use substrates with different short-chain fatty acid compositions for PHA synthesis, significantly improving the ability of PHA-synthesizing microbial communities to utilize different short-chain fatty acids and the enrichment efficiency of PHA-synthesizing microbial communities.

[0130] In summary, the mixed microbial communities constructed by the method of enriching single microbial communities with single short-chain fatty acids and then mixing and proportioning the single microbial communities proposed in the present invention can adapt to substrates with different short-chain fatty acid compositions and efficiently carry out PHA synthesis and accumulation.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for constructing a PHA - synthesizing microbial community, characterized in that, The method includes: enriching PHA-synthesizing microbial communities from activated sludge by using culture media containing different single short-chain fatty acids or their salts respectively, and then mixing the PHA-synthesizing microbial communities enriched by the culture media containing different single short-chain fatty acids or their salts.

2. The method for constructing a PHA - synthesizing microbial community according to claim 1, characterized in that, The culture medium contains the following components: short-chain fatty acids or their salts, amino acids, macronutrients and micronutrients; Preferably, the culture medium contains the following components: 1-5 g / L of short-chain fatty acids or their salts, 1-5 mg / L of amino acids, 0.5-1.2 g / L of macronutrients, and 1-3 mg / L of micronutrients.

3. The method for constructing a PHA - synthesizing microbial community according to claim 2, characterized in that, The amino acids are lysine, isoleucine, tyrosine, histidine, phenylalanine and tryptophan; Preferably, the amino acids contain the following components: 0.1-0.3 mg / L of lysine, 0.2-0.5 mg / L of isoleucine, 0.1-0.4 mg / L of tyrosine, 0.3-0.6 mg / L of histidine, 0.3-0.5 mg / L of phenylalanine, and 0.2-0.4 mg / L of tryptophan; And / or, the short-chain fatty acid is one of acetic acid, propionic acid, butyric acid, valeric acid, lactic acid; And / or, the macronutrients include one or more of magnesium, calcium, phosphorus, potassium; And / or, the micronutrients include one or more of iron, boron, copper, iodine, manganese, molybdenum, zinc, cobalt.

4. The method for constructing a PHA - synthesizing microbial community according to any one of claims 1 to 3, characterized in that, The culture medium further contains a chelating agent and a reducing agent; Preferably, the chelating agent is EDTA, and / or the reducing agent is thiourea; More preferably, the culture medium contains 1-5 g / L of short-chain fatty acids or their salts, 0.5-0.8 g / L of MgSO4·7H2O, 0.1-0.2 g / L of EDTA, 0.05-0.1 g / L of CaCl2·2H2O, 0.04-0.06 g / L of K2HPO4, 0.02-0.05 g / L of KH2PO4, 5-15 mg / L of thiourea, 0.1-0.3 mg / L of lysine, 0.2-0.5 mg / L of isoleucine, 0.1-0.4 mg / L of tyrosine, 0.3-0.6 mg / L of histidine, 0.3-0.5 mg / L of phenylalanine, 0.2-0.4 mg / L of tryptophan, 1-2 mg / L of FeCl3·6H2O, 0.1-0.2 mg / L of H3BO3, 0.02-0.04 mg / L of CuSO4·5H2O, 0.02-0.04 mg / L of KI, 0.1-0.2 mg / L of MnCl2·4H2O, 0.05-0.07 mg / L of Na2MoO4·2H2O, 0.1-0.2 mg / L of ZnSO4·7H2O, and 0.1-0.2 mg / L of CoCl2·6H2O.

5. The method for constructing a PHA - synthesizing microbial community according to any one of claims 1 to 4, characterized in that, The enrichment adopts an aerobic instantaneous feeding method, and feeding, aeration, sedimentation and drainage are carried out in sequence in each enrichment cycle; After every 2-3 enrichment cycles, there is also an aeration stage without adding a carbon source; Preferably, the time of the aeration stage without adding a carbon source is 620-680 min; and / or, the time of the aeration stage in each enrichment cycle is 620-680 min; More preferably, after 1.5 - 2.5 h of feeding, an ammonium salt is added, and the addition amount of the ammonium salt is 0.2 - 0.5 g / L.

6. The method for constructing a PHA - synthesizing microbial community according to any one of claims 1 to 5, characterized in that, The activated sludge is the aerobic treatment sludge of winery cellar bottom wastewater.

7. A PHA - synthesizing microbial community constructed by the method for constructing a PHA - synthesizing microbial community according to any one of claims 1 to 6.

8. Use of the PHA - synthesizing microbial community according to claim 7 in PHA production or in constructing a PHA - synthesizing microbial community.

9. A culture medium for enriching a PHA - synthesizing microbial community, characterized in that, The culture medium comprises the following components: short-chain fatty acid or its salt 1 - 5 g / L, amino acid 1 - 5 mg / L, macronutrient 0.5 - 1.2 g / L, micronutrient 1 - 3 mg / L; Among them, the amino acid is lysine, isoleucine, tyrosine, histidine, phenylalanine, and tryptophan; Preferably, the amino acid comprises the following components: lysine 0.1 - 0.3 mg / L, isoleucine 0.2 - 0.5 mg / L, tyrosine 0.1 - 0.4 mg / L, histidine 0.3 - 0.6 mg / L, phenylalanine 0.3 - 0.5 mg / L, tryptophan 0.2 - 0.4 mg / L; And / or, the short-chain fatty acid is one of acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid; And / or, the macronutrient includes one or more of magnesium, calcium, phosphorus, and potassium; And / or, the micronutrient includes one or more of iron, boron, copper, iodine, manganese, molybdenum, zinc, and cobalt; More preferably, the culture medium comprises short-chain fatty acid or its salt 1 - 5 g / L, MgSO4·7H2O 0.5 - 0.8 g / L, EDTA 0.1 - 0.2 g / L, CaCl2·2H2O 0.05 - 0.1 g / L, K2HPO4 0.04 - 0.06 g / L, KH2PO4 0.02 - 0.05 g / L, thiourea 5 - 15 mg / L, lysine 0.1 - 0.3 mg / L, isoleucine 0.2 - 0.5 mg / L, tyrosine 0.1 - 0.4 mg / L, histidine 0.3 - 0.6 mg / L, phenylalanine 0.3 - 0.5 mg / L, tryptophan 0.2 - 0.4 mg / L, FeCl3·6H2O 1 - 2 mg / L, H3BO3 0.1 - 0.2 mg / L, CuSO4·5H2O 0.02 - 0.04 mg / L, KI 0.02 - 0.04 mg / L, MnCl2·4H2O 0.1 - 0.2 mg / L, Na2MoO4·2H2O 0.05 - 0.07 mg / L, ZnSO4·7H2O 0.1 - 0.2 mg / L, CoCl2·6H2O 0.1 - 0.2 mg / L.

10. Use of the culture medium according to claim 9 in enriching a PHA - synthesizing microbial community; Preferably, the use is to enrich a PHA - synthesizing microbial community from activated sludge.

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