A high-density fermentation process using mixed microbial communities to simultaneously achieve rapid biomass growth and high PHA synthesis.

Abstract

A high-density fermentation process using mixed microbial communities to simultaneously achieve rapid biomass growth and high-content PHA synthesis is disclosed. This process uses microbial sludge discharged from the SBR reactor in the PHA-producing bacteria enrichment stage as inoculum and high-COD wastewater as substrate, achieving simultaneous rapid biomass growth and high-content PHA synthesis in a single fermentation. The proposed high-density fermentation process using mixed microbial communities can rapidly proliferate PHA-producing mixed microbial communities within a short time and maintain their high PHA synthesis capacity, resulting in a significant increase in PHA yield. This process can operate stably under high organic loads and can utilize high-concentration organic wastewater, reducing wastewater dilution costs and effectively lowering the cost of PHA production from mixed microbial communities. The proposed high-density fermentation process achieves a PHA volumetric productivity of 4.968 ± 0.048 g / L·d. When integrated into a traditional three-stage PHA synthesis process, it significantly improves the overall PHA yield, ultimately reaching 5.39 g PHA / g seed sludge, which is 8-9 times the yield of traditional batch processes.

Description

Technical Field

[0001] This invention belongs to the field of biodegradable plastic synthesis and waste resource recycling technology, specifically involving a high-density fermentation process of mixed microbial communities that simultaneously achieves rapid biomass growth and high-content PHA synthesis. Background Technology

[0002] PHA is an energy storage substance synthesized within cells under conditions of abundant carbon sources and limited nitrogen, phosphorus, and other resources. It possesses physical properties similar to traditional petroleum-based plastics, exhibiting excellent thermal processability, biodegradability, and biocompatibility. Therefore, it holds promise as a replacement for traditional chemically synthesized plastics, alleviating the increasingly serious problem of "white pollution." The environment contains a large amount of waste carbon sources, and the resource utilization of these waste carbon sources has become one of the directions for technological development in the environmental field. The scientific and rational utilization of waste carbon sources to synthesize PHA has become an important means of carbon reduction. Currently, the commercial promotion of PHA mainly relies on pure microbial fermentation, where engineered microorganisms can achieve rapid PHA synthesis. However, pure microbial fermentation incurs high costs for raw materials, sterilization, and genetic modification, resulting in a high price for the final PHA product, severely restricting its large-scale application. In contrast, the mixed microbial community PHA production process uses existing microbial communities as screening targets, operates in a completely open system, requires no sterilization, and can fully utilize sludge fermentation broth, kitchen waste fermentation broth, and waste animal and vegetable oils as substrates, achieving resource utilization of waste and showing broad application prospects.

[0003] Mixed microbial communities are often operated at low loading (1000-4000 mg COD / L) during the enrichment stage to screen for strains capable of synthesizing PHA. However, low loading results in slow biomass growth, while high loading leads to system imbalance and ultimately system collapse. Low biomass is a significant reason for the low yield in the PHA synthesis stage, severely limiting the large-scale industrial application of mixed microbial communities and PHA. Furthermore, the separation of microbial proliferation and PHA synthesis in traditional processes complicates the production flow. Therefore, rapidly increasing the biomass of PHA-producing bacteria while simultaneously achieving PHA synthesis would greatly improve the yield of mixed-microbial PHA synthesis and reduce production costs, which is of great significance for promoting the large-scale application of mixed-microbial PHA synthesis. Summary of the Invention

[0004] The purpose of this invention is to solve the problems of low biomass in mixed microbial communities restricting PHA production, and the separation of biomass growth and PHA synthesis phases. This invention provides a method for simultaneously achieving rapid biomass growth and high-content PHA synthesis in a one-step fermentation process, thereby improving the overall yield of PHA synthesis in mixed microbial communities.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A high-density fermentation process using mixed microbial communities to simultaneously achieve rapid biomass growth and high PHA synthesis, the method comprising the following steps:

[0007] Step 1: Establish a high-density fermentation system: such as Figure 1 As shown, the high-density fermentation system includes a fermentation tank (which can be made of glass, stainless steel, or other materials), a stirring device, a temperature control device (which can be a water bath, electric temperature control, or other methods), an aeration device, a sampling port, a feeding port, and an exhaust valve.

[0008] Step 2: Collect the bacterial liquid in the PHA-producing mixed bacteria enrichment reactor, and aerate it to ensure that the carbon and nitrogen sources are fully consumed. The time is usually 12-36 hours to obtain seed mud.

[0009] Step 3: Place the seed mud and fermentation liquid in a fermentation tank for fermentation. The volume of the fermentation tank is not limited; taking a 1L actual volume and a 0.8L working volume as an example, the aeration rate during fermentation is 1.5-3L / min, the dissolved oxygen concentration is 30%-50% higher than the saturated dissolved oxygen concentration in water at that temperature, the pressure in the fermentation tank is maintained at 0-0.025MPa, the fermentation temperature is controlled at 25±5℃, and the stirring speed is 500-800rpm. The pressure of the fermentation tank is adjusted by regulating the aeration rate.

[0010] Furthermore, in step two, the MLVSS in the enrichment reactor is 3-5 g / L.

[0011] Furthermore, in step two, the dominant bacteria in the mixed microbial community of the PHA-producing bacteria enrichment reactor and seed mud are Thaurera, Paracoccus, Halomonas, Corynebacterium, and Amaricoccus.

[0012] Furthermore, in step two, the PHA-producing mixed bacteria enrichment reactor is a salt-tolerant system that has been acclimated to salinity (0%-1.8% salinity) or a PHA-producing bacteria enrichment system that has not been acclimated to salinity.

[0013] Furthermore, in step three, the fermentation broth is artificially prepared and mainly composed of small molecule volatile acids, including carbon sources such as glucose, glycerol, protein, ethanol, etc., or acid-producing broth from actual anaerobic fermentation, with a substrate concentration of 10000±100mgCOD / L-30000±100mgCOD / L.

[0014] Furthermore, the small molecule volatile acid is one or more of acetic acid, propionic acid, butyric acid, and valeric acid.

[0015] Furthermore, the salinity of the fermentation broth is 0%-2.8%, and the initial pH is 6.0-8.0; ammonium chloride and potassium dihydrogen phosphate are added to the fermentation broth as nitrogen and phosphorus sources, respectively, so that the C / N / P ratio in the substrate is 100:5-10:1-1.5, and a certain amount of mineral salt solution is used to provide trace elements.

[0016] Furthermore, in step three, the amount of seed mud inoculated is 10±1% to 20±1% of the total volume of the fermentation liquid.

[0017] Furthermore, in step three, the fermentation time is 18 to 20 hours, at which point the PHA content in the microorganisms reaches its maximum.

[0018] Furthermore, the process also includes step four, whereby the remaining fermentation broth after high-density fermentation, after centrifugation and filtration to remove bacteria, is used as the substrate for the front-end PHA-producing bacteria enrichment reactor.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The mixed-culture high-density fermentation process proposed in this invention can achieve PHA synthesis while the biomass of PHA-producing bacteria rapidly increases in one-step fermentation;

[0021] 2. The mixed-culture high-density fermentation process proposed in this invention combines the expansion culture stage of PHA-producing bacteria with the PHA synthesis stage. After being embedded into the traditional three-stage process, it can significantly improve the PHA yield of the entire process. Under the condition of inoculating the same volume (or mass) of seed mud, its PHA yield (g PHA / g seed mud) is about 8-9 times that of the original process.

[0022] 3. From the perspective of the overall process, the high-density fermentation process proposed in this invention can utilize high COD carbon sources (10000±100mg COD / L-30000±100mg COD / L) as substrates, enabling the microbial community to operate stably under high organic loads and achieving simultaneous biomass growth and PHA synthesis. Compared with the original process with lower organic loads (1000-4000mg COD / L for the enrichment stage and 3000-6000mg COD / L for the PHA synthesis stage), the high-density fermentation process will significantly increase the cell growth density and greatly reduce the cost of diluting the substrate.

[0023] 4. The substrate carbon source used in this invention can be a high-concentration organic waste carbon source that is nitrogen-deficient or nitrogen-free, such as acidified liquid of molasses wastewater, acidified liquid of anaerobic fermentation of residual sludge, and acidified liquid of anaerobic fermentation of kitchen waste. While utilizing waste carbon sources to achieve resource recovery, it can also bring certain environmental benefits by reducing biochemical oxygen demand. Attached Figure Description

[0024] Figure 1This is a diagram of the apparatus for the high-density fermentation process in this invention. In the diagram: 1 is a fermentation tank, 2 is a stirring device, 3 is a temperature control device, 4 is an aeration device, 5 is a sampling port, 6 is a feeding port, and 7 is an exhaust valve.

[0025] Figure 2 This is a schematic diagram comparing the high-density fermentation embedded three-stage process with the traditional batch process. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0027] The process of this invention uses the bacterial sludge discharged from the SBR reactor in the PHA-producing bacteria enrichment section as inoculum and high-COD wastewater as substrate, achieving simultaneous rapid biomass growth and high-content PHA synthesis in a single fermentation. The mixed-culture high-density fermentation process proposed in this invention can rapidly proliferate the PHA-producing mixed bacteria and maintain its high PHA synthesis capacity in a short time, resulting in a significant increase in PHA yield. This process can operate stably under high organic loads, utilizes high-concentration organic wastewater, reduces wastewater dilution costs, and effectively lowers the cost of PHA production from mixed bacteria. The mixed-culture high-density fermentation process proposed in this invention achieves a PHA volumetric productivity of 4.968±0.048 g / L·d. When integrated into the traditional three-stage PHA synthesis process, it can significantly improve the overall PHA yield, ultimately reaching 5.39 g PHA / g seed sludge, which is 8-9 times the yield of traditional batch processes.

[0028] Example 1:

[0029] A high-density fermentation method based on mixed microbial communities to simultaneously achieve biomass growth and PHA synthesis in a one-step fermentation process comprises the following steps:

[0030] Step 1: Establish a high-density fermentation system: The high-density fermentation system consists of a fermentation tank system, such as... Figure 1 As shown, the fermentation tank system includes a fermentation tank 1 (which can be made of glass, stainless steel, or other materials) and a stirring device 2, a temperature control device 3 (which can be a water bath, electric temperature control, or other methods), an aeration device 4, a sampling port 5, a feeding port 6, an exhaust valve 7, etc., provided on the fermentation tank 1. The fermentation tank has a volume of 1L and a working volume of 0.8L. The temperature control device is in water bath mode.

[0031] Step 2: Collect the PHA-producing bacteria enrichment reactor and aerate it fully for 24 hours to completely deplete the nitrogen source. The MLVSS is 3.45±0.02g / L.

[0032] Step 3: When starting the high-density fermentation system in Step 1, place the bacterial sludge from Step 2 into the reactor, with a volume of 160 ml (20% of the working volume of the fermenter). Use the high-density fermentation mode, that is: add 640 ml (80% of the working volume of the fermenter) of fermentation broth to the fermenter. The salinity of the fermentation broth is 0.8% (calculated as NaCl), the pH is 8.0±0.2, and the substrate concentration is 20000±100 mg COD / L. Its COD composition is: acetate / propionate / butyrate / valerate = 20 / 10 / 60 / 10, 0.37 g / L NH4Cl and 0.11 g / L KH2PO4 as carbon and nitrogen sources. In addition, add 1 g / L MgSO4, 0.4 g / L CaCl2, 0.4 g / L thiourea, and 1 mL / L mineral salt solution. Adjust the aeration flow rate to 1.5 L / min, temperature to 25℃, stirring speed to 700 rpm, and fermenter pressure to 0.02 MPa. Defoamers such as polyethers can be used to suppress foaming. At this point, the MLVSS in the fermenter is 0.690 ± 0.002 g / L.

[0033] Step 4: Begin the high-density fermentation process. After 18 hours of fermentation, the PHA content within the microbial cells reaches its maximum, at which point fermentation ends. The fermentation broth is discharged and can be used for subsequent PHA purification, etc.

[0034] Step 5: After centrifugation and filtration to remove bacteria, the remaining fermentation broth contains mainly acetate, with a COD of approximately 1300-1800 mg COD / L. This can be used as a substrate in the upstream PHA-producing bacteria enrichment reactor. Figure 2 As shown.

[0035] Experimental conclusion:

[0036] (1) After 18 hours of fermentation, the MLVSS in the fermenter was 6.636 g / L, the volumetric biomass productivity was 8.856 g / L·d, the maximum accumulation capacity of PHA was 0.560±0.004 g PHA / g VSS, the volumetric biomass productivity was 4.968±0.048 g / L·d, and the yield was 5.39 g PHA / g seed mud.

[0037] (2) During the fermentation process, the microorganisms maintain a good growth trend and make full use of the VFA in the substrate for growth metabolism and PHA synthesis. After the fermentation is completed, the concentration of the fermentation broth is about 1600 mg COD / L.

[0038] (3) The PHA obtained after fermentation consists of two monomers, HB and HV, with relative contents of 84.2% and 15.8%, respectively. The composition of PHA can be achieved by adjusting the ratio of even-carbon VFAs (acetate and butyrate) and odd-carbon VFAs (propionate and valerate) in the substrate.

[0039] Comparative Example 1:

[0040] Step 1: Perform standard batch process to synthesize PHA. Specifically, take 160ml of bacterial sludge from the enrichment reactor and place it in a 500ml beaker. Add 160ml of carbon-source-only fermentation broth, with the same COD composition as in Example 1. Three control groups with different conditions were set up: R1 (pH 7.0, salinity 0.8%), R2 (pH 8.0, salinity 0.8%), and R3 (pH 7.0, salinity 1.8%). Aeration and stirring are performed. One cycle of the batch process includes: running for 1.5 hours, then stopping aeration and stirring, letting it stand for 0.5 hours, and removing 160ml of supernatant. The second, third, and fourth cycles also require removing 160ml of supernatant. Additionally, before the start of the second, third, fourth, and fifth cycles, 160ml of carbon-source-only fermentation broth needs to be added. In the fifth cycle, no supernatant is removed (final volume is 320mL), at which point the PHA content reaches its highest level.

[0041] Step 2: Perform high-density mixed-culture fermentation to synthesize PHA, with the specific setup being the same as described in Example 1.

[0042] Experimental conclusion:

[0043] (1) The yield comparison between the conventional batch process and the mixed-culture high-density fermentation process is shown in Table 1. It can be seen that the biomass did not change in the conventional batch process, and its maximum PHA accumulation capacity was slightly higher than that of the high-density fermentation process. However, since the high-density fermentation process involves a biomass growth process, its overall yield is higher than that of the conventional batch process.

[0044] Table 1 Comparison between high-density fermentation process and conventional batch process

[0045]

[0046] Example 2:

[0047] Step 1: Anaerobic fermentation of kitchen waste yields an acid-producing broth with a SCOD of 55000±300 mg COD / L. The volatile fatty acid (VFA) concentration is 50-70 mg COD / mg COD, with acetate comprising 21%, propionate 3%, butyrate 67%, and valerate 9%. The acid-producing fermentation broth is diluted to 20000±100 mg COD / L for high-density fermentation with mixed microbial communities; it is also diluted to 6400±10 mg COD / L for conventional batch processes.

[0048] Step 2: Perform high-density mixed-culture fermentation to synthesize PHA, with the specific setup being the same as described in Example 1.

[0049] Step 3: Perform conventional batch process synthesis of PHA, with the specific settings consistent with those described in Example 2.

[0050] Experimental conclusion:

[0051] Table 2 Comparison of high-density fermentation process and conventional batch process based on actual wastewater.

[0052]

[0053] (1) Table 2 shows a comparison of the yields of the conventional batch process and the mixed-culture high-density fermentation process based on actual wastewater. It can be seen that the biomass in the conventional batch process remained unchanged, and its maximum PHA accumulation capacity was slightly higher than that of the high-density fermentation process. Compared with the simulated wastewater in Example 2, the PHA content, PHA volumetric productivity, and yield of both the conventional batch process and the mixed-culture high-density fermentation process decreased slightly. However, in terms of biomass growth, the mixed-culture high-density fermentation process based on actual wastewater showed a slight improvement. Overall, the mixed-culture high-density fermentation process based on actual wastewater still has good PHA production capacity, with a yield nine times that of the conventional batch process.

[0054] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for high-density fermentation of mixed microbial communities that simultaneously achieves rapid biomass growth and high PHA synthesis, characterized in that: The method includes the following steps: Step 1: Establish a high-density fermentation system: The high-density fermentation system includes a fermenter (1), a stirring device (2), a temperature control device (3), an aeration device (4), a sampling port (5), a feeding port (6), and an exhaust valve (7). Step 2: Acclimate the PHA-producing mixed bacteria to a salinity of 0.5%~1.8%, collect the bacterial solution from the PHA-producing mixed bacteria enrichment reactor after salinity acclimation, and aerate it to ensure that the carbon and nitrogen sources are fully consumed to obtain seed mud. The dominant bacteria in the mixed bacterial community in the PHA-producing bacteria enrichment reactor and seed mud are *Daucus*, *Paragonimus*, *Halomonas*, *Corynebacterium*, and *Proteus*. Step 3: Place the seed mud and fermentation broth in a fermentation tank for fermentation. The aeration rate during fermentation is 1.5~3 L / min, the dissolved oxygen concentration is greater than 30% of the saturated dissolved oxygen concentration in water at this temperature, the pressure in the fermentation tank is maintained at 0~0.025 MPa, the fermentation temperature is controlled at 25±5℃, the fermentation time is 18~20 hours, and the stirring speed is 500~800 rpm. The fermentation broth is a carbon source mainly composed of small molecule volatile acids or acid-producing liquid from actual anaerobic fermentation. The substrate concentration of the fermentation broth is 10000±100 mg COD / L~30000±100 mg. COD / L; the small molecule volatile acid is one or more of acetic acid, propionic acid, butyric acid, and valeric acid; the salinity of the fermentation broth is 0.8%~2.8%, and the initial pH is 6.0~8.0; ammonium chloride and potassium dihydrogen phosphate are added to the fermentation broth as nitrogen and phosphorus sources, respectively, so that the C / N / P ratio in the substrate is 100:5~10:1~1.5, and a certain amount of mineral salt solution is used to provide trace elements.

2. The method for high-density fermentation of mixed microbial communities to simultaneously achieve rapid biomass growth and high PHA synthesis according to claim 1, characterized in that: In step two, the MLVSS in the enrichment reactor is 3~5 g / L.

3. The method for high-density fermentation of mixed microbial communities to simultaneously achieve rapid biomass growth and high PHA synthesis according to claim 1, characterized in that: In step three, the carbon source may also include glucose, glycerol, protein, or ethanol.

4. The method for high-density fermentation of mixed microbial communities to simultaneously achieve rapid biomass growth and high PHA synthesis according to claim 1, characterized in that: In step three, the amount of seed mud inoculated is 10±1% to 20±1% of the total volume of the fermentation liquid.

5. A high-density fermentation method for mixed microbial communities that simultaneously achieves rapid biomass growth and high PHA synthesis according to any one of claims 1 to 4, characterized in that: The process also includes step four, whereby the remaining fermentation broth after high-density fermentation, after centrifugation and filtration to remove bacteria, is used as the substrate for the front-end PHA-producing bacteria enrichment reactor.

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