Method for synergistically synthesizing medium-chain polyhydroxyalkanoate based on functional microbial community

Abstract

The invention relates to the field of biological material engineering, in particular to a method for synergistically synthesizing medium-chain polyhydroxyalkanoate (mcl-PHA) based on functional microbial communities. The method comprises the following specific steps: converting organic matters into lactic acid and ethanol by utilizing microorganisms; then, the carbon chain extension flora is converted into medium-chain fatty acid (MCFAs), and COs and Hs are released; and under the aerobic condition, the endogenous MCFAs and COs are synergistically converted into the mcl-PHA by the pre-cultured hydrogen oxidizing bacteria. The method can be realized by constructing a microbial space ecological niche through a single reactor or adopting a multi-reactor series system. According to the strategy, the requirement of traditional mcl-PHA synthesis for additional addition of precursors (such as MCFAs) is avoided, sterilization is not needed in other processes except for hydrogen oxidizing bacteria culture, and then the raw material and operation cost is greatly reduced. According to the method, organic waste and CO are efficiently and synergistically utilized, carbon emission reduction is achieved, and a new way is opened up for industrialization of degradable bioplastics.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial engineering, and particularly relates to a method for co-synthesizing medium-chain polyhydroxyalkanoates based on functional microbial communities. Background Art

[0002] Polyhydroxyalkanoates (PHA) are a class of completely biodegradable bioplastics that have received much attention in recent years due to their sustainability and environmental friendliness. Medium-chain PHAs (mcl-PHAs) are applicable to almost all application fields of traditional plastics due to their excellent physical and chemical properties. However, the existing medium-chain PHA synthesis technologies have the following main defects:

[0003] 1. High raw material cost: The synthesis of medium-chain PHA requires medium-chain fatty acids (MCFAs) as precursor substances, and most microorganisms cannot naturally synthesize MCFAs. Existing methods usually require additional addition of MCFAs or medium-chain alkanes, resulting in high raw material costs.

[0004] 2. Limited strain selection: The strains currently found capable of synthesizing medium-chain PHA mainly focus on the genus Pseudomonas, with a single species, and the metabolic burden of the single-strain fermentation process is heavy, affecting the synthesis efficiency.

[0005] 3. Difficulty in utilizing organic waste: Organic waste (such as food waste) has the potential as a cheap raw material, but its complex composition is not suitable for the growth of single strains, and it usually does not contain medium-chain fatty acid precursors. Existing methods are difficult to directly utilize organic waste to synthesize medium-chain PHA under open conditions.

[0006] 4. Low fermentation efficiency: The synthesis of medium-chain PHA involves multiple metabolic steps, including organic carbon degradation, medium-chain fatty acid synthesis, and PHA polymerization, etc. The metabolic burden of a single strain to complete these steps is heavy, resulting in low synthesis efficiency.

[0007] 5. Synthesis problems in an open environment: Existing technologies are difficult to achieve continuous synthesis of medium-chain PHA in an open environment, mainly due to high risks of contamination by miscellaneous bacteria and complex metabolic pathways.

[0008] In summary, the existing medium-chain PHA synthesis technologies have significant defects in terms of raw material cost, strain selection, metabolic efficiency, and applicability in an open environment. Summary of the Invention

[0009] In view of the deficiencies of the prior art, the present invention provides a method for co-synthesizing medium-chain polyhydroxyalkanoates based on functional microbial communities, a green method for continuously and efficiently synthesizing medium-chain PHA using renewable raw materials in an open environment.

[0010] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0011] The present invention discloses a method for the collaborative synthesis of medium-chain polyhydroxyalkanoates based on spatial microbial communities, which realizes the efficient conversion of organic waste into medium-chain polyhydroxyalkanoates (mcl-PHA) by constructing a biotransformation system composed of four functional microbial populations. Specifically, it includes the following steps:

[0012] (1) Use microorganisms that convert carbohydrate organic matter to convert carbohydrate organic matter into lactic acid; the microorganisms that convert carbohydrate organic matter include, but are not limited to, lactic acid bacteria, yeasts, glycolytic bacteria, starch-decomposing bacteria, and one or more of lactic acid enzymes, yeast enzymes, glycolytic enzymes, and starch-decomposing enzymes without specifying the specific genus and species. The inoculation amount of the microorganisms that convert carbohydrate organic matter is 2-5%.

[0013] (2) Use yeasts to convert carbohydrate organic matter into ethanol; without specifying the specific genus and species, the inoculation amount is 2-5%.

[0014] (3) Use medium-chain fatty acid functional flora to extend and convert the lactic acid in step (1) and the ethanol in step (2) into C6-C8 medium-chain fatty acids (MCFAs), and release CO2 and H2, with an inoculation amount of 20-50%. The medium-chain fatty acid functional flora includes a flora with Caproiciproducens genus, Anaerococcus genus, Megasphaera genus, and Clostridium genus as the core, and Clostridium genus includes, but is not limited to, Clostridium klyuveri.

[0015] (4) Under aerobic conditions, add hydrogen-oxidizing bacteria to the medium-chain fatty acids in step (3). The hydrogen-oxidizing bacteria utilize the CO2 and H2 released in step (3) for growth, and synthesize medium-chain polyhydroxyalkanoates from medium-chain fatty acids under nitrogen-limiting conditions in the later stage of growth. The hydrogen-oxidizing bacteria include, but are not limited to, Paracoccus denitrificans.

[0016] Preferably, inoculate the hydrogen-oxidizing bacteria into the BPM medium at a ratio of 0.1%, and culture at 30 °C and 180 r / min for 72 hours. The BPM medium includes 5 g / L of beef extract, 10 g / L of peptone, 5 g / L of NaCl, and pH 7.0. Except for the culture of hydrogen-oxidizing bacteria, the rest of the processes involved in the present invention are carried out under non-sterile conditions.

[0017] The above process disclosed by the present invention is realized in two ways.

[0018] One is: the synthesis of the medium-chain polyhydroxyalkanoate is completed in a single reactor, and the ecological niches of microorganisms with different functions are constructed in the single reactor to form a spatial microbial community. The specific steps are as follows:

[0019] (1) The reactor comprises a feed zone, a reaction zone and a discharge zone which are arranged in sequence. The reaction zone is filled with a porous carrier material which is in a granular or blocky form.

[0020] (2) Microorganisms, yeasts and medium-chain fatty acid functional bacteria that convert carbohydrate organic matter are respectively colonized on porous carrier materials at different positions in the reaction zone, that is, they are colonized in sequence along the reaction zone, and no porous carrier material is added to the hydrogen oxidizing bacteria area.

[0021] (3) A filter is provided between the porous carrier materials colonized with microorganisms, yeasts and medium-chain fatty acid functional bacteria. A filter with a pore size of 100-500 μm is used when the particles are suspended at a high concentration, and a filter with a pore size of 20-100 μm is used when the particles are suspended at a low concentration. As one embodiment, the microorganisms and yeasts that convert carbohydrate organic matter can be colonized in the same ecological niche and separated by the filter.

[0022] (4) Carbohydrate organic matter is introduced through the feed port on the feed zone, air is introduced into the hydrogen oxidizing bacteria area, and an ORP sensor is provided to maintain the redox potential between 50-100 mV. The CO2 and H2 produced by the medium-chain fatty acid functional bacteria naturally diffuse in the reactor and are utilized by the hydrogen oxidizing bacteria. A gas collection system is provided on the reactor to collect the exhaust gas and return it to the reactor.

[0023] (5) A plurality of sub-areas are provided in the hydrogen oxidizing bacteria area. As one embodiment, the volume of the hydrogen oxidizing bacteria area is 4 times the volume of the medium-chain fatty acid functional bacteria colonization area, and the interior is equally divided into 4 switchable sub-areas. The number of open sub-areas is determined according to the concentration of caproic acid in the medium-chain fatty acids. If the caproic acid concentration is high, the number of open sub-areas is increased to increase the dilution multiple. The sub-areas are separated by partitions, and control valves are provided on the partitions to allow the sub-areas to be connected. Nitrogen-restricted culture medium is added to the hydrogen oxidizing bacteria area, and the hydrogen oxidizing bacteria are cultured in BPM culture medium in advance, and inoculated at a ratio of 40-45% of the volume of the nitrogen-restricted culture medium after centrifugal cleaning. After the fermentation liquid of the medium-chain fatty acids flows into the hydrogen oxidizing bacteria area, the caproic acid concentration therein is maintained at ≤4 g / L. Then, the gas generated during the fermentation of the medium-chain fatty acids is continuously introduced, and synthesized for 72 hours at 30-35° C. in a batch manner, and then the hydrogen oxidizing bacteria are discharged and collected.

[0024] The nitrogen-limited medium comprises KH2PO4 2 g / L, K2HPO4 2 g / L, NaCl 2 g / L, CaCl2 10 mg / L, MgSO4·7H2O 200 mg / L, FeSO4·7H2O 13.9 mg / L, NiCl2·6H2O 0.36 mg / L, trace element solution 8 mL / L, and vitamin solution 8 mL / L.

[0025] Another one is that the synthesis of the medium-chain polyhydroxyalkanoate is completed in multiple reactors connected in series to form a segmented functional microbial system. The specific steps are as follows:

[0026] (1) The conversion of carbohydrate organic matter into lactic acid and ethanol is carried out in the first reactor.

[0027] (2) The conversion of lactic acid and ethanol into medium-chain fatty acids by the medium-chain fatty acid functional flora is carried out in the second reactor.

[0028] (3) The synthesis of medium-chain polyhydroxyalkanoate (mcl-PHA) by the hydrogen-oxidizing bacteria using CO2, H2, and medium-chain fatty acids is carried out in the third reactor; a gas collection system is arranged on the second reactor to continuously collect the CO2 and H2 gases discharged from the second reactor, and the collected gases are continuously pumped into the third reactor by an air pump to form a gas circulation system.

[0029] (4) In the third reactor, the hydrogen-oxidizing bacteria are pre-cultured in the BPM medium, centrifuged and washed, and then added to the nitrogen-limited medium at a ratio of 40-45%. Then, the fermentation broth of medium-chain fatty acids is added to keep the concentration of caproic acid therein at ≤4 g / L. The synthesis is carried out in a batch mode at 30 °C for 72-120 h, and the gases generated during the fermentation of medium-chain fatty acids are continuously introduced and used for gas stirring. The present invention has the following beneficial effects:

[0030] 1. Solve the problem of raw material cost for the synthesis of medium-chain PHA: By constructing a functional microbial community, using microorganisms (such as lactic acid bacteria) that convert carbohydrate organic matter, yeast, and medium-chain fatty acid functional flora to convert carbohydrate organic matter into medium-chain fatty acids, there is no need to add expensive medium-chain fatty acids or medium-chain alkanes as precursors, greatly reducing the raw material cost.

[0031] 2. Expand the selection range of strains for the synthesis of medium-chain PHA: Different from the traditional synthesis of medium-chain PHA mainly relying on Pseudomonas, the present invention disassembles the synthesis task of medium-chain PHA and distributes the metabolic steps to different functional microorganisms, greatly increasing the types of selectable strains and enhancing the flexibility and robustness of the system.

[0032] 3. Improved synthesis efficiency and system stability: By reducing the metabolic burden of a single microorganism, each functional microorganism focuses on specific metabolic tasks, significantly improving the synthesis efficiency. At the same time, the synergistic effect of the microbial community enhances the system's adaptability to environmental changes and resistance to external pollution.

[0033] 4. Achieved synthesis under non-sterile conditions: Except for the cultivation of hydrogen-oxidizing bacteria, the rest of the process is carried out under non-sterile conditions, significantly reducing the energy consumption and equipment investment required for sterilization and lowering the production cost.

[0034] 5. Provided two flexible implementation methods: The two technical routes of the single reactor method and the multi-reactor series system can be flexibly selected according to actual application requirements to adapt to production needs under different scales and conditions.

[0035] 6. Achieved resource recycling: By converting carbohydrate organic matter into high-value medium-chain PHA, waste resource utilization is realized, promoting the development of circular economy. At the same time, the CO2 and H2 produced by the medium-chain fatty acid functional flora are utilized by hydrogen-oxidizing bacteria, reducing greenhouse gas emissions.

[0036] 7. Improved the flexibility of process control: In the single reactor method, the hydrogen-oxidizing bacteria area is equally divided into switchable areas, which can be flexibly adjusted according to the concentration of medium-chain fatty acids to adapt to different fermentation conditions. In the multi-reactor series system, each reactor can independently optimize operating parameters, improving the accuracy of process control. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the single reactor of Embodiment 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] If not specifically specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0040] To make the purpose, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to specific embodiments.

[0041] Embodiment 1 Construction of Spatial Microbial Community in a Single Reactor and Synthesis of mcl-PHA

[0042] 1. Material Preparation

[0043] 1.1 Strain Preparation

[0044] Lactic acid bacteria: Isolated from spoiled vegetables and cultured on MRS medium for 24 hours.

[0045] Yeast: Commercial bread yeast, activated and cultured on YPD medium for 12 hours.

[0046] Medium-chain fatty acid functional flora: Domesticated from anaerobic digestion sludge, mainly including the genera Caproiciproducens, Megasphaera elsdenii, and Clostridium klyuveri.

[0047] Hydroxide bacteria: Paracoccus denitrificans, isolated from soil, and cultured in BPM medium (5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, pH 7.0) at an inoculation amount of 0.1% for 72 hours.

[0048] 1.2 Porous Carrier Material

[0049] Select polyurethane foam material or carbon fiber felt, cut it into 1 cm × 1 cm × 1 cm cubes, soak it in sterile water for 24 hours, and then sterilize it by high-pressure steam at 121 °C for 15 minutes.

[0050] 1.3 Reactor Design

[0051] Refer to Figure 1 As shown, an organic glass rectangular reactor is made, with a total volume of 14 L, divided into three regions: Feed zone: Located at the bottom of the reactor, with a volume of 1 L; Reaction zone: Located in the middle, with a volume of 8 L, divided into three functional regions: Lactic acid bacteria and yeast region (2 L), Medium-chain fatty acid functional flora region (2 L), and Hydroxide bacteria region (8 L); Discharge zone: Located at the top, with a volume of 1 L.

[0052] 2. Reactor Construction

[0053] 2.1 Spatial Niche Construction

[0054] In the lactic acid bacteria and yeast region, fill the treated polyurethane foam / carbon fiber felt, with a filling rate of 70%. In the medium-chain fatty acid functional flora region, fill the treated polyurethane foam / carbon fiber felt, with a filling rate of 80%. Set a filter screen with a pore size of 100 μm between the lactic acid bacteria and yeast region and the medium-chain fatty acid functional flora region. Set a filter screen with a pore size of 50 μm between the medium-chain fatty acid functional flora region and the hydroxide bacteria region. The hydroxide bacteria region is not filled with porous material and is equally divided into 4 independently controllable sub-regions. During the domestication period of each unit, separate them with stainless steel plates.

[0055] 2.2 Parameter Monitoring System

[0056] Install pH probes, ORP probes and temperature sensors in each area; install dissolved oxygen probes in the area of hydrogen-oxidizing bacteria; set up a gas collection and reflux system at the top of the reactor. Set up a flow rate control device (such as a valve, etc.) at the bottom of the reactor.

[0057] 3. Operation Process

[0058] 3.1 Colonization of Functional Bacterial Communities

[0059] Lactic acid bacteria and yeast: Mix and inoculate the activated cultured lactic acid bacteria and yeast into the areas of lactic acid bacteria and yeast respectively at an inoculation amount of 3%, and culture them at 28°C for 48 hours to colonize them on the porous carrier.

[0060] Functional bacterial community of medium-chain fatty acids: Inoculate the domesticated functional bacterial community of medium-chain fatty acids into the area of the functional bacterial community of medium-chain fatty acids at an inoculation amount of 35%, and culture it under anaerobic conditions at 30°C for 72 hours.

[0061] Do not inoculate the area of hydrogen-oxidizing bacteria temporarily.

[0062] 3.2 Reactor Startup and Operation

[0063] Continuously introduce simulated food waste (mainly composed of rice, noodles, and vegetable residues) through the feed inlet, with a total organic carbon concentration of 15 g / L and an initial flow rate of 0.5 L / h; monitor the caproic acid concentration at the outlet of the area of the functional bacterial community of medium-chain fatty acids, and adjust the feed flow rate and organic load to keep the caproic acid concentration stable at 3 - 4 g / L. After the system has been operating stably for 7 days, start preparing for the inoculation of hydrogen-oxidizing bacteria.

[0064] 3.3 Operation of the Area of Hydrogen-oxidizing Bacteria

[0065] Centrifuge the Paracoccus denitrificans bacterial solution cultured in the BPM medium (6000 rpm, 10 minutes) to collect it, and wash it twice with sterile PBS solution; determine the number of sub-areas to open in the area of hydrogen-oxidizing bacteria according to the caproic acid production in the area of the functional bacterial community of medium-chain fatty acids (for example, if the caproic acid production can support 2 sub-areas, then open 2). It is necessary to maintain the caproic acid concentration at 4 g / L and below in the area of hydrogen-oxidizing bacteria. For example: if the caproic acid is 12 g / L, then open 3 sub-areas to increase the dilution factor by increasing the area.

[0066] Add nitrogen-limited medium (KH2PO4 2 g / L, K2HPO4 2 g / L, NaCl 2 g / L, CaCl2 10 mg / L, MgSO4·7H2O 200 mg / L, FeSO4·7H2O 13.9 mg / L, NiCl2·6H2O 0.36 mg / L, trace element solution 8 mL / L, vitamin solution 8 mL / L) to the open sub-region, and inoculate the washed hydrogen-oxidizing bacteria into the nitrogen-limited medium at a ratio of 40% of the volume of the nitrogen-limited medium; adjust the reactor flow rate so that after the fermentation broth of medium-chain fatty acids flows into the hydrogen-oxidizing bacteria region, the concentration of caproic acid in the nitrogen-limited medium remains at about 3.5 g / L; then introduce air into the hydrogen-oxidizing bacteria region to maintain the ORP at 75 - 85 mV; collect the CO2 and H2 gases produced by the medium-chain fatty acid functional flora, and introduce them into the hydrogen-oxidizing bacteria region through an air pump to form a gas circulation system.

[0067] 3.4 mcl-PHA synthesis and harvesting

[0068] After inoculating the hydrogen-oxidizing bacteria, perform batch culture at 30 °C for 72 hours; during the culture process, continuously monitor the consumption of caproic acid and cell growth; after 72 hours, drain the culture broth in the hydrogen-oxidizing bacteria region, and collect the cells by centrifugation (8000 rpm, 15 minutes); extract mcl-PHA using a chloroform-methanol mixed solvent (2:1, v / v), and purify it by precipitation with cold methanol.

[0069] 4. Results and analysis

[0070] 4.1 System stability evaluation

[0071] Operate continuously for 30 days, and monitor the pH changes in each region: the pH in the lactic acid bacteria and yeast regions is stable at 5.2 - 5.6, the pH in the medium-chain fatty acid functional flora region is stable at 6.0 - 6.5, and the pH in the hydrogen-oxidizing bacteria region is stable at 7.0 - 7.5; monitor the changes in the main flora composition in each region to evaluate the stability of the microbial community.

[0072] 4.2 mcl-PHA yield and properties

[0073] mcl-PHA yield: 10 - 15% of the dry cell mass.

[0074] mcl-PHA composition: Analyzed by gas chromatography-mass spectrometry (GC-MS), it is determined that the monomer composition is mainly 3-hydroxyhexanoate (3HHx, 85%), 3-hydroxyoctanoate (3HO, 10%), and 3-hydroxydecanoate (3HD, 5%).

[0075] Physical properties: Melting point 42 - 48 °C, glass transition temperature -46 °C, tensile strength 8.5 MPa, elongation at break 320%.

[0076] 4.3 Resource Utilization Efficiency Analysis

[0077] Conversion rate of organic waste: 85% of the input organic carbon is utilized.

[0078] Utilization rates of CO2 and H2: 73% of CO2 and 82% of H2 are utilized by hydrogen-oxidizing bacteria.

[0079] Conversion rate of medium-chain fatty acids: 92% of caproic acid is converted into mcl-PHA.

[0080] Example 2 Construction of a Spatial Microbial Community in a Single Reactor Based on Brewer's Wastewater as Raw Material and Synthesis of mcl-PHA

[0081] 1. Material Preparation

[0082] 1.1 Strain Preparation

[0083] Lactic acid bacteria: A mixed flora of Lactobacillus oenos and Lactobacillus plantarum isolated from traditional fermented distiller's grains, activated and cultured on MRS medium for 36 hours to adapt to the alcohol and acidic environment.

[0084] Medium-chain fatty acid functional flora: Domesticallyated from anaerobic digestion sludge that has been treating brewer's wastewater for a long time, mainly containing Caproiciproducens, with strains with enhanced acid and ethanol tolerance specifically strengthened.

[0085] Hydrogen-oxidizing bacteria: Paracoccus denitrificans, cultured in BPM medium and then gradually domesticated to adapt to an environment containing low concentrations of ethanol and phenolic substances. The domestication period is 14 days.

[0086] 1.2 Porous Carrier Material

[0087] Modified volcanic rock fillers are selected, with a particle size of 2 - 3 mm and a specific surface area of 240 m 2 / g. After sintering and surface modification at 950 ± 20 °C, the surface hydrophilicity is improved, which is beneficial for biofilm attachment. Before use, it is treated in hot water at 95 °C for 8 hours and then autoclaved at 121 °C for 30 minutes.

[0088] The surface modification process is as follows: Hydrophilic polymer coating: Prepare a 1% aqueous solution of polyvinyl alcohol (PVA), put the volcanic rock into the coating solution, evacuate for 30 minutes to allow the solution to fully penetrate the pores, add 0.5% glutaraldehyde as a cross-linking agent, react at 70 °C for 2 hours, wash with deionized water, and vacuum dry at 50 °C for 12 hours.

[0089] 1.3 Reactor Design

[0090] A rectangular reactor is made of plexiglass, with a total volume of 12 L, a length of 65 cm, a width of 16 cm, and a height of 12 cm. It is divided into three regions: Feed zone: Located at the bottom of the reactor, with a volume of 1.2 L, equipped with an automatic pH adjustment system; Reaction zone: Located in the middle, with a volume of 9.6 L, divided into three functional regions: Lactobacillus region (2.4 L), medium-chain fatty acid functional flora region (2.4 L), and hydrogen-oxidizing bacteria region (4.8 L); Discharge zone: Located at the top, with a volume of 1.2 L.

[0091] 2. Reactor construction

[0092] 2.1 Spatial niche construction

[0093] In the rectangular reactor, three functional regions are arranged in sequence along the liquid flow direction: In the Lactobacillus region, modified volcanic rock fillers are filled, with a filling rate of 65%; in the medium-chain fatty acid functional flora region, modified volcanic rock fillers are filled, with a filling rate of 70%; a stainless steel filter with a pore size of 80 μm is set between the Lactobacillus region and the medium-chain fatty acid functional flora region; a stainless steel filter with a pore size of 40 μm is set between the medium-chain fatty acid functional flora region and the hydrogen-oxidizing bacteria region; the hydrogen-oxidizing bacteria region is not filled with porous materials and is equally divided into 4 independently controllable sub-regions along the width direction, and each sub-region can be individually opened or closed through a control valve. During the domestication period of each unit, it is separated by a stainless steel plate.

[0094] 2.2 Parameter monitoring system

[0095] pH probes, ORP probes, temperature sensors, and conductivity sensors are installed in each region; an online ethanol concentration monitoring system is set in the Lactobacillus region; an online volatile fatty acid monitoring system is set in the medium-chain fatty acid functional flora region; a dissolved oxygen probe and a microbial activity fluorescence monitoring device are set in the hydrogen-oxidizing bacteria region; a gas collection, analysis, and reflux system is set at the top of the reactor and equipped with a gas flow meter; a hydraulic load and organic load automatic adjustment system is set at the bottom of the reactor.

[0096] 3. Operation process

[0097] 3.1 Colonization of functional flora

[0098] Lactobacillus: The activated and cultured Lactobacillus kefiranofaciens and Lactobacillus plantarum are mixed in a ratio of 2:3 (v / v) and inoculated into the Lactobacillus region at a high inoculation amount of 7%, and cultured at 32 °C for 48 hours; subsequently, a dilution containing 10% brewing wastewater (pH adjusted to 5.8) is introduced and circulated at a flow rate of 30 mL / h for 24 hours to enable the flora to adapt to the brewing wastewater environment.

[0099] Medium-chain fatty acid functional bacteria: Inoculate the domesticated medium-chain fatty acid functional bacteria into the medium-chain fatty acid functional bacteria area at an inoculation amount of 40%, and statically culture for 24 hours under anaerobic conditions at 33°C; then introduce lactic acid bacteria fermentation broth (containing 5% brewing wastewater) with a flow rate of 25 mL / h and circulate for 48 hours to promote biofilm formation.

[0100] Do not inoculate the hydrogen-oxidizing bacteria area for the time being.

[0101] 3.2 Reactor startup and operation

[0102] Mix brewing wastewater (COD 200 g / L) and tap water at a ratio of 1:3 (v / v) (i.e., the addition ratio of brewing wastewater is 25%, and the final COD is about 50 g / L), and supplement trace elements (FeSO4·7H2O 10 mg / L, MgSO4·7H2O 100 mg / L, CaCl2 50 mg / L); adjust the pH of the mixed solution to 5.5 - 6.0, inject through the feed port, and the initial flow rate is 0.4 L / h; monitor the pH change in the lactic acid bacteria area and keep it at 4.8 - 5.2; if the pH is too low, inject 5% NaHCO3 solution through the automatic regulation system for adjustment.

[0103] Monitor the caproic acid concentration at the outlet of the medium-chain fatty acid functional bacteria area and keep it at 3.0 - 3.5 g / L; if it is lower than 2.5 g / L, increase the addition ratio of brewing wastewater to 30%; if it is higher than 4.0 g / L, reduce the addition ratio to 20%; after the system runs for 14 days, evaluate the system stability and prepare for the inoculation of hydrogen-oxidizing bacteria.

[0104] 3.3 Operation of the hydrogen-oxidizing bacteria area

[0105] Centrifuge and collect the domesticated Paracoccus denitrificans bacterial solution and wash it twice with PBS solution; decide to open 3 sub-areas according to the caproic acid production in the medium-chain fatty acid functional bacteria area (if the caproic acid is stable above 3.5 g / L, all 4 sub-areas can be opened).

[0106] Add nitrogen-limited medium to the opened sub-areas, and inoculate the washed hydrogen-oxidizing bacteria into the nitrogen-limited medium at a ratio of 45% of the volume of the nitrogen-limited medium; adjust the reactor flow rate so that the flow rate of the medium-chain fatty acid fermentation broth flowing into the hydrogen-oxidizing bacteria area is 0.3 - 0.35 L / h; introduce air into the hydrogen-oxidizing bacteria area to maintain the dissolved oxygen concentration at 1.5 - 2.0 mg / L; turn on the gas circulation system, introduce the collected CO2 and H2 gases into the hydrogen-oxidizing bacteria area, and adjust the reflux ratio to 70%.

[0107] 3.4 mcl-PHA synthesis and harvesting

[0108] After inoculating with hydrogen-oxidizing bacteria, batch culture was carried out at 30 °C for 80 hours (extended culture time to adapt to the raw material of brewing wastewater); during the culture process, samples were taken every 12 hours to detect the consumption of caproic acid, cell growth and PHA accumulation; after 80 hours, the culture solution in the hydrogen-oxidizing bacteria area was drained, and the bacteria were collected by centrifugation (8000 rpm, 15 minutes); after collecting the bacteria, they were first washed once with 0.9% NaCl solution and then once with distilled water to remove possible residual phenolic substances; mcl-PHA was extracted by the modified chloroform-methanol extraction method, purified by cold methanol precipitation, weighed after vacuum drying and analyzed.

[0109] 4. Results and Analysis

[0110] 4.1 System Stability Evaluation

[0111] Continuous operation was carried out for 35 days, and the parameter changes in each area were monitored: the average pH in the lactic acid bacteria area was 4.9, and the ORP was -50 mV; the average pH in the medium-chain fatty acid functional bacteria area was 5.9, and the ORP was -150 mV; the average pH in the hydrogen-oxidizing bacteria area was 7.2, and the ORP was +60 mV.

[0112] Long-term stability tests showed that when the COD of brewing wastewater fluctuated in the range of 180 - 220 g / L, the system could automatically adjust and maintain stable operation; microbial community analysis showed that the proportion of Lactobacillus kefiranofaciens in the lactic acid bacteria area increased with the running time (from 40% to 65%), indicating that it had stronger adaptability to the alcohol component in brewing wastewater; in the medium-chain fatty acid functional bacteria area, the proportion of Caproiciproducens acidigenes was 40 - 80%, dominating the conversion process from lactic acid to medium-chain fatty acids.

[0113] 4.2 Yield and Characteristics of mcl-PHA

[0114] Yield of mcl-PHA: 18 - 25% of the dry cell mass, higher than that in Example 1 using food waste.

[0115] Composition of mcl-PHA: 3-hydroxyhexanoate (3HHx, 82%), 3-hydroxyoctanoate (3HO, 12%) and 3-hydroxydecanoate (3HD, 6%).

[0116] Physical properties: melting point 44 - 49 °C, glass transition temperature -42 °C, tensile strength 9.2 MPa, elongation at break 285%. Compared with the mcl-PHA obtained in Example 1, the product obtained in this example had an increased tensile strength but a slightly reduced elasticity.

[0117] 4.3 Resource Utilization Efficiency Analysis

[0118] Utilization rate of brewing wastewater: The COD removal rate of the influent into the system is 80%.

[0119] Lactic acid conversion rate: 92% of the lactic acid produced in the lactic acid bacteria area is utilized by the medium-chain fatty acid functional flora.

[0120] Yield of medium-chain fatty acids: The average is 5.3 g / L, among which caproic acid accounts for 62%, caprylic acid accounts for 28%, and butyric acid accounts for 10%.

[0121] Utilization rates of CO2 and H2: 76% of CO2 and 85% of H2 are utilized by the hydrogen-oxidizing bacteria.

[0122] Recycling of water resources: 75% of the water discharged from the system can be recycled for dilution water in a new batch.

[0123] Example 3 Construction of a single-reactor spatial microbial community based on partial conversion of sucrose and synthesis of mcl-PHA

[0124] 1. Material preparation

[0125] 1.1 Preparation of strains

[0126] Lactic acid bacteria: A mixed flora of Streptococcus thermophilus and Lactobacillus paracasei was selected and cultured in MRS medium for 24 hours respectively, and then mixed in a ratio of 1:1 (v / v); These two strains were selected because their utilization efficiency of sucrose can be regulated by temperature and pH.

[0127] Medium-chain fatty acid functional flora: Enriched and domesticated from anaerobic sludge treating molasses wastewater, mainly including Megasphaera and Caproiciproducens, and these strains can utilize both lactic acid and monosaccharides to convert into medium-chain fatty acids.

[0128] Hydrogen-oxidizing bacteria: A mutant strain of Cupriavidus necator with the sucrase gene knocked out was used to reduce its direct utilization ability of sucrose, so as to promote its preferential utilization of medium-chain fatty acids as a carbon source to synthesize PHA.

[0129] 1.2 Porous carrier material

[0130] A kaolin-cellulose composite porous material was used to make spherical particles with a diameter of 6 mm, a porosity of 65%, and an average pore diameter of 15 μm, which has good buffering performance and biocompatibility. It was soaked in distilled water for 48 hours and sterilized at 121 °C for 30 minutes before use.

[0131] 1.3 Reactor design

[0132] A rectangular reactor was made of plexiglass, with a total volume of 15 L, a length of 70 cm, a width of 18 cm, and a height of 12 cm. It was divided into the following areas: Feed area: Located at the bottom of the reactor, with a volume of 1.5 L, equipped with an automatic pH adjustment and temperature control system; Reaction area: With a volume of 12 L, divided into three functional areas: Lactobacillus area (3 L): Designed as a "flow rate adjustable area", controlling the sucrose conversion rate by changing the flow rate; Medium-chain fatty acid functional flora area (3 L): Designed as a "dual-substrate adaptation area", optimized to utilize both lactic acid and sucrose simultaneously; Hydroxide bacteria area (6 L): Divided into 4 independently controlled sub-areas, each with a volume of 1.5 L; Discharge area: Located at the top, with a volume of 1.5 L, equipped with a product collection system.

[0133] 2. Reactor construction

[0134] 2.1 Spatial niche construction

[0135] Lactobacillus area: Designed with three temperature gradient sub-areas (34 °C → 38 °C → 42 °C) along the flow direction, filled with kaolin-cellulose composite porous material, with a filling rate of 60%, and a variable-speed peristaltic pump was installed to control the flow rate; An on-line monitoring system was set at the outlet of the Lactobacillus area to detect the sucrose and lactic acid concentrations in real time, and the flow rate was controlled through a feedback mechanism to ensure that about 1 / 3 of the sucrose remained unconverted; Medium-chain fatty acid functional flora area: Filled with kaolin-cellulose composite porous material, with a filling rate of 70%, and 5% activated carbon was added to adsorb potential inhibitors; A stainless steel filter with a pore size of 75 μm was set between the Lactobacillus area and the medium-chain fatty acid functional flora area; A stainless steel filter with a pore size of 45 μm was set between the medium-chain fatty acid functional flora area and the hydroxide bacteria area; A shunt pipeline was set in the hydroxide bacteria area to ensure uniform liquid distribution to each sub-area.

[0136] 2.2 Parameter monitoring system

[0137] A saccharimeter, a pH probe, and a temperature sensor were installed in the feed area; Three temperature control units, three pH probes (located in different temperature sub-areas respectively), and an on-line analyzer for sucrose and lactic acid at the outlet were installed in the Lactobacillus area; A pH probe, an ORP probe, a temperature sensor, and an on-line analysis system for volatile fatty acids were installed in the medium-chain fatty acid functional flora area; A dissolved oxygen probe, a pH probe, a temperature sensor, and an on-line monitoring system for PHA fluorescence staining were installed in the hydroxide bacteria area; A gas collection and analysis system was set at the top of the reactor to be able to measure the contents of CO2, H2, and O2 respectively.

[0138] 3. Operation process

[0139] 3.1 Colonization of functional flora

[0140] Colonization of the Lactobacillus area: First, Streptococcus thermophilus and Lactobacillus paracasei were cultured offline until OD 600= 1.8, inoculate it into the lactic acid bacteria region at an inoculation amount of 8%, control the three temperature sub-regions to be 34 °C, 38 °C and 42 °C respectively, introduce MRS medium containing 1% sucrose, the flow rate is 50 mL / h, and the colonization time is 36 hours.

[0141] Colonization in the medium-chain fatty acid functional flora region: Inoculate the enriched and domesticated medium-chain fatty acid functional flora into this region at an inoculation amount of 45%, statically culture it under anaerobic conditions at 35 °C for 24 hours, and then introduce MRS medium containing 3% lactic acid and 1% sucrose, the flow rate is 40 mL / h, and carry out 48-hour adaptive culture.

[0142] Preparation of the hydrogen-oxidizing bacteria region: Offline culture the Cupriavidus necator mutant strain with gene knockout to the mid-logarithmic growth phase, and centrifuge and collect it for standby.

[0143] 3.2 Reactor startup and control of partial sucrose conversion

[0144] Prepare a 10% sucrose solution (supplemented with 1.0 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 0.2 g / L of MgSO4·7H2O and 5 mL / L of trace element solution) as the initial medium; at the initial stage of system startup, set a higher flow rate (80 mL / h) in the lactic acid bacteria region to make the residence time of sucrose short and the conversion rate low, and monitor the sucrose and lactic acid concentrations at the outlet; after the system runs stably (about 24 hours), start the control of sucrose conversion: According to the online monitoring data, when the lactic acid / sucrose molar ratio is lower than 2:1, reduce the flow rate by 10%; when the ratio is higher than 2:1, increase the flow rate by 10%. Through this feedback control, about 2 / 3 of the sucrose is converted into lactic acid.

[0145] Temperature gradient control: If the sucrose conversion rate is too high, reduce the temperature of the third temperature sub-region to 40 °C; if the conversion rate is too low, increase the temperature of the first temperature sub-region to 36 °C.

[0146] pH control: The pH in the lactic acid bacteria region is maintained within the range of 5.2 - 5.8, and it is controlled by automatically adding a 5% Na2CO3 solution.

[0147] After stable operation for 72 hours, confirm that the sucrose conversion rate at the outlet of the lactic acid bacteria region remains about 66% (that is, the sucrose to lactic acid molar ratio is about 1:2).

[0148] 3.3 Optimization of medium-chain fatty acid synthesis

[0149] The regional temperature of the medium-chain fatty acid functional flora is set at 35 ± 0.5 °C, and the pH is controlled within the range of 5.8 - 6.2; monitor the utilization of sucrose and lactic acid by the medium-chain fatty acid functional flora, and promote the synergistic utilization of the two carbon sources by adjusting the ratio of trace elements added in the region: if the consumption of lactic acid is slow, add additional FeSO4·7H2O (to a total concentration of 20 mg / L); if the consumption of sucrose is slow, add additional MnSO4·H2O (to a total concentration of 15 mg / L); after the system has been operating stably for 7 days, the concentration of medium-chain fatty acids at the outlet of the medium-chain fatty acid functional flora region is stabilized at 12.0 - 12.5 g / L, and the main components are caproic acid (85%), caprylic acid (10%) and heptanoic acid (5%).

[0150] 3.4 Inoculation of hydrogen-oxidizing bacteria and PHA synthesis

[0151] According to the production of medium-chain fatty acids, decide to open 3 sub-regions of hydrogen-oxidizing bacteria; add a sucrose-free medium (KH2PO4 2.0 g / L, K2HPO4 2.5 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, trace element solution 10 mL / L) to each sub-region; inoculate the Cupriavidus necator mutant strain collected by centrifugation into each sub-region at a volume ratio of 40%; adjust the system flow rate to 50 mL / h to make the fermentation broth of medium-chain fatty acids flow into the hydrogen-oxidizing bacteria region, and keep the caproic acid concentration entering the hydrogen-oxidizing bacteria region at 3.8 - 4.0 g / L; introduce air into the hydrogen-oxidizing bacteria region to maintain the dissolved oxygen at 2.0 - 2.5 mg / L; set up a gas circulation system to recycle the CO2 and H2 generated in the first two regions and introduce them into the hydrogen-oxidizing bacteria region to improve the carbon utilization efficiency.

[0152] 3.5 mcl-PHA synthesis and batch harvesting

[0153] After inoculation of the hydrogen-oxidizing bacteria, the first sub-region is harvested after culturing at 30 °C for 72 hours; the second sub-region is harvested after culturing for 96 hours after inoculation; the third sub-region is harvested after culturing for 120 hours after inoculation; through the batch harvesting strategy, evaluate the effects of different culture times on the yield and monomer composition of mcl-PHA; at each batch harvest, drain the culture solution of the corresponding sub-region, and collect the cells by centrifugation (8000 rpm, 15 minutes); extract mcl-PHA by the chloroform-methanol mixed solvent (2:1, v / v) extraction method, and after purification by cold methanol precipitation, vacuum dry at 60 °C for 10 hours.

[0154] 4. Results and analysis

[0155] 4.1 Sucrose conversion control effect

[0156] The reactor operated continuously for 21 days. The average sucrose conversion rate at the outlet of the lactic acid bacteria region was 67.3 ± 2.5%, which basically met the design goal (about 2 / 3 of sucrose was converted into lactic acid); the response time of the flow rate control system was 15 - 20 minutes, which could effectively cope with sucrose concentration fluctuations; the temperature gradient system was adjusted an average of 2 - 3 times a day to ensure appropriate fermentation; the analysis of the lactic acid bacteria flora composition showed that the ratio of Lactobacillus paracasei to Streptococcus thermophilus tended to be 1.5:1 after the system operated stably, indicating that Lactobacillus paracasei was dominant in this system.

[0157] 4.2 Carbon source utilization of the functional flora of medium-chain fatty acids

[0158] The utilization rate of lactic acid by the functional flora of medium-chain fatty acids was 93.5%, and the utilization rate of residual sucrose was 42.7%; the analysis of the dual-substrate utilization pattern showed that when the system was stable, about 75% of the medium-chain fatty acids came from lactic acid conversion, and about 25% came from direct sucrose conversion; the residual sucrose at the final outlet was about 19% of the feed sucrose, indicating that the overall utilization rate of sucrose by the system reached 81%; the flora analysis of the functional flora region of medium-chain fatty acids showed that the proportion of Megasphaera increased to 45% because it had good utilization abilities for both sucrose and lactic acid.

[0159] 4.3 Effects of mcl-PHA production and different culture times

[0160] The first batch harvested at 72 hours: the mcl-PHA content was 13.5% of the dry cell mass.

[0161] The second batch harvested at 96 hours: the mcl-PHA content was 11.2% of the dry cell mass.

[0162] The third batch harvested at 120 hours: the mcl-PHA content was 13.8% of the dry cell mass.

[0163] Analysis of the mcl-PHA monomer composition:

[0164] 72-hour batch: 3HHx (78%), 3HO (18%), 3HD (4%); 96-hour batch: 3HHx (84%), 3HO (10%), 3HD (6%); 120-hour batch: 3HHx (80%), 3HO (12%), 3HD (18%).

[0165] The increase in the proportion of long-chain monomers was caused by the gradual utilization of residual sucrose as an auxiliary carbon source.

[0166] 4.4 Physical properties of the product

[0167] 72 - hour batch: melting point 40 - 45 °C, glass transition temperature - 48 °C, tensile strength 7.8 MPa, elongation at break 340%.

[0168] 96 - hour batch: melting point 42 - 47 °C, glass transition temperature - 46 °C, tensile strength 8.5 MPa, elongation at break 310%.

[0169] 120 - hour batch: melting point 44 - 49 °C, glass transition temperature - 44 °C, tensile strength 9.2 MPa, elongation at break 280%.

[0170] As the cultivation time extends, the crystallinity of the obtained mcl - PHA increases, the mechanical strength improves, but the elasticity decreases slightly.

[0171] 4.5 System stability and resource utilization efficiency

[0172] System pH stability: The average pH in the lactic acid bacteria region is 5.5 ± 0.3, the average pH in the medium - chain fatty acid functional flora region is 6.0 ± 0.2, and the average pH in the hydrogen - oxidizing bacteria region is 7.1 ± 0.2.

[0173] Carbon source utilization efficiency: The efficiency of converting the total sucrose carbon entering the system into mcl - PHA is 28.5%, which is higher than that of the traditional single - strain fermentation process (about 20%).

[0174] Energy utilization efficiency: Compared with the traditional step - by - step fermentation process, the comprehensive energy consumption is reduced by about 35%.

[0175] Water resource recycling: 70% of the system - discharged water can be recycled as dilution water for new batches.

[0176] Waste gas utilization rate: The fixation rate of CO2 generated by the system by hydrogen - oxidizing bacteria is 35%, and the utilization rate of H2 is 80%.

[0177] 4.6 Special application performance

[0178] Due to the sucrose partial conversion strategy, the obtained mcl - PHA has a more uniform monomer distribution and better thermal stability than the products of traditional processes; the products of the 96 - hour harvest batch show the best comprehensive performance and are suitable for application scenarios that require a balance of strength and elasticity; in the biodegradation test, the complete degradation time of the product in soil is 120 - 150 days, meeting the standards of controllable biodegradable materials.

[0179] Example 4 Multi - reactor coupled mcl - PHA synthesis system based on expired food raw materials

[0180] 1. System overview

[0181] In this embodiment, a multi-reactor coupled mcl-PHA synthesis system was constructed. Expired foods (bread, biscuits, fruit mixture) were used as raw materials, and step-by-step conversion was achieved through four independently operating functional reactors. The system includes: (1) a pretreatment and saccharification reactor; (2) a lactic acid fermentation reactor; (3) a medium-chain fatty acid synthesis reactor; (4) a PHA accumulation reactor. Each reactor is coupled through pipelines, pump sets, and an intelligent control system to form a complete process flow.

[0182] 2. Raw material preparation and characteristics

[0183] 2.1 Raw material composition and pretreatment

[0184] Composition of the expired food mixture: bread (40%), biscuits (30%), fruit (30%); the bread and biscuits mainly come from expired recyclables in local supermarkets, and the fruit includes overripe bananas, apples, and seasonal fruits.

[0185] Initial pretreatment of raw materials: Remove the packaging, cut into pieces 2-3 cm in size, and mix evenly.

[0186] Component analysis: Dry matter content is 46-52%, carbon-nitrogen ratio is 25-35:1, and total sugar content is 38-45% (including 31-36% starch and 7-9% soluble sugar).

[0187] 3. Reactor design and process conditions

[0188] 3.1 Pretreatment and saccharification reactor (R1)

[0189] Reactor type: 50L vertical stainless steel reactor with a double-layer jacket and a top stirring system. Stirring system: Adopt a double propeller blade with a rotation speed of 50-120 rpm and automatic adjustment. Temperature control: A water bath heating system accurate to ±0.5°C. Enzymatic hydrolysis system: Equipped with an automatic addition device for three enzymes: α-amylase (for starch hydrolysis, activity 100,000 U / g), pectinase (for pectin hydrolysis, activity 25,000 U / g), cellulase (for cellulose hydrolysis, activity 15,000 U / g).

[0190] Operating conditions: Temperature 50-55°C, pH adjusted to 5.5-6.0, water-solid ratio 4:1, v / m, saccharification time 4-6 hours.

[0191] Detection system: Online pH meter, thermometer, viscometer, refractometer.

[0192] 3.2 Lactic acid fermentation reactor (R2)

[0193] Reactor type: 60L stainless steel anaerobic fermenter, equipped with a gas discharge valve and a pressure sensor at the top. Design features: The bottom is designed as a 60° cone to reduce dead zones; three layers of sampling ports are designed on the side wall. Stirring system: Low-shear blade agitator, rotation speed 30 - 80 rpm. Temperature control: Jacket water circulation system, maintaining 37 ± 1 °C.

[0194] Microbial inoculation: A mixed flora of Lactobacillus acidophilus and Lactobacillus plantarum with strong acid resistance is selected, and the inoculation amount is 5%. Nutrient addition: Supplement (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, and yeast extract 1.0 g / L.

[0195] Operating conditions: Temperature 37 °C, initial pH 6.2, fermentation time 18 - 24 hours.

[0196] On-line monitoring: pH, temperature, ORP, lactic acid concentration.

[0197] 3.3 Medium-chain fatty acid synthesis reactor (R3)

[0198] Reactor type: 80L stainless steel anaerobic reactor, with a biofilm fixing device. Biofilm carrier: High-specification ceramic rings, specific surface area 300 m 2 / m 3 , filling rate 40%. Temperature control: Maintain 33 ± 0.5 °C. Microbial inoculation: Acid-resistant medium-chain fatty acid-producing flora enriched in pit mud, mainly containing bacteria of the family Ruminococcaceae.

[0199] Operating conditions: pH is controlled at 5.8 - 6.2, ORP is controlled at -250 to -300 mV, hydraulic retention time (HRT) 30 - 36 hours.

[0200] Monitoring system: On-line volatile fatty acid analyzer, automatically sampling and analyzing every 4 hours.

[0201] 3.4 PHA accumulation reactor (R4)

[0202] Reactor type: 100L stainless steel aerobic fermentation tank, equipped with microporous aeration discs; Aeration system: variable frequency blower, dissolved oxygen automatically controlled at 20 - 40% saturation; Temperature control: 30 ± 1°C; Strain: Domesticated Pseudomonas is used, which has good tolerance to medium-chain fatty acids and can efficiently synthesize mcl-PHA. Nutritional formula: Basic inorganic salt medium, containing KH2PO4 2.0g / L, K2HPO4 3.0g / L, (NH4)2SO4 (concentration dynamically adjusted according to batches, 0.2 - 0.5g / L), MgSO4·7H2O 0.4g / L.

[0203] Operating conditions: Batch fermentation, each batch runs for 48 - 60 hours.

[0204] Process control: Maintain a high nitrogen source level in the initial 24 hours to promote biomass growth, and then reduce the nitrogen source concentration and maintain a C / N ratio > 40 to promote PHA accumulation.

[0205] Monitoring system: Online flow cytometer combined with Nile red staining to monitor PHA accumulation in real time.

[0206] 3.5 Coupling control system

[0207] Central control unit: An automatic control system based on industrial-grade PLC to achieve coordinated operation between reactors; Intermediate storage tanks (each 20L) are set up between reactors, equipped with liquid level sensors and temperature control; Pumping system: A peristaltic pump is used to achieve precise flow control, with a flow rate range of 0.5 - 5L / h; Automatic cleaning system: Each reactor is equipped with a CIP cleaning circuit.

[0208] 4. Operating procedures

[0209] 4.1 Pretreatment and saccharification process

[0210] Mix the shredded expired food with water at a ratio of 1:4 (m / v), slowly heat up to 52°C while stirring evenly; Add 0.3% α-amylase, 0.1% pectinase and 0.2% cellulase based on the dry weight of the raw materials; Conduct enzymatic hydrolysis for 2 hours at pH 5.8, then heat up to 65°C to inactivate for 10 minutes; Cool to 40°C, filter through a 100-mesh sieve to remove unhydrolyzed solid residues (about 15 - 20% of the dry weight of the raw materials); Adjust the pH of the filtrate to 6.2, and online detect the total reducing sugar concentration to ensure it reaches 80 - 100g / L; Transport the saccharified liquid to the intermediate storage tank of the lactic acid fermentation reactor through a peristaltic pump and keep it at a constant temperature.

[0211] 4.2 Lactic acid fermentation process

[0212] Inoculate the reactor R2 with 5% of the lactic acid bacteria mixed flora and start the temperature control system; pump in the saccharified liquid from the intermediate storage tank at an initial rate of 0.5 L / h, and maintain the stirring speed at 50 rpm; monitor the pH change, and when the pH drops below 5.0, automatically add 10% Na2CO3 solution to adjust the pH to 5.0; when the reactor liquid level reaches 80% of the working volume, stop feeding and start batch fermentation; ferment for 18 - 24 hours to ensure that the total reducing sugar conversion rate > 90% and the lactic acid concentration reaches 40 - 50 g / L; judge the fermentation end point according to the online lactic acid concentration monitoring results and pump the fermented liquid into the next-stage intermediate storage tank.

[0213] 4.3 Medium-chain fatty acid synthesis process

[0214] Introduce the lactic acid fermentation broth into the medium-chain fatty acid synthesis reactor through a pipeline; operate in a continuous feeding mode, and control the hydraulic retention time to be 34 hours; maintain the pH of the first zone of the reactor at 5.9 - 6.1 and the pH of the second zone at 5.7 - 5.9; monitor the concentration changes of butyric acid, caproic acid and caprylic acid through an online volatile fatty acid analyzer; when the total concentration of medium-chain fatty acids at the outlet stabilizes at 8 - 10 g / L (where caproic acid accounts for 60 - 70%), determine that the system reaches a stable state; continuously pump the product to the intermediate storage tank and maintain the pH above 5.5 to prevent the precipitation of medium-chain fatty acids.

[0215] 4.4 PHA accumulation process

[0216] Inoculate the PHA accumulation reactor with Pseudomonas putida KT2440, and the initial OD 600 is controlled at 0.6 - 0.8; operate in two stages: growth stage (0 - 24 hours): dilute the fermentation broth of medium-chain fatty acids at a ratio of 1:5, and maintain the nitrogen source level ((NH4)2SO4 0.5 g / L) to promote biomass increase; accumulation stage (24 - 60 hours): no longer add nitrogen source, and increase the medium-chain fatty acid concentration to 3 - 4 g / L; maintain the dissolved oxygen at 30% of the saturation, the temperature at 30 °C, and the pH at 7.0 - 7.2; monitor the PHA accumulation every 8 hours by flow cytometry combined with Nile red staining; when the PHA content reaches more than 20% of the dry weight and the cell density OD 600 > 4, stop fermentation.

[0217] 4.5 Product harvesting and treatment

[0218] Collect the bacterial cells by a continuous flow centrifuge (10,000×g) and separate the supernatant; wash the collected bacterial cells twice with phosphate buffer (pH 7.0); prepare the dried bacterial cells by freeze-drying; separate mcl-PHA by a modified chloroform extraction method: add a 3% sodium hypochlorite solution and digest for 30 minutes, add chloroform and extract for 6 hours, filter and separate the chloroform phase, precipitate mcl-PHA with cold methanol, and dry it in vacuum at 60°C for 12 hours.

[0219] Product detection and analysis: Determine the monomer composition by GC-MS, measure the thermal properties by DSC, and test the mechanical properties.

[0220] 5. Results and Analysis

[0221] 5.1 Raw material conversion efficiency

[0222] Conversion rate of expired food to fermentable sugars: 72 - 78% (fluctuates according to the raw material composition).

[0223] Conversion rate of sugars to lactic acid: 92 - 95%.

[0224] Conversion rate of lactic acid to medium-chain fatty acids: 65 - 70%, main product composition: caproic acid (85%), octanoic acid (10%), heptanoic acid (15%).

[0225] Conversion rate of medium-chain fatty acids to mcl-PHA: 38 - 42%.

[0226] 5.3 mcl-PHA product characteristics

[0227] Yield: 12 - 18% of the dry cell mass.

[0228] Monomer composition: 3-hydroxyhexanoate (3HHx, 85 - 90%), 3-hydroxyoctanoate (3HO, 5 - 10%) and 3-hydroxydecanoate (3HD, 5 - 10%).

[0229] Physical properties: melting point 42 - 47°C, glass transition temperature -46°C, tensile strength 8.5 MPa, elongation at break 320%.

[0230] Molecular weight: weight-average molecular weight (Mw) is 200,000 - 250,000 Daltons, polydispersity index (PDI) is 1.8 - 2.2.

[0231] Influence of raw material differences: The proportion of 3HO in PHA obtained from batches with high fruit content is slightly higher, and the proportion of 3HHx in batches with high bread content is slightly higher.

[0232] 5.4 Resource utilization efficiency

[0233] Carbon source utilization efficiency: The total efficiency of converting carbon elements in expired food into PHA is 18 - 22%.

[0234] Energy efficiency: Compared with the traditional step-by-step method, the total energy consumption is reduced by about 25%.

[0235] Water resource utilization: The water recycling rate of the system reaches 65%.

[0236] Residue treatment: The remaining solid residue of R1 is used for composting; the waste liquid of R2 and R3 is used for producing organic fertilizers.

[0237] 6. Environmental benefit analysis

[0238] Carbon footprint: Compared with petroleum-based plastics, the carbon emission is reduced by about 70%.

[0239] Waste reduction: For every 1 kg of mcl-PHA produced, about 4.5 kg of expired food can be consumed.

[0240] Biodegradability: The degradation rate is >90% within 90 days under composting conditions and >85% within 180 days in the marine environment.

[0241] Reducing landfill pressure: The system can reduce about 450 tons of expired food entering landfills annually (calculated based on a production capacity of 100 tons).

[0242] The embodiments described above are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities, characterized in that: The following steps are involved: (1) using a microorganism that converts carbohydrate organic matter to convert the carbohydrate organic matter into lactic acid; (2) using yeast to convert carbohydrate organic matter into ethanol; (3) converting the lactic acid from step (1) and the ethanol from step (2) into medium-chain fatty acids using a medium-chain fatty acid functional bacterial community, and releasing CO2 and H2; (4) Under aerobic conditions, hydrogen oxidizing bacteria are added to the medium-chain fatty acids in step (3). The hydrogen oxidizing bacteria utilize the CO2 and H2 released in step (3) to grow, and synthesize medium-chain polyhydroxyalkanoates from the medium-chain fatty acids under nitrogen-limited conditions in the late growth stage.

2. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 1, characterized in that: The microorganisms that convert carbohydrate organic matter include one or more of lactic acid bacteria, yeast, glycolytic bacteria, amylolytic bacteria, lactic acid enzymes, yeast enzymes, glycolytic enzymes, and amylolytic enzymes.

3. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 1, characterized in that: The medium-chain fatty acid functional bacterial flora includes bacterial flora with Caproiciproducens, Anaerococcus, Megasphaera and Clostridium as the core.

4. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 1, characterized in that: The inoculation amount of the microorganisms for converting carbohydrate organic matter is 2-5%, the inoculation amount of the yeast in step (2) is 2-5%, and the inoculation amount of the medium-chain fatty acid functional bacteria is 20-50%.

5. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 1, characterized in that: The hydrogen oxidizing bacteria were inoculated into the BPM medium at a ratio of 0.1%, and cultured at 30°C and 180 r / min for 72 hours.

6. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to any one of claims 1 to 5, characterized in that: The synthesis of the medium-chain polyhydroxyalkanoate is completed in a reactor, which includes a feed zone, a reaction zone and a discharge zone. The reaction zone is filled with a porous carrier material, and microorganisms, yeasts and medium-chain fatty acid functional bacteria for converting carbohydrate organic matter are respectively colonized on the porous carrier materials at different positions in the reaction zone. No porous carrier material is added to the hydrogen oxidizing bacteria area, carbohydrate organic matter is introduced through the feed port, air is introduced into the hydrogen oxidizing bacteria area, and the redox potential is maintained between 50-100mV.

7. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 6, characterized in that: A filter is arranged between the porous carrier materials colonized with microorganisms for converting carbohydrate organic matter, yeast and medium-chain fatty acid functional bacteria, and the pore size of the filter is 20-500 μm.

8. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 6, characterized in that: The hydrogen oxidizing bacteria area is provided with a plurality of sub-areas, and a nitrogen-restricted culture medium is added. After the hydrogen oxidizing bacteria are cultured in the BPM culture medium, they are centrifuged and cleaned and inoculated at a ratio of 40-45% of the volume of the nitrogen-restricted culture medium. After the fermentation liquid of the medium-chain fatty acid in step (3) flows into the hydrogen oxidizing bacteria area, the hexanoic acid concentration therein is maintained at ≤4 g / L. Then, the gas generated during the fermentation of the medium-chain fatty acid is continuously introduced, and the gas is synthesized at 30-35° C. for 72 hours in a batch manner, and then the liquid is discharged to collect the hydrogen oxidizing bacteria.

9. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to any one of claims 1 to 5, characterized in that: The synthesis of the medium-chain polyhydroxyalkanoate is completed in multiple reactors connected in series, the conversion of carbohydrate organic matter into lactic acid and ethanol is carried out in the first reactor, the conversion of lactic acid and ethanol into medium-chain fatty acids by the medium-chain fatty acid functional bacteria is carried out in the second reactor, and the synthesis of medium-chain polyhydroxyalkanoate by hydrogen oxidizing bacteria using CO2, H2 and medium-chain fatty acids is carried out in the third reactor; the second reactor is provided with a gas collection system and is connected to the third reactor through an air pump.

10. The method for collaboratively synthesizing medium-chain polyhydroxyalkanoates based on space microbial communities according to claim 9, characterized in that: In the third reactor, after the hydrogen oxidizing bacteria are cultured in the BPM medium, they are centrifuged and washed and then added to the nitrogen-limited medium at a ratio of 40-45%, and then the fermentation broth of medium-chain fatty acids is added to keep the concentration of caproic acid therein at ≤4g / L. The mixture is synthesized in a batch mode at 30°C for 72-120h, and the gas produced during the fermentation of the medium-chain fatty acids is continuously introduced and stirred with the gas.

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