WO2022012101A1 - 一种厌氧固定化菌剂、制备方法及其应用 - Google Patents

一种厌氧固定化菌剂、制备方法及其应用 Download PDF

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WO2022012101A1
WO2022012101A1 PCT/CN2021/087449 CN2021087449W WO2022012101A1 WO 2022012101 A1 WO2022012101 A1 WO 2022012101A1 CN 2021087449 W CN2021087449 W CN 2021087449W WO 2022012101 A1 WO2022012101 A1 WO 2022012101A1
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anaerobic
functional
bacterial
bacterial agent
anaerobic digestion
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PCT/CN2021/087449
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French (fr)
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何品晶
段皓文
吕凡
章骅
邵立明
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同济大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/084—Polymers containing vinyl alcohol units
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/023—Methane
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • the invention belongs to the field of environmental engineering materials, and particularly relates to an anaerobic immobilized bacterial agent, a preparation method and an application thereof.
  • anaerobic digestion can not only control the environmental pollution of easily degradable biomass waste, but also convert the organic matter contained in it into methane-based energy gas.
  • the organic acid mainly acetic acid is easy to accumulate rapidly in the anaerobic digestion reactor, causing The acid inhibition of methanogens causes the instability of the methanation process and even the failure of the reactor.
  • ammonia inhibition will further aggravate the problem of organic acid accumulation, resulting in dual inhibition of ammonia and organic acids.
  • bioaugmentation measures can increase the rate of lipid degradation, consume excess organic acids, shorten the lag period of the methanation process, and finally restore the normal operation capacity of the reactor by adding bacterial flora to the anaerobic digestion reactor.
  • Suspension culture mode is easy to cause the loss of additional functional flora; 2.
  • the additional flora is difficult to adapt to the physical and chemical conditions inside the reactor, and it will die quickly, or its competitive advantage will be lost by the reactor.
  • the internal indigenous microorganisms surpass; 3.
  • the structure of the external flora is not specific, a single strain is easy to die, and the compound strain lacks synergistic metabolism, resulting in low efficiency, and it is necessary to increase the amount of inoculation and the frequency of inoculation; 4. Difficulty in culturing pure anaerobic strains Large, long generation time, can not meet the large-scale needs of large-scale reactors; 5. The additional bacteria cannot tolerate high ammonia concentration, and cannot solve the problem of organic acid accumulation under the condition of ammonia inhibition. In recent years, a series of methods have been developed in China to relieve organic acid inhibition in anaerobic digestion reactors.
  • Chinese patents with publication numbers CN202089962U, CN110184171A, CN201644487U and CN201644487U design new anaerobic digestion reactors, but such technologies are not applicable in anaerobic digestion reactors where organic acid suppression has occurred.
  • the Chinese invention patents with publication numbers CN107475304A, CN109554402A and CN109182390A add carbon materials, but such technologies need to continuously supplement carbon materials to make up for the loss of carbon materials, and carbon material leaching also has potential toxic effects on microorganisms.
  • the Chinese invention patent with publication number CN102992478B adds an ion exchange resin device, but this type of technology needs to regenerate the ion exchange resin, and also needs to deal with the regeneration waste liquid, which increases the processing cost.
  • the Chinese invention patent with the publication number of CN103014070B has prepared a composite enzyme agent based on liquefaction enzyme, saccharification enzyme, cellulase and lipase, and the Chinese invention patent with the publication number of CN106085926A has prepared a compound enzyme with Pelotomaculum schinkii (Shi's dark anaerobic sausage).
  • the present invention is made to solve the above problems, and aims to provide an anaerobic immobilized bacterial agent, a preparation method and an application thereof.
  • the present invention provides a method for preparing an anaerobic immobilized bacterial agent, which has the characteristics of including the following steps: Step 1: Perform anaerobic culture on four different anaerobic functional bacterial species at a certain temperature to obtain corresponding Cultivate the bacterial solution, mix different cultured bacterial solutions according to a certain volume ratio to obtain a composite functional bacterial solution; step 2, concentrate the composite functional bacterial solution to obtain functional bacterial group precipitation; step 3, dissolve the functional bacterial group precipitation in polyethylene The functional bacteria group polyvinyl alcohol solution is obtained in the alcohol aqueous solution, and then the functional bacteria group polyvinyl alcohol solution is dropped into the first buffer solution to obtain polyvinyl alcohol gel beads; step 4, the polyvinyl alcohol gel beads are placed in the In the second buffer solution of sulfate, sulfate-modified polyvinyl alcohol gel beads are obtained, that is, anaerobic immobilized bacterial agent, wherein the anaerobic functional bacteria are respectively Faecalibacterium
  • the anaerobic immobilized bacterial agent provided by the present invention, it can also have the following characteristics: wherein, the mass fraction of polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 10%-15%, and the composite functional bacteria in step 1
  • the volume ratio of the liquid to the polyvinyl alcohol aqueous solution in step 3 is 10:1-20:1.
  • each liter of the first buffer solution contains 0.15mol-0.2mol Na2HPO4, 0.2mol-0.25mol NaH2PO4, and 50g-60g of H 3 BO 3
  • each liter of the second buffer solution contains 1 mol-1.5 mol of Na 2 SO 4
  • the diameter of the anaerobic immobilized bacterial agent is 0.5 cm-1 cm.
  • the present invention also provides an anaerobic immobilized bacterial agent, which is prepared by the preparation method of the anaerobic immobilized bacterial agent, wherein the anaerobic immobilized bacterial agent is a circular condensed bead formed by wrapping a gel with a film, and the circular The diameter of the shaped beads is 0.5cm-1cm.
  • the invention also provides the application of an anaerobic immobilized bacterial agent in anaerobic digestion.
  • anaerobic immobilized bacterial agent provided by the present invention in anaerobic digestion, it can also have the following characteristics: wherein, at a certain temperature, four anaerobic functional bacterial species are respectively cultivated by using pure bacterial culture technology to obtain The OD600 of the corresponding cultured bacterial solution is 15-20, and then the different cultured bacterial solutions are mixed according to a certain volume ratio to obtain a composite functional bacterial solution.
  • the specific operation of anaerobic digestion is to add anaerobic immobilized bacterial agent to Go to the anaerobic digestion reactor for anaerobic digestion.
  • the culture temperature of anaerobic functional bacteria is the same as the temperature of anaerobic immobilized bacteria for anaerobic digestion in the anaerobic digestion reactor.
  • the anaerobic functional bacteria include proteolytic bacteria. Thermofaecalis, Acetobacter brown thermophilus, Methanosarcina pasteurii, and Methanobacterium thermoautotrophicum.
  • the minimum dosage of the composite functional bacterial liquid is calculated by the following formula:
  • V 0 the minimum usage amount (L) of the composite functional bacterial liquid
  • C total the total concentration of organic acids inside the anaerobic digestion reactor (in terms of acetate, mM);
  • pH pH inside the anaerobic digestion reactor
  • V 1 the effective working volume (L) of the anaerobic digestion reactor.
  • the anaerobic immobilized bacterial agent provided by the present invention in anaerobic digestion, it can also have the following characteristics: wherein, when the ammonia nitrogen concentration in the anaerobic digestion reactor is less than or equal to 4g/L, the temperature is 30°C- At 43 °C, the volume ratio of Faecalibacterium thermophilum proteolyticus: Acetobacter brown thermophilus: Methanosarcina pasteuri and Methanothermoautotrophicus in the composite functional bacterial solution is 2-1:3-1:5 -1:2-1, when the ammonia nitrogen concentration in the anaerobic digestion reactor is less than or equal to 4g/L and the temperature is 50°C-65°C, the thermophilic Faecalibacterium proteolyticum in the composite functional bacterial solution: brown thermophilic acetic acid production Bacteria: The volume ratio of Methanosarcina pasteuri and Methanothera thermoautotrophic bacteria is 3-1:4-1:2-1:4-1.
  • the anaerobic immobilized bacterial agent provided in the present invention in anaerobic digestion, it can also have the following characteristics: wherein, when the ammonia nitrogen concentration in the anaerobic digestion reactor is 4g/L-7g/L, and the temperature is At 30°C-43°C, the volume ratio of Faecalibacterium proteolytica: Thermoacetic acetogens brown: Methanosarcina pasteurii and Methanothera thermoautotrophic bacteria in the composite functional bacterial solution is 2-1:2- 1:5-1:3-1, when the ammonia nitrogen concentration in the anaerobic digestion reactor is 4g/L-7g/L and the temperature is 50°C-65°C, the proteolytic thermophilic Faecalibacterium : Acetobacter brown thermophilus: Methanosarcina pasteurii and Methanococcus thermoautotrophs in a volume ratio of 3-1:3-1:4-1:5-1.
  • the anaerobic immobilized bacterial agent provided in the present invention in anaerobic digestion, it can also have the following characteristics: wherein, after drying the anaerobic immobilized bacterial agent, dried beads are obtained, and the dried condensed Before the beads are put into the anaerobic digestion reactor, they are activated for 12h-24h, and the nutrient solution used in the activation is the culture solution used for culturing anaerobic functional strains by pure bacteria culture technology.
  • anaerobic immobilized bacterial agent provided by the present invention, four different anaerobic functional bacterial species are selected, and the corresponding bacterial culture solution is obtained by using the pure bacterial culture technology, and then the four cultured bacteria are combined according to a certain volume ratio.
  • the mixed functional bacteria liquid is mixed to obtain a composite functional bacteria liquid, which is then concentrated into a functional bacterial group precipitation, and then the functional bacterial group precipitate is dissolved in a polyvinyl alcohol aqueous solution, and then the solution is dropped into the first buffer solution to obtain polyvinyl alcohol gel beads, The gel beads are put into a second buffer solution containing sulfate for modification to obtain sulfate-modified polyvinyl alcohol gel beads, that is, an anaerobic immobilized bacterial agent.
  • the anaerobic immobilized bacterial agent is formed by gelation of a polyvinyl alcohol aqueous solution, and is a circular condensate with a diameter of 0.5cm-1cm formed by wrapping the gel in a film.
  • the condensed beads can be directly added to the anaerobic digestion reactor for anaerobic digestion without modification of the equipment, which can effectively relieve the organic acid inhibition of anaerobic digestion in situ and the accumulation of organic acids under the condition of ammonia inhibition. .
  • Faecalibacterium proteolyticus rapidly decomposes cell debris, proteins and lipids; acetic acid is decomposed into carbon dioxide and hydrogen using the homoacetic acid oxidation pathway of thermophilic acetogens; thermoautotrophic methanotrophic bacteria produce hydrogenotrophic products.
  • the methane process consumes hydrogen to create a favorable thermodynamic environment for thermophilic acetogens brown. Therefore, the mixed flora can accelerate the reduction of organic acid load, restore the pH of the anaerobic digestion reactor, and then relieve the toxic effect of organic acid inhibition on the original flora inside the reactor, and restore the functional activity of the original flora;
  • the use of an acid separation unit, the addition of buffer materials and the retrofitting of existing reactors reduces disposal costs.
  • Faecalibacterium proteolyticus, Thermoacetogenes brown, Methanothermothermoautotrophicum and Methanosarcina pasteuri are all ammonia-resistant strains, so the compound functional bacterial solution can relieve the accumulation of organic acids under the condition of ammonia inhibition. . It can be seen from the above that the composite functional bacterial solution is not only acid-resistant but also ammonia-resistant, and has a wide range of applications.
  • the present invention uses sulfate as modifier for modification, and overcomes the disadvantage that traditional polyvinyl alcohol-boric acid gel beads cannot be stored in drying.
  • the sulfate-boronic acid core has sufficient structural flexibility and structural strength to support the gel beads to maintain the structural integrity of the immobilized bacterial agent and resistance to external force shocks.
  • immobilizing the external mixed functional bacterial agent instead of suspending it in the reactor in the form of free cells can make the external bacterial agent better colonize the original flora inside the reactor, and avoid the added bacterial agent.
  • a large amount of water is lost along with the reaction effluent, so there is no need to supplement the added bacterial agent for a long time; the cell density of the external bacterial agent is also increased, and the treatment effect is enhanced.
  • modified gel beads can be stored after drying, so they can be mass-produced and transported over long distances to be applied to anaerobic digestion reactors in different regions.
  • the preparation method provided by the present invention is simple and low in energy consumption, and the obtained immobilized bacterial agent is resistant to acid and ammonia, which can significantly enhance the processing ability of the bacteria in the anaerobic digestion reactor to the accumulated organic acid, and does not require long-term treatment. add.
  • the effect is stable and the cost is low, no large-scale transformation of the existing reactor is required, the problems of the loss of functional bacteria and the poor self-recovery ability of the anaerobic digestion bacteria group are solved, and the organic waste and sewage can be effectively removed.
  • Fig. 1 is the preparation flow chart of the anaerobic immobilized bacterial agent of the present invention.
  • the anaerobic functional bacterial species used in the present invention are Coprothermobacter proteolyticus (DSM 5265), Thermoacetogenium phaeum (DSM 26808), Methanosarcina barkeri (DSM 26808). 800) and Methanothermobacter thermoautotrophicus (DSM 1053), the four anaerobic functional strains were all purchased through general commercial channels.
  • Fig. 1 is the preparation flow chart of anaerobic immobilized bacterial agent of the present invention, as shown in Fig. 1, the preparation method of anaerobic immobilized bacterial agent is as follows:
  • Step 1 Perform anaerobic cultivation on four different anaerobic functional bacterial species at a certain temperature to obtain corresponding cultured bacterial solutions, and mix the different cultured bacterial solutions according to a certain volume ratio to obtain a composite functional bacterial solution.
  • step 2 the composite functional bacterial liquid is centrifuged and concentrated to obtain functional bacterial precipitation.
  • Step 3 Dissolving the functional flora precipitation in the polyvinyl alcohol aqueous solution to obtain a functional flora polyvinyl alcohol solution, then dropping the functional flora polyvinyl alcohol solution into the first buffer solution, and letting it stand for 24 hours to obtain a polyvinyl alcohol coagulation solution. glue beads.
  • Step 4 put the polyvinyl alcohol gel beads into the second buffer solution containing sulfate, and let stand for 48 hours to obtain sulfate-modified polyvinyl alcohol gel beads with a diameter of 0.5 cm-1 cm, that is, anaerobic immobilization Bacterial agent.
  • the four anaerobic functional bacterial species are respectively Faecalibacterium proteolyticus, Thermoacetogenes brown, Methanosarcina pasteuri and Methanothermoautotrophicus.
  • the activated bacterial liquid of the anaerobic functional strains the four anaerobic functional strains were respectively anaerobic cultured through the pure bacterial culture technology to obtain their respective cultured bacterial liquids.
  • the redox potential of the culture liquid is below -300mV, and the OD600 is 15-20.
  • the OD600 of each bacterial solution was the same.
  • the specific operation of the pure bacteria culture technology is as follows: in an anaerobic sterile environment, the activated bacteria liquid is inoculated into the culture liquid of the fermenter with an inoculation amount of 2%-5%, and the stirring speed is 150r/min.
  • the bacterial liquid was concentrated until the OD600 reached 15-20.
  • the fermenter adopts a 3.8L high borosilicate glass tank, which is injected with 2L of liquid culture medium.
  • the cultures of Faecalibacterium proteolyticus, Acetobacter thermophilus brown, Methanosarcina pasteurii and Methanothera thermoautotrophs were mixed to obtain a composite functional bacterial solution.
  • the culture solution for culturing anaerobic functional strains is per liter containing CH 3 COONa, sodium acetate, 2 g; NH 4 Cl, ammonium chloride, 1 g; K 2 HPO 4 3H 2 O, dipotassium hydrogen phosphate trihydrate, 0.4 g; MgCl 2 ⁇ 6H 2 O, magnesium chloride hexahydrate, 0.2 g; CaCl 2 ⁇ 2H 2 O, calcium chloride dihydrate, 0.1 g; Na 2 S ⁇ 9H 2 O, sodium sulfide nonahydrate, 0.2 g; FeCl 2 ⁇ 4H 2 O, ferrous chloride tetrahydrate, 4 mg; H 3 BO 3 , boric acid, 0.2 mg; ZnCl 2 , zinc chloride, 1 mg; CuCl 2 ⁇ 2H 2 O, cupric chloride dihydrate, 0.2 mg; MnCl 2 ⁇ 4H 2 O, manganese chloride tetrahydrate, 1mg; Na 2 Mo
  • step 3 the mass fraction of polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 10%-15%, and the volume ratio of the composite functional bacterial solution in step 1 to the polyvinyl alcohol aqueous solution in step 3 is 10:1-20:1 .
  • each liter of the first buffer solution contains 0.15mol-0.2mol of Na 2 HPO 4 , 0.2-0.25 mol of NaH 2 PO 4 and 50-60 g of H 3 BO 3 , and the balance is sterile water .
  • Each liter of the second buffer solution contains 1 mol-1.5 mol of Na 2 SO 4 , and the balance is sterile water.
  • the anaerobic immobilized bacterial agent is formed by gelation of a polyvinyl alcohol aqueous solution, and the shape is a round bead formed by wrapping the gel with a film, and the diameter of the round bead is 0.5cm-1cm.
  • the anaerobic functional strains are encapsulated in the beads.
  • the newly prepared gel beads can be used directly, or dried to obtain dried beads for transportation and storage.
  • the dried beads need to be activated before use, which is counted as the first drying-activation.
  • the gel beads after the first drying-activation can be dried and activated again, and the activity after the second drying-activation remains at 80-90%, providing greater flexibility in use.
  • the drying method used is as follows: the gel beads are placed in an environment of 80° C., and dried for 24 hours to obtain the dried gel beads.
  • the anaerobic immobilized bacterial agent is applied to the anaerobic digestion technology, which can not only effectively relieve the organic acid inhibition but also effectively relieve the organic acid accumulation under the condition of ammonia inhibition.
  • the specific operation is to put it into the anaerobic digestion reactor, the usage amount of the anaerobic immobilized bacterial agent is determined by the amount of the composite functional bacterial liquid in step 1, and the minimum value of the composite functional bacterial liquid
  • the dosage is calculated by the following formula:
  • V 0 the minimum usage amount (L) of the composite functional bacterial liquid
  • C total the total concentration of organic acids inside the anaerobic digestion reactor (in terms of acetate, mM);
  • pH pH inside the anaerobic digestion reactor
  • V 1 the effective working volume (L) of the anaerobic digestion reactor.
  • the culture temperature of the four anaerobic functional bacteria is the same as the temperature at which the anaerobic immobilized bacterial agent performs anaerobic digestion in the anaerobic digestion reactor, that is, the same as the operating temperature of the anaerobic digestion reactor.
  • the volume ratio of the four anaerobic functional bacteria in the composite bacterial solution is determined by the physical and chemical parameters of the anaerobic digestion reactor, specifically:
  • thermophilic Faecalibacterium proteolyticum in the composite functional bacterial solution Brown thermophilic acetogen: Pasteurella methane
  • the volume ratio of Sarcinus and thermoautotrophic Methanothermia is 2-1:3-1:5-1:2-1;
  • thermophilic Faecalibacterium proteolyticum in the composite functional bacteria solution Brown thermophilic acetogenic bacteria: Pasteurella methane
  • the volume ratio of Sarcinus and thermoautotrophic Methanothermia is 3-1:4-1:2-1:4-1;
  • thermophilic Faecalibacterium proteolyticum in the composite functional bacterial solution Brown thermophilic acetogen: The volume ratio of M. pasteurii and M. thermoautotrophic bacteria is 2-1:2-1:5-1:3-1;
  • thermophilic Faecalibacterium proteolyticum in the compound functional bacterial solution Brown thermophilic acetogen: The volume ratio of Methanosarcina pasteuri and Methanothermoautotrophicum was 3-1:3-1:4-1:5-1.
  • the dried anaerobic immobilized bacterial agent is used, so before being put into the anaerobic digestion reactor, it needs to be activated for 12h-24h to restore the microbial activity in the immobilized bacterial agent and activate the
  • the nutrient solution used is the culture solution used for culturing anaerobic functional strains by pure bacteria culture technology. But in practice, the same technical effect can be achieved using directly prepared gel beads.
  • the concentration unit mM in which the examples of the present invention appear is mmol/L.
  • OD600 is measured by spectrophotometer
  • volatile suspended solid concentration is measured according to "Water and Wastewater Monitoring and Analysis Method (Fourth Edition)”
  • ammonia nitrogen concentration is measured by Kjeldahl analyzer
  • volatile Organic acids were measured by gas chromatography.
  • This example specifically describes the preparation and application of the anaerobic immobilized bacterial agent.
  • thermophilic faecalis proteolyticum Take 180mL, 240mL, 120mL and 240mL of the culture solution of thermophilic faecalis proteolyticum, acetonitrile brown thermophilus, methanosarcina pasteurii and methanotherm thermoautotrophic bacteria and mix them, then centrifuge and concentrate, centrifugal force 5000g , the centrifugation temperature was 4 °C, and the centrifugation time was 5 min to obtain functional bacterial precipitation.
  • anaerobic digestion reactor Four 16.3L anaerobic digestion reactors were selected as the treatment objects.
  • the parameters are: effective working volume 10.5L, operating temperature 55°C, volatile suspended solid concentration 5g/L, pH 6.0, organic acid concentration 104mM , the ammonia nitrogen concentration is 1.5g/L.
  • the reactor stopped feeding according to the dosing situation of the immobilized bacterial agent, it was divided into the control group (without adding the bacterial agent) and the experimental group (adding the bacterial agent).
  • the experimental group began to restore the methane production capacity, and the maximum acetic acid consumption rate was 15.6 mM/d; the control group began to restore the methane production capacity after 20 days, and the maximum acetic acid consumption rate was 9.2 mM/d.
  • Both the control group and the experimental group have two anaerobic digestion reactors, and the above data are the average values of the two groups of data.
  • This example specifically describes the preparation and application of the anaerobic immobilized bacterial agent.
  • This functional bacterial group precipitation was added to 60 mL of polyvinyl alcohol aqueous solution with a mass fraction of 12% and stirred evenly, and the stirred polyvinyl alcohol aqueous solution was dropped into each liter containing Na 2 HPO 4 , 0.2 mol; NaH 2 PO 4 , 0.23 mol; H 3 BO 3 , in the first buffer solution of 55 g, let stand for 24 h to form polyvinyl alcohol gel beads; put the polyvinyl alcohol gel beads into the second buffer solution containing 1.5 mol of Na 2 SO 4 per liter. In the buffer solution, let stand for 48 hours to form sulfate-modified polyvinyl alcohol gel beads with a diameter of 0.8 cm, and the gel beads are anaerobic immobilized bacterial agents.
  • the effective working volume is 5.6L
  • the operating temperature is 55°C
  • the volatile suspended solid concentration is 3g/L
  • the pH is 5.5
  • the organic acid concentration is 96.5mM
  • the ammonia nitrogen concentration is 4.5g/L.
  • the reactor stopped feeding according to the dosing situation of the immobilized bacterial agent, it was divided into the control group (without adding the bacterial agent) and the experimental group (adding the bacterial agent).
  • the experimental group began to recover the methane production capacity, and the maximum acetic acid consumption rate was 9.9 mM/d; the control group began to restore the methane production capacity after 28 days, and the maximum acetic acid consumption rate was 6.8 mM/d.
  • Both the control group and the experimental group have three anaerobic digestion reactors, and the above data are the average values of the three groups of data.
  • This example specifically describes the preparation and application of the anaerobic immobilized bacterial agent.
  • This functional bacterial group precipitate was added to 100 mL of polyvinyl alcohol aqueous solution with a mass fraction of 15% and stirred evenly, and the stirred polyvinyl alcohol aqueous solution was added dropwise into each liter containing Na 2 HPO 4 , 0.2 mol; NaH 2 PO 4 , 0.23 mol; H 3 BO 3 , in the first buffer solution of 60 g, let stand for 24 h to form polyvinyl alcohol gel beads; put the polyvinyl alcohol gel beads into the second buffer solution containing 1.2 mol of Na 2 SO 4 per liter. In the buffer solution, let stand for 48 hours to form sulfate-modified polyvinyl alcohol gel beads with a diameter of 1.0 cm, and the gel beads are the anaerobic immobilized bacterial agent.
  • the treatment object is a 45.2L anaerobic digestion reactor, the effective working volume is 30.2L, the operating temperature is 35°C, the volatile suspended solid concentration is 1g/L, the pH is 6.5, the organic acid concentration is 50.3mM, and the ammonia nitrogen concentration is 3.6g/L. L.
  • the reactor stopped feeding according to the dosing situation of the immobilized bacterial agent, it was divided into the control group (without adding the bacterial agent) and the experimental group (adding the bacterial agent).
  • the experimental group began to restore the methane production capacity, and the maximum acetic acid consumption rate was 12.3 mM/d; the control group began to restore the methane production capacity after 15 days, and the maximum acetic acid consumption rate was 7.9 mM/d.
  • This example specifically describes the preparation and application of the anaerobic immobilized bacterial agent.
  • This functional bacterial group precipitate was added to 20 mL of polyvinyl alcohol aqueous solution with a mass fraction of 15% and stirred evenly, and the stirred polyvinyl alcohol aqueous solution was dropped into each liter containing Na 2 HPO 4 , 0.2 mol; NaH 2 PO 4 , 0.23 mol; H 3 BO 3 , 50 g of the first buffer solution, let stand for 24 h to form polyvinyl alcohol gel beads; put the polyvinyl alcohol gel beads into the second buffer solution containing 1.2 mol of Na 2 SO 4 per liter. In the buffer solution, stand for 48 hours to form sulfate-modified polyvinyl alcohol gel beads with a diameter of 0.7 cm, and the gel beads are the anaerobic immobilized bacterial agent.
  • the reactor stopped feeding according to the dosing situation of the immobilized bacterial agent, it was divided into the control group (without adding the bacterial agent) and the experimental group (adding the bacterial agent).
  • the experimental group began to recover the methane production capacity, and the maximum acetic acid consumption rate was 13.2 mM/d; the control group began to recover the methane production capacity after 10 days, and the maximum acetic acid consumption rate was 9.8 mM/d.
  • Both the control group and the experimental group have three anaerobic digestion reactors, and the above data are the average values of the three groups of data.
  • the preparation method and application of the anaerobic immobilized bacterial agent provided by the embodiment of the present invention, four different anaerobic functional bacterial species are selected, and the corresponding bacterial culture solution is obtained by using the pure bacterial culture technology, and then the The four kinds of bacterial cultures are mixed to obtain a composite functional bacterial solution, which is then concentrated into functional bacterial precipitation, and then the functional bacterial precipitation is dissolved in the polyvinyl alcohol aqueous solution, and then the solution is dropped into the first buffer solution to obtain polyvinyl alcohol.
  • the gel beads are put into a second buffer solution containing sulfate for modification to obtain sulfate-modified polyvinyl alcohol gel beads, that is, an anaerobic immobilized bacterial agent.
  • the anaerobic immobilized bacterial agent is formed by gelation of a polyvinyl alcohol aqueous solution, and is a circular condensate with a diameter of 0.5cm-1cm formed by wrapping the gel in a film.
  • the beads can be directly added to the anaerobic digestion reactor for anaerobic digestion without modification of the equipment, which can effectively relieve the organic acid inhibition of anaerobic digestion in situ and the accumulation of organic acids under the condition of ammonia inhibition. .
  • Faecalibacterium proteolyticus rapidly decomposes cell debris, proteins and lipids; acetic acid is decomposed into carbon dioxide and hydrogen using the homoacetic acid oxidation pathway of thermophilic acetogens; thermoautotrophic methanotrophic bacteria produce hydrogenotrophic products.
  • the methane process consumes hydrogen to create a favorable thermodynamic environment for thermophilic acetogens brown. Therefore, the mixed flora can accelerate the reduction of organic acid load, restore the pH of the anaerobic digestion reactor, and then relieve the toxic effect of organic acid inhibition on the original flora inside the reactor, and restore the functional activity of the original flora;
  • the use of an acid separation unit, the addition of buffer materials and the retrofitting of existing reactors reduces disposal costs.
  • Faecalibacterium proteolyticus, Thermoacetogenes brown, Methanothermothermoautotrophicum and Methanosarcina pasteuri are all ammonia-resistant strains, so the compound functional bacterial solution can relieve the accumulation of organic acids under the condition of ammonia inhibition. . It can be seen from the above that the composite functional bacterial solution is not only acid-resistant but also ammonia-resistant, and has a wide range of applications.
  • sulfate as modifier for modification overcomes the disadvantage that traditional polyvinyl alcohol-boric acid gel beads cannot be stored in drying.
  • the sulfate-boronic acid core has sufficient structural flexibility and structural strength to support the gel beads to maintain the structural integrity of the immobilized bacterial agent and resistance to external force shocks.
  • immobilizing the external mixed functional bacterial agent instead of suspending it in the reactor in the form of free cells can make the external bacterial agent better colonize the original flora inside the reactor, and avoid the added bacterial agent.
  • a large amount of water is lost along with the reaction effluent, so there is no need to supplement the added bacterial agent for a long time; the cell density of the external bacterial agent is also increased, and the treatment effect is enhanced.
  • the modified beads can be stored after drying, so they can be used for large-scale production and long-distance transportation, and can be applied to anaerobic digestion reactors in different regions.
  • the disadvantage is that the preparation is carried out near the oxygen reactor and it is put into use immediately.
  • the minimum usage amount of the composite functional bacterial solution can be determined; at the same time, the mixing ratio of the four anaerobic functional bacterial species can be determined under different working conditions, which is economical and effective. to relieve organic acid inhibition.
  • the pH in the anaerobic digestion reactor is generally greater than 5, and the anaerobic immobilized bacterial agent prepared in the embodiment of the present invention is suitable for the anaerobic digestion reactor with pH greater than 5 (including pH 5), so there is no need for anaerobic digestion.
  • the digestion reactor was pH adjusted.
  • Each liter of the first buffer solution contains 0.15mol-0.2mol of Na 2 HPO 4 , 0.2mol-0.25mol of NaH 2 PO 4 and 50g-60g of H 3 BO 3
  • each liter of the second buffer solution contains 1mol-1.5 mol of Na 2 SO 4 , NaH 2 PO 4 are beneficial to maintain the steady state of microbial cells
  • the addition of H 3 BO 3 forms a boric acid core
  • Na 2 SO 4 modifies the boronic core to obtain modified gel beads .
  • thermophilic Faecalibacterium proteolyticus in the composite functional bacterial solution Brown thermophilus acetogen: Pasteurella m.
  • the volume ratio of cocci and thermoautotrophic methanotrophic bacteria is 2-1:3-1:5-1:2-1.
  • thermophilic Faecalibacterium proteolyticus in the composite functional bacterial solution Brown thermophilic acetogen: Pasteurella methane
  • the volume ratio of cocci and thermoautotrophic methanotrophic bacteria is 3-1:4-1:2-1:4-1.
  • thermophilic Faecalibacterium proteolyticum in the composite functional bacterial solution Brown thermophilic acetogens: Pasteurella
  • the volume ratio of Methanosarcina and Methanothermoautotrophicum was 2-1:2-1:5-1:3-1.
  • thermophilic Faecalibacterium proteolyticus in the composite functional bacterial solution Brown thermophilic acetogens: Pasteurella The volume ratio of Methanosarcina and M.
  • thermoautotrophic bacteria was 3-1:3-1:4-1:5-1. The selection of these ratios is based on experimental data. Specifically, by simulating the above four working conditions, and setting up multiple groups of parallel reactors under each working condition, combined with microbial sequencing analysis, we found that the abundance of these four microorganisms was relatively high. High, is the dominant microorganism, so we choose the abundance ratio of these four microorganisms as the mixed volume ratio. If the volume of a certain microorganism is lower than this ratio, the mutual support relationship in the inoculant will be destroyed, and then it will not be able to relieve the inhibition of organic acids. If the quantity is low, the hydrogen gas produced by F.
  • thermophilus proteolyticus cannot be consumed, and the thermodynamic results are changed, thereby creating a thermodynamic barrier for F. thermophilic proteolyticum, and the reaction stops. Higher than this ratio will cause waste of bacterial agent, and increase the use of polyvinyl alcohol aqueous solution, which is less economical. Therefore, these are obtained based on the results of the self-reaction of the flora, taking into account both the reaction effect and the economy.
  • the anaerobic immobilized inoculum should be activated for 12h-24h before being put into the anaerobic digestion reactor.
  • the activation can restore the activity of the microbial inoculum to better relieve acid inhibition.
  • the microbial reaction is relatively sensitive to temperature conditions. If the anaerobic functional strains are not cultivated at a specific temperature in advance, the immobilized bacterial agent cannot be used, and even a large number of microorganisms in the bacterial agent die. For example, if the microbial inoculum cultured at 35 °C is immediately put into the reactor at 55 °C, it will cause a longer stagnation period, and a large number of microorganisms in the inoculum will die, changing the proportion of microorganisms in the inoculum, which will lead to Bacterial agent ineffective.
  • the culture conditions of the same temperature are selected, that is, the culture temperature of the anaerobic functional strains is the same as the temperature of the anaerobic digestion of the anaerobic immobilized bacterial agent in the anaerobic digestion reactor.
  • the preparation method provided by the present invention is simple and low in energy consumption, and the obtained immobilized bacterial agent is resistant to acid and ammonia, which can significantly enhance the processing ability of the bacteria in the anaerobic digestion reactor to the accumulated organic acid, and does not require long-term treatment. add.
  • the effect is stable and the cost is low, no large-scale transformation of the existing reactor is required, the problems of the loss of functional bacteria and the poor self-recovery ability of the anaerobic digestion bacteria group are solved, and the organic waste and sewage can be effectively removed.

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Abstract

提供了一种厌氧固定化菌剂、制备方法及其应用。该菌剂的制备方法是，选用四种不同的厌氧功能菌种，利用纯菌培养技术得到相应的培养菌液，然后按照一定体积比将四种培养菌液进行混合获得复合功能菌液，随后浓缩为功能菌群沉淀，再将功能菌群沉淀溶解在聚乙烯醇水溶液中，将该溶液滴入第一缓冲溶液中得到聚乙烯醇凝胶珠，得到的胶珠放入含有硫酸盐的第二缓冲溶液中得到硫酸盐改性聚乙烯醇凝胶珠，即厌氧固定化菌剂。

Description

一种厌氧固定化菌剂、制备方法及其应用 技术领域
本发明属于环境工程材料领域，具体涉及一种厌氧固定化菌剂、制备方法及其应用。
背景技术
作为有效的生物处理技术之一，厌氧消化不仅可以控制易降解生物质废物的环境污染，也可以将其含有的有机物转化为以甲烷为主的能源气体。当进料为高含固率的生物质废物(如餐厨垃圾、禽畜粪便和污水厂污泥等)时，以乙酸为主的有机酸很容易在厌氧消化反应器中快速积累，引起对产甲烷菌的酸抑制现象，造成甲烷化过程不稳定，甚至反应器运行失败等问题。特别是当同时发生氨抑制的情况下，氨抑制会进一步加剧有机酸积累问题，形成氨和有机酸双重抑制。
针对上述有机酸快速积累的问题，生物强化措施通过外加菌群至厌氧消化反应器，以提高脂类降解速率、消耗过量有机酸、缩短甲烷化过程迟滞期，最终恢复反应器正常运行能力。目前，生物强化措施主要存在四大问题：1.悬浮培养模式易造成外加功能菌群的流失；2.外加菌群难以适应反应器内部的物化条件，会迅速死亡，或者其竞争优势被反应器内部的土著微生物超越；3.外加菌群结构不特异，单一菌种容易死亡，复合菌种缺乏协同代谢，导致效率不高，需要提高接种量和接种频率；4.厌氧纯菌种培养难度大，世代时间长，无法满足大型反应器的大规模需要；5.外加菌群不耐受高氨浓度，无法解除氨抑制条件下的有机酸积累问题。近年来，国内发展了一系列方法来解除厌氧消化反应器有机酸抑制，例如公开号为CN202089962U、CN110184171A、CN201644487U和CN201644487U的中国专利设计了新式厌氧消化反应器，但此类技术并不适用于已发生有机酸抑制的厌氧消化反应器。公开号为CN107475304A、CN109554402A和CN109182390A的中国发明专利投加了碳材料，但此类技术需不断补充碳材料来弥补碳材料的流失，同时碳材料浸出物对微生物也有潜在的毒害作用。公开号为CN102992478B的中国发明专利添加了离子交换树脂装置，但此类技术需对离子交换树脂进行再生，也需对再生废液进行处理，增加了处理成本。公开号为CN103014070B的中国发明专利制备了以液化酶、糖化酶、纤维素酶和脂肪酶为主的复合酶剂，公开号为CN106085926A的中国发明专利制备了以Pelotomaculum schinkii(施氏暗色厌氧香肠状菌)为主的复合菌剂，此类技术通过促进微生物代谢作用，进而缓解有机酸抑制，但此类技术均没有实现酶剂和厌氧菌剂的固定化，致使酶剂和菌剂流失，削弱作用效果。而利用固定化菌剂原位解除厌氧消化有机酸抑制的方法则未见报道。
发明内容
本发明是为了解决上述问题而进行的，目的在于提供一种厌氧固定化菌剂、制备方法及其应用。
本发明提供了一种厌氧固定化菌剂的制备方法，具有这样的特征，包括以下步骤：步骤1，在一定温度下对四种不同的厌氧功能菌种分别进行厌氧培养得到相应的培养菌液，按照一定体积比将不同的培养菌液进行混合获得复合功能菌液；步骤2，将复合功能菌液进行浓缩获得功能菌群沉淀；步骤3，将功能菌群沉淀溶解在聚乙烯醇水溶液中得到功能菌群聚乙烯醇溶液，然后将功能菌群聚乙烯醇溶液滴入第一缓冲溶液中，得到聚乙烯醇凝胶珠；步骤4，将聚乙烯醇凝胶珠放在含有硫酸盐的第二缓冲溶液中，得到硫酸盐改性聚乙烯醇凝胶珠， 即厌氧固定化菌剂，其中，厌氧功能菌种分别为解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌以及热自养甲烷热杆菌。
在本发明提供的厌氧固定化菌剂的制备方法中，还可以具有这样的特征：其中，聚乙烯醇水溶液中聚乙烯醇的质量分数为10％-15％，步骤1中的复合功能菌液与步骤3中的聚乙烯醇水溶液的体积比为10：1-20：1。
在本发明提供的厌氧固定化菌剂的制备方法中，还可以具有这样的特征：其中，每升第一缓冲溶液中包含0.15mol-0.2mol的Na2HPO4，0.2mol-0.25mol的NaH2PO4，及50g-60g的H 3BO 3，每升第二缓冲溶液中包含1mol-1.5mol的Na 2SO 4，厌氧固定化菌剂的直径为0.5cm-1cm。
本发明还提供了一种厌氧固定化菌剂，由厌氧固定化菌剂的制备方法制备得到，其中，厌氧固定化菌剂为由薄膜包裹凝胶物形成的圆形凝珠，圆形凝珠的直径为0.5cm-1cm。
本发明还提供了一种厌氧固定化菌剂在厌氧消化中的应用。
在本发明提供的厌氧固定化菌剂在厌氧消化中的应用中，还可以具有这样的特征：其中，在一定温度下，利用纯菌培养技术将四种厌氧功能菌种分别培养得到相应的培养菌液，培养菌液的OD600为15-20，然后按照一定体积比将不同的培养菌液进行混合获得复合功能菌液，厌氧消化的具体操作为将厌氧固定化菌剂加入到厌氧消化反应器进行厌氧消化，厌氧功能菌种的培养温度与厌氧固定化菌剂在厌氧消化反应器中进行厌氧消化的温度相同，厌氧功能菌种包括解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌以及热自养甲烷热杆菌。
在本发明提供的厌氧固定化菌剂在厌氧消化中的应用中，还可以具有这样的特征：其中，复合功能菌液的最小用量由下式计算得到：
Figure PCTCN2021087449-appb-000001
V 0:复合功能菌液的最小使用量(L)；
C total：厌氧消化反应器内部有机酸总浓度(以乙酸根计，mM)；
pH:厌氧消化反应器内部pH；
C vs:厌氧消化反应器内部挥发性悬浮固体浓度(g/L)；
V 1：厌氧消化反应器有效工作体积(L)。
在本发明提供的厌氧固定化菌剂在厌氧消化中的应用中，还可以具有这样的特征：其中，当厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为30℃-43℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为2-1：3-1：5-1：2-1，当厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为50℃-65℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为3-1：4-1：2-1：4-1。
在本发明提供的厌氧固定化菌剂在厌氧消化中的应用中，还可以具有这样的特征：其中，当厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为30℃-43℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为2-1：2-1：5-1：3-1，当厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为50℃-65℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为3-1：3-1：4-1：5-1。
在本发明提供的厌氧固定化菌剂在厌氧消化中的应用中，还可以具有这样的特征：其中，将厌氧固定化菌剂干化后得到干化的凝珠，干化的凝珠在投入厌氧消化反应器前，进行活化12h-24h，活化时所用的营养液为利用纯菌培养技术培养厌氧功能菌种时所用的培养液。
发明的作用与效果
根据本发明提供的厌氧固定化菌剂的制备方法及应用，选用四种不同的厌氧功能菌种，利用纯菌培养技术得到相应的培养菌液，然后按照一定体积比将四种培养菌液进行混合获得复合功能菌液，随后浓缩为功能菌群沉淀，再将功能菌群沉淀溶解在聚乙烯醇水溶液中，然后将该溶液滴入第一缓冲溶液中得到聚乙烯醇凝胶珠，将该凝胶珠放入含有硫酸盐的第二缓冲溶液中进行改性，得到硫酸盐改性聚乙烯醇凝胶珠，即厌氧固定化菌剂。厌氧固定化菌剂由聚乙烯醇水溶液凝胶化形成，为由薄膜包裹凝胶物形成的直径为0.5cm-1cm的圆形凝珠。该凝珠能够直接加入到厌氧消化反应器中进行厌氧消化，无需对设备进行改造，既能够有效解除原位有效解除厌氧消化有机酸抑制又能够有效解除氨抑制条件下的有机酸积累。
解蛋白嗜热粪杆菌快速分解细胞碎屑、蛋白质和脂质；利用棕色嗜热产醋菌的同型乙酸氧化途径，将乙酸分解为二氧化碳及氢气；热自养甲烷热杆菌则进行氢营养型产甲烷过程，消耗氢气进而为棕色嗜热产醋菌创造有利的热力学环境；巴氏甲烷八叠球菌通过乙酸发酵型和氢营养型产甲烷途径，分别消耗乙酸和氢气。因此，混合菌群可以加速降低有机酸负荷，恢复厌氧消化反应器pH，进而解除有机酸抑制对反应器内部原有菌群的毒害作用，恢复原有菌群的功能活性；且避免了有机酸分离装置的使用、缓冲材料的添加和现有反应器的改造，降低了处理成本。并且解蛋白嗜热粪杆菌、棕色嗜热产醋菌、热自养甲烷热杆菌及巴氏甲烷八叠球菌均属于耐氨菌种，所以复合功能菌液能够解除氨抑制条件下的有机酸积累。由以上可知，该复合功能菌液不仅耐酸而且耐氨，适用范围广。
本发明利用硫酸盐作为改性剂进行改性，克服了传统聚乙烯醇-硼酸凝胶珠无法干化保存的缺点。硫酸盐-硼酸内核具有足够的结构柔性和结构强度来支撑凝胶珠，以维持固定化 菌剂的结构完整和对外力冲击的抗性。
同时，对外加混合功能菌剂进行固定化处理，而不是以游离的细胞形态悬浮于反应器中，可以使外加菌剂更好地定殖于反应器内部原有菌群，避免了外加菌剂随着反应出水而大量流失，进而无需长期补充外加菌剂；也提高了外加菌剂的细胞密度，增强了处理效果。
此外，改性凝胶珠能够被干化处理后保存，因此能够进行大规模生产和长途运输，以应用于不同区域的厌氧消化反应器，克服了普通凝胶珠无法干化，所以必须在厌氧反应器附近进行制备，并立马投入使用的缺点。
由以上可知，本发明所提供的制备方法简单、能耗低，得到的固定化菌剂耐酸耐氨，可以显著增强厌氧消化反应器内部菌群对已积累有机酸的处理能力，且无需长期投加。与现有技术相比，效果稳定且成本低廉，无需对现有反应器进行大规模改造，解决了功能菌种流失和厌氧消化菌群自我恢复能力差的问题，可以有效解除有机垃圾和污水厌氧消化过程中的有机酸抑制以及氨抑制条件下的有机酸积累。
附图说明
图1是本发明的厌氧固定化菌剂的制备流程图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，以下结合实施例及附图对本发明一种厌氧固定化菌剂、制备方法及其应用作具体阐述。
除特别说明外，本发明的实施例所用的原料及试剂均通过一般商业途径购买得到。
本发明所用的厌氧功能菌种分别为解蛋白嗜热粪杆菌(Coprothermobacter proteolyticus,DSM 5265)、棕色嗜热产醋菌(Thermacetogenium phaeum,DSM 26808)、巴氏甲烷八叠球菌(Methanosarcina barkeri,DSM 800)和热自养甲烷热杆菌(Methanothermobacter thermautotrophicus,DSM 1053)，该四种厌氧功能菌种均通过一般商业途径购买得到。
图1是本发明的厌氧固定化菌剂的制备流程图，如图1所示，厌氧固定化菌剂的制备方法如下：
步骤1，在一定温度下对四种不同的厌氧功能菌种分别进行厌氧培养得到相应的培养菌液，按照一定体积比将不同的培养菌液进行混合获得复合功能菌液。
步骤2，将复合功能菌液离心浓缩获得功能菌群沉淀。
步骤3，将功能菌群沉淀溶解在聚乙烯醇水溶液中得到功能菌群聚乙烯醇溶液，然后将功能菌群聚乙烯醇溶液滴入第一缓冲溶液中，静置24h，得到聚乙烯醇凝胶珠。
步骤4，将聚乙烯醇凝胶珠放入含有硫酸盐的第二缓冲溶液中，静置48h，得到直径为0.5cm-1cm的硫酸盐改性聚乙烯醇凝胶珠，即厌氧固定化菌剂。
步骤1中，四种厌氧功能菌种分别为解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌以及热自养甲烷热杆菌。利用厌氧功能菌种的活化菌液，通过纯菌培养技术分别对该四种厌氧功能菌种进行厌氧培养，得到各自的培养菌液。培养菌液的氧化还原电位在-300mV 以下，OD600为15-20。每种菌液的OD600相同。
纯菌培养技术的具体操作为：在厌氧无菌环境下，将活化菌液以2％-5％的接种量分别接种于发酵罐的培养液中，搅拌速度为150r/min。通过在发酵罐的培养液出口安装无菌聚醚砜中空纤维细胞回收模块，浓缩菌液，直至OD600达到15-20。发酵罐采用3.8L高硼硅玻璃罐体，注入2L液体培养基，罐体进出料口和进出气口均用0.22μm无菌滤膜封闭，在121℃下灭菌20min。
按照一定体积比对解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌和热自养甲烷热杆菌的培养菌液进行混合，获得复合功能菌液。
培养厌氧功能菌种的培养液为每升包含CH 3COONa，乙酸钠，2g；NH 4Cl，氯化铵，1g；K 2HPO 4 3H 2O，三水合磷酸氢二钾，0.4g；MgCl 2·6H 2O，六水合氯化镁，0.2g；CaCl 2·2H 2O，二水合氯化钙，0.1g；Na 2S·9H 2O，九水合硫化钠，0.2g；FeCl 2·4H 2O，四水合氯化亚铁，4mg；H 3BO 3，硼酸，0.2mg；ZnCl 2，氯化锌，1mg；CuCl 2·2H 2O，二水合氯化铜，0.2mg；MnCl 2·4H 2O，四水合氯化锰，1mg；Na 2MoO 4·4H 2O，四水合钼酸钠，0.1mg；AlCl 3·6H 2O，六水合氯化铝，1mg；CoCl 2·6H 2O，六水合氯化钴，2mg；NiCl 2·6H 2O，六水合氯化镍，0.2mg；Na 2SeO 3·5H 2O，五水合亚硒酸钠，2mg；EDTA-2Na，乙二胺四乙酸二钠，10mg；pyridoxine hydrochloride，盐酸吡哆素，0.5mg；thiamine hydrochloride，盐酸硫胺素，0.3mg；D-calcium pantothenate，D-泛酸半钙盐，0.3mg；folic acid，叶酸，0.1mg；riboflavin，核黄素，0.3mg；nicotinic acid，烟酸，0.3mg；P-aminobenzoic acid，4-氨基苯甲酸，0.3mg；vitamin B 12，维生素B 12，0.1mg的水溶液，pH＝6.8-7.0。
步骤3中，聚乙烯醇水溶液中聚乙烯醇的质量分数为10％-15％，步骤1中的复合功能菌液与步骤3中的聚乙烯醇水溶液的体积比为10：1-20：1。
步骤3中，每升第一缓冲溶液中包含0.15mol-0.2mol的Na 2HPO 4，0.2mol-0.25mol的NaH 2PO 4及50g-60g的H 3BO 3，余量为无菌水 。
每升第二缓冲液中包含1mol-1.5mol的Na 2SO 4，余量为无菌水。
厌氧固定化菌剂由聚乙烯醇水溶液凝胶化形成，形态为由薄膜包裹凝胶物形成的圆形凝珠，圆形凝珠的直径为0.5cm-1cm。其中厌氧功能菌种被包裹在凝珠内。
在本发明中，刚制备出的凝胶珠可以直接使用，也可以进行干化后得到干化的凝珠以便运输和保存。干化的凝珠在使用前需要进行活化，计作第一次干化-活化。第一次干化-活化后的凝胶珠还可以再进行一次干化-活化，并且第二次干化-活化后的活性保持在80-90％，提供了更大的使用灵活性。
在本发明的实施例中，所用的干化方法为：将凝胶珠放入80℃的环境中，干燥24小时得到干化的凝珠。
将该厌氧固定化菌剂应用到厌氧消化技术中，既能够有效解除有机酸抑制又能够有效解除氨抑制条件下的有机酸积累。在本发明的实施例中，具体操作为将其投入到厌氧消化反应器中，厌氧固定化菌剂的使用量由步骤1中的复合功能菌液的量决定，复合功能菌液的最小用量由下式计算得到：
Figure PCTCN2021087449-appb-000002
V 0:复合功能菌液的最小使用量(L)；
C total:厌氧消化反应器内部有机酸总浓度(以乙酸根计，mM)；
pH:厌氧消化反应器内部pH；
C vs:厌氧消化反应器内部挥发性悬浮固体浓度(g/L)；
V 1:厌氧消化反应器有效工作体积(L)。
四种厌氧功能菌的培养温度与厌氧固定化菌剂在厌氧消化反应器中进行厌氧消化的温度相同，也就是与厌氧消化反应器的运行温度相同。同时，复合菌液中的四种厌氧功能菌的体积比由厌氧消化反应器的物化参数决定，具体为：
1.当厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为30℃-43℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为2-1：3-1：5-1：2-1；
2.当厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为50℃-65℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为3-1：4-1：2-1：4-1；
3.当厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为30℃-43℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为2-1：2-1：5-1：3-1；
4.当厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为50℃-65℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为3-1：3-1：4-1：5-1。
在本发明的实施例中，使用的为干化的厌氧固定化菌剂，因此在投入厌氧消化反应器前，需要进行活化12h-24h，以恢复固定化菌剂中的微生物活性，活化时所用的营养液为利用纯菌培养技术培养厌氧功能菌种时所用的培养液。但在实际应用中，使用直接制备的凝胶珠可以达到同样的技术效果。
本发明的实施例出现的浓度单位mM为mmol/L。
本发明的实施例中，OD600通过分光光度计测得，挥发性悬浮固体浓度根据《水和废水监测分析方法(第四版)》测得，氨氮浓度通过凯氏定氮仪测得，挥发性有机酸通过气相 色谱仪测得。
<实施例1>
本实施例对厌氧固定化菌剂的制备及应用做具体阐述。
在55℃条件下，分别利用纯菌培养技术培养不同的厌氧功能菌种，使其培养菌液OD600为15。分别取180mL、240mL、120mL和240mL的解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌和热自养甲烷热杆菌的培养菌液进行混合，然后离心浓缩，离心力5000g，离心温度4℃，离心时间5min，获得功能菌群沉淀。
将此功能菌群沉淀加入到50mL质量分数为15％的聚乙烯醇水溶液中搅拌均匀，得到功能菌群聚乙烯醇溶液，然后将该功能菌群聚乙烯醇溶液滴入每升包含Na 2HPO 4，0.15mol；NaH 2PO 4，0.2mol；H 3BO 3，50g的第一缓冲溶液中，静置24h，形成聚乙烯醇凝胶珠；将此聚乙烯醇凝胶珠放入每升包含1mol Na 2SO 4的第二缓冲溶液中，静置48h，形成直径为0.5cm的硫酸盐改性聚乙烯醇凝胶珠，此凝胶珠即为厌氧固定化菌剂。
选取四个16.3L的厌氧消化反应器作为处理对象，参数均为：有效工作体积10.5L，运行温度为55℃，挥发性悬浮固体浓度为5g/L，pH为6.0，有机酸浓度为104mM，氨氮浓度1.5g/L。
反应器停止进料后，根据固定化菌剂投加情况，分为对照组(未投加菌剂)和实验组(投加菌剂)。菌剂投加8天后，实验组开始恢复产甲烷能力，最大乙酸消耗速率为15.6mM/d；对照组则在20天后，开始恢复产甲烷能力，最大乙酸消耗速率为9.2mM/d。
对照组和实验组均为两个厌氧消化反应器，以上数据为两组数据的平均值。
<实施例2>
本实施例对厌氧固定化菌剂的制备及应用做具体阐述。
在55℃条件下，分别利用纯菌培养技术培养不同的厌氧功能菌种，使其培养菌液OD600为18。分别取150mL、150mL、200mL和250mL的解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌和热自养甲烷热杆菌的培养菌液进行混合，然后离心浓缩，离心力5000g，离心温度4℃，离心时间5min，获得功能菌群沉淀。
将此功能菌群沉淀加入到60mL质量分数为12％的聚乙烯醇水溶液中搅拌均匀，将搅拌后的聚乙烯醇水溶液滴入每升包含Na 2HPO 4，0.2mol；NaH 2PO 4，0.23mol；H 3BO 3，55g的第一缓冲溶液中，静置24h，形成聚乙烯醇凝胶珠；将此聚乙烯醇凝胶珠放入每升包含1.5mol的Na 2SO 4的第二缓冲溶液中，静置48h，形成直径为0.8cm的硫酸盐改性聚乙烯醇凝胶珠，此凝胶珠即为厌氧固定化菌剂。
选取六个8.2L的厌氧消化反应器作为处理对象，参数均为：
有效工作体积5.6L，运行温度为55℃，挥发性悬浮固体浓度为3g/L，pH为5.5，有机酸浓度为96.5mM，氨氮浓度为4.5g/L。
反应器停止进料后，根据固定化菌剂投加情况，分为对照组(未投加菌剂)和实验组(投加菌剂)。菌剂投加15天后，实验组开始恢复产甲烷能力，最大乙酸消耗速率为9.9mM/d；对照组则在28天后，开始恢复产甲烷能力，最大乙酸消耗速率为6.8mM/d。
对照组和实验组均为三个厌氧消化反应器，以上数据为三组数据的平均值。
<实施例3>
本实施例对厌氧固定化菌剂的制备及应用做具体阐述。
在35℃条件下，分别利用纯菌培养技术培养不同的厌氧功能菌种，使其培养菌液OD600 为20。分别取180mL、270mL、450mL和180mL的解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌和热自养甲烷热杆菌的培养菌液进行混合，然后离心浓缩，离心力5000g，离心温度4℃，离心时间5min，获得功能菌群沉淀。
将此功能菌群沉淀加入到100mL质量分数为15％的聚乙烯醇水溶液中搅拌均匀，将搅拌后的聚乙烯醇水溶液滴入每升包含Na 2HPO 4，0.2mol；NaH 2PO 4，0.23mol；H 3BO 3，60g的第一缓冲溶液中，静置24h，形成聚乙烯醇凝胶珠；将此聚乙烯醇凝胶珠放入每升包含1.2mol的Na 2SO 4的第二缓冲溶液中，静置48h，形成直径为1.0cm的硫酸盐改性聚乙烯醇凝胶珠，此凝胶珠即为厌氧固定化菌剂。
处理对象为45.2L厌氧消化反应器，有效工作体积30.2L，运行温度为35℃，挥发性悬浮固体浓度为1g/L，pH为6.5，有机酸浓度为50.3mM，氨氮浓度为3.6g/L。
反应器停止进料后，根据固定化菌剂投加情况，分为对照组(未投加菌剂)和实验组(投加菌剂)。菌剂投加10天后，实验组开始恢复产甲烷能力，最大乙酸消耗速率为12.3mM/d；对照组则在15天后，开始恢复产甲烷能力，最大乙酸消耗速率为7.9mM/d。
<实施例4>
本实施例对厌氧固定化菌剂的制备及应用做具体阐述。
在35℃条件下，分别利用纯菌培养技术培养不同的厌氧功能菌种，使其培养菌液OD600为20。分别取50mL、50mL、125mL和75mL的解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌和热自养甲烷热杆菌的培养菌液进行混合，然后离心浓缩，离心力5000g，离心温度4℃，离心时间5min，获得功能菌群沉淀。
将此功能菌群沉淀加入到20mL质量分数为15％的聚乙烯醇水溶液中搅拌均匀，将搅拌后的聚乙烯醇水溶液滴入每升包含Na 2HPO 4，0.2mol；NaH 2PO 4，0.23mol；H 3BO 3，50g的第一缓冲溶液中，静置24h，形成聚乙烯醇凝胶珠；将此聚乙烯醇凝胶珠放入每升包含1.2mol的Na 2SO 4的第二缓冲溶液中，静置48h，形成直径为0.7cm的硫酸盐改性聚乙烯醇凝胶珠，此凝胶珠即为厌氧固定化菌剂。
选取六个2.5L的厌氧消化反应器作为处理对象，参数均为：有效工作体积1.5L，运行温度为35℃，挥发性悬浮固体浓度为4g/L，pH为6.8，有机酸浓度为100.6mM，氨氮浓度为6.5g/L。
反应器停止进料后，根据固定化菌剂投加情况，分为对照组(未投加菌剂)和实验组(投加菌剂)。菌剂投加6天后，实验组开始恢复产甲烷能力，最大乙酸消耗速率为13.2mM/d；对照组则在10天后，开始恢复产甲烷能力，最大乙酸消耗速率为9.8mM/d。
对照组和实验组均为三个厌氧消化反应器，以上数据为三组数据的平均值。
实施例的作用与效果
根据本发明的实施例所提供的厌氧固定化菌剂的制备方法及应用，选用四种不同的厌氧功能菌种，利用纯菌培养技术得到相应的培养菌液，然后按照一定体积比将四种培养菌液进行混合获得复合功能菌液，随后浓缩为功能菌群沉淀，再将功能菌群沉淀溶解在聚乙烯醇水溶液中，然后将该溶液滴入第一缓冲溶液中得到聚乙烯醇凝胶珠，将该凝胶珠放入含有硫酸盐的第二缓冲溶液中进行改性，得到硫酸盐改性聚乙烯醇凝胶珠，即厌氧固定化菌剂。厌氧固定化菌剂由聚乙烯醇水溶液凝胶化形成，为由薄膜包裹凝胶物形成的直径为0.5cm-1cm的圆形凝珠。该凝珠能够直接加入到厌氧消化反应器中进行厌氧消化，无需对设备进行改造， 既能够有效解除原位有效解除厌氧消化有机酸抑制又能够有效解除氨抑制条件下的有机酸积累。
解蛋白嗜热粪杆菌快速分解细胞碎屑、蛋白质和脂质；利用棕色嗜热产醋菌的同型乙酸氧化途径，将乙酸分解为二氧化碳及氢气；热自养甲烷热杆菌则进行氢营养型产甲烷过程，消耗氢气进而为棕色嗜热产醋菌创造有利的热力学环境；巴氏甲烷八叠球菌通过乙酸发酵型和氢营养型产甲烷途径，分别消耗乙酸和氢气。因此，混合菌群可以加速降低有机酸负荷，恢复厌氧消化反应器pH，进而解除有机酸抑制对反应器内部原有菌群的毒害作用，恢复原有菌群的功能活性；且避免了有机酸分离装置的使用、缓冲材料的添加和现有反应器的改造，降低了处理成本。并且解蛋白嗜热粪杆菌、棕色嗜热产醋菌、热自养甲烷热杆菌及巴氏甲烷八叠球菌均属于耐氨菌种，所以复合功能菌液能够解除氨抑制条件下的有机酸积累。由以上可知，该复合功能菌液不仅耐酸而且耐氨，适用范围广。
利用硫酸盐作为改性剂进行改性，克服了传统聚乙烯醇-硼酸凝胶珠无法干化保存的缺点。硫酸盐-硼酸内核具有足够的结构柔性和结构强度来支撑凝胶珠，以维持固定化菌剂的结构完整和对外力冲击的抗性。
同时，对外加混合功能菌剂进行固定化处理，而不是以游离的细胞形态悬浮于反应器中，可以使外加菌剂更好地定殖于反应器内部原有菌群，避免了外加菌剂随着反应出水而大量流失，进而无需长期补充外加菌剂；也提高了外加菌剂的细胞密度，增强了处理效果。
此外，改性凝珠能够被干化处理后保存，因此能够进行大规模生产和长途运输，以应用于不同区域的厌氧消化反应器，克服了普通凝胶珠无法干化，所以必须在厌氧反应器附近进行制备，并立马投入使用的缺点。
此外，根据厌氧消化反应器的主要物化参数，能够确定复合功能菌液的最小使用量；同时在明确的不同工况条件下，能够确定四种厌氧功能菌种的混合比例，可以经济有效地解除有机酸抑制。
厌氧消化反应器中的pH一般大于5，而本发明的实施例所制备的厌氧固定化菌剂适用于pH大于5(包含pH为5)的厌氧消化反应器，因此无需对厌氧消化反应器进行pH调整。
由于反应器中的有机酸的积累导致了酸抑制的形成，因此厌氧固定化菌剂与反应底物混合(即有机酸组分)均匀后，进行静置反应，降解有机酸，进而解除抑制。
每升第一缓冲溶液中包含0.15mol-0.2mol的Na 2HPO 4，0.2mol-0.25mol的NaH 2PO 4及50g-60g的H 3BO 3，每升第二缓冲溶液中包含1mol-1.5mol的Na 2SO 4，NaH 2PO 4有利于保持微生物细胞的稳态，H 3BO 3的加入形成了硼酸内核，而Na 2SO 4对硼酸内核进行改性，得到改性的凝胶珠。
当厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为30℃-43℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为2-1：3-1：5-1：2-1。当厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为50℃-65℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为3-1：4-1：2-1：4-1。当厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温 度为30℃-43℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为2-1：2-1：5-1：3-1。当厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为50℃-65℃时，复合功能菌液中的解蛋白嗜热粪杆菌：棕色嗜热产醋菌：巴氏甲烷八叠球菌及热自养甲烷热杆菌的体积比为3-1：3-1：4-1：5-1。这些比例的选择是通过实验数据得出，具体为通过模拟上述四种工况，并在每种工况条件下设置多组平行反应器，结合微生物测序分析，我们发现这四种微生物丰度较高，为优势微生物，因此我们选择这四种微生物的丰度比例作为混合体积比。如果某种微生物的体积低于这个比例，会导致菌剂中的互营关系被破坏，进而无法起到解除有机酸抑制的作用，例如：热自养甲烷热杆菌和巴氏甲烷八叠球菌的数量较低，则无法消耗解蛋白嗜热粪杆菌产生的氢气，改变热力学结果，进而对解蛋白嗜热粪杆菌造成热力学屏障，反应停止。高于这个比例，会造成菌剂的浪费，并且增加聚乙烯醇水溶液的使用量，经济性较差。因此，这些是根据菌群自身反应结果得到的，兼顾反应效果和经济性。
厌氧固定化菌剂在投入厌氧消化反应器前，进行活化12h-24h，活化可以恢复微生物菌剂的活性，以更好地解除酸抑制。
微生物反应对温度条件比较敏感，如果不预先在特定温度下培养厌氧功能菌种，则无法发挥固定化菌剂的作用，甚至造成菌剂中微生物的大量死亡。例如在35℃条件下培养的微生物菌剂，如果立马投放到55℃的反应器中，会造成更长的停滞期，以及菌剂中微生物的大量死亡，改变菌剂中的微生物比例，进而导致菌剂失效。因此，为达到最优处理效果，选择温度相同的培养条件，也就是厌氧功能菌种的培养温度与厌氧固定化菌剂在厌氧消化反应器的进行厌氧消化的温度相同。
由以上可知，本发明所提供的制备方法简单、能耗低，得到的固定化菌剂耐酸耐氨，可以显著增强厌氧消化反应器内部菌群对已积累有机酸的处理能力，且无需长期投加。与现有技术相比，效果稳定且成本低廉，无需对现有反应器进行大规模改造，解决了功能菌种流失和厌氧消化菌群自我恢复能力差的问题，可以有效解除有机垃圾和污水厌氧消化过程中的有机酸抑制以及氨抑制条件下的有机酸积累。
上述对实施例的描述是为便于该技术领域的普通技术人员理解和使用本发明。熟悉本领域技术的人员可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的修改都应该在本发明的保护范围之内。

Claims (10)

  1. 一种厌氧固定化菌剂的制备方法，其特征在于，包括以下步骤：
    步骤1，在一定温度下对四种不同的厌氧功能菌种分别进行厌氧培养得到相应的培养菌液，按照一定体积比将不同的所述培养菌液进行混合获得复合功能菌液；
    步骤2，将所述复合功能菌液进行浓缩获得功能菌群沉淀；
    步骤3，将所述功能菌群沉淀溶解在聚乙烯醇水溶液中得到功能菌群聚乙烯醇溶液，然后将所述功能菌群聚乙烯醇溶液滴入第一缓冲溶液中，得到聚乙烯醇凝胶珠；
    步骤4，将所述聚乙烯醇凝胶珠放入含有硫酸盐的第二缓冲溶液中，得到硫酸盐改性聚乙烯醇凝胶珠，即厌氧固定化菌剂，
    其中，所述厌氧功能菌种分别为解蛋白嗜热粪杆菌、棕色嗜热产醋菌、巴氏甲烷八叠球菌以及热自养甲烷热杆菌。
  2. 根据权利要求1所述的厌氧固定化菌剂的制备方法，其特征在于：
    其中，步骤3中，所述聚乙烯醇水溶液中聚乙烯醇的质量分数为10％-15％，
    步骤1中的所述复合功能菌液与步骤3中的所述聚乙烯醇水溶液的体积比为10：1-20：1。
  3. 根据权利要求1所述的厌氧固定化菌剂的制备方法，其特征在于：
    其中，每升所述第一缓冲溶液中包含0.15mol-0.2mol的Na 2HPO 4，0.2mol-0.25mol的NaH 2PO 4及50g-60g的H 3BO 3，
    每升所述第二缓冲溶液中包含1mol-1.5mol的Na 2SO 4，
    所述厌氧固定化菌剂的直径为0.5cm-1cm。
  4. 一种厌氧固定化菌剂，其特征在于，由权利要求1-3中任一项所述的厌氧固定化菌剂的制备方法制备得到，其中，所述厌氧固定化菌剂为由薄膜包裹凝胶物形成的圆形凝珠，所述圆形凝珠的直径为0.5cm-1cm。
  5. 一种如权利要求4所述的厌氧固定化菌剂在厌氧消化中的应用。
  6. 根据权利要求5所述的厌氧固定化菌剂在厌氧消化中的应用，其特征在于：
    其中，在一定温度下，利用纯菌培养技术将四种厌氧功能菌种分别培养得到相应的培养菌液，所述培养菌液的OD600为15-20，然后按照一定体积比将不同的所述培养菌液进行混合获得复合功能菌液，
    所述厌氧消化的具体操作为将所述厌氧固定化菌剂加入到厌氧消化反应器进行厌氧消化，所述厌氧功能菌种的培养温度与所述厌氧固定化菌剂在所述厌氧消化反应器中进行厌氧消化的温度相同，
    四种所述厌氧功能菌种分别为解蛋白嗜热粪杆菌、棕色嗜热产醋 菌、巴氏甲烷八叠球菌以及热自养甲烷热杆菌。
  7. 根据权利要求6所述的厌氧固定化菌剂在厌氧消化中的应用，其特征在于：
    其中，所述复合功能菌液的最小用量由下式计算得到：
    Figure PCTCN2021087449-appb-100001
    V 0：复合功能菌液的最小使用量(L)；
    C total：厌氧消化反应器内部有机酸总浓度(以乙酸根计，mM)；
    pH：厌氧消化反应器内部pH；
    C vs：厌氧消化反应器内部挥发性悬浮固体浓度(g/L)；
    V 1：厌氧消化反应器有效工作体积(L)。
  8. 根据权利要求6所述的厌氧固定化菌剂在厌氧消化中的应用，其特征在于：
    其中，当所述厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为30℃-43℃时，所述复合功能菌液中的所述解蛋白嗜热粪杆菌∶所述棕色嗜热产醋菌∶所述巴氏甲烷八叠球菌及所述热自养甲烷热杆菌的体积比为2-1∶3-1∶5-1∶2-1；
    当所述厌氧消化反应器内的氨氮浓度小于等于4g/L，温度为 50℃-65℃时，所述复合功能菌液中的所述解蛋白嗜热粪杆菌：所述棕色嗜热产醋菌：所述巴氏甲烷八叠球菌及所述热自养甲烷热杆菌的体积比为3-1：4-1：2-1：4-1。
  9. 根据权利要求6所述的厌氧固定化菌剂在厌氧消化中的应用，其特征在于：
    其中，当所述厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为30℃-43℃时，所述复合功能菌液中的所述解蛋白嗜热粪杆菌：所述棕色嗜热产醋菌：所述巴氏甲烷八叠球菌及所述热自养甲烷热杆菌的体积比为2-1：2-1：5-1：3-1；
    当所述厌氧消化反应器内的氨氮浓度为4g/L-7g/L，温度为50℃-65℃时，所述复合功能菌液中的所述解蛋白嗜热粪杆菌：所述棕色嗜热产醋菌：所述巴氏甲烷八叠球菌及所述热自养甲烷热杆菌的体积比为3-1：3-1：4-1：5-1。
  10. 根据权利要求6所述的厌氧固定化菌剂在厌氧消化中的应用，其特征在于：
    其中，将所述厌氧固定化菌剂干化后得到干化的凝珠，所述干化的凝珠在投入所述厌氧消化反应器前，进行活化12h-24h，活化时所用的营养液为利用纯菌培养技术培养所述厌氧功能菌种时所用的所述培养液。
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XUE RONG 士, HUANG GUOL ,GUO YAN : "Nitrogen and Phosphorus Removals by Algae and Bacterium Immobilized Using Modified PVA-sulfate Method", ENVIRONMENTAL POLLUTION & CONTROL, 31 October 2005 (2005-10-31), XP055888785, ISSN: 1001-3865 *

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