CN112029681A - Preparation of special liquid composite microbial inoculum for decomposing vinasse - Google Patents

Preparation of special liquid composite microbial inoculum for decomposing vinasse Download PDF

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CN112029681A
CN112029681A CN202010872338.1A CN202010872338A CN112029681A CN 112029681 A CN112029681 A CN 112029681A CN 202010872338 A CN202010872338 A CN 202010872338A CN 112029681 A CN112029681 A CN 112029681A
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trichoderma reesei
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左勇
何颂捷
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Sichuan Normal University
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Abstract

The invention belongs to the technical field of vinasse degradation, and particularly relates to a preparation method of a special liquid composite microbial inoculum for vinasse decomposition. The preparation method comprises the following steps: s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei; s2, primary seed culture; s3, secondary seed culture; s4, respectively inoculating the secondary seed solution of the Bacillus beijerinckii UN-5 and the secondary seed solution of the Trichoderma reesei obtained in the S3 to a sodium carboxymethylcellulose fermentation culture medium for culture; s5, mixing and culturing the Bacillus beiLeisi UN-5 microbial inoculum and the trichoderma reesei microbial inoculum according to a proportion to prepare the composite microbial inoculum. The liquid composite microbial inoculum belongs to a viable bacteria preparation, improves the activity of cellulase when the microbial inoculum is used for vinasse, improves the acid resistance of the composite microbial inoculum, has high substrate conversion efficiency, improves the substrate utilization rate under the combined action of bacteria and fungi in the composite microbial inoculum at different stages of vinasse decomposition, shortens the vinasse decomposition fermentation period, and improves the product quality.

Description

Preparation of special liquid composite microbial inoculum for decomposing vinasse
Technical Field
The invention belongs to the technical field of vinasse degradation, and particularly relates to a preparation method of a special liquid composite microbial inoculum for vinasse decomposition.
Background
Vinasse appears in a unique solid state fermentation mode and distillation production of solid state white spirit, and the annual yield of the vinasse in China reaches thousands of tons. The distiller's grains contain crude protein, crude starch, crude fat, crude fiber (including lignin, cellulose and hemicellulose), ashless and nitrogen-free extract, and in addition, the distiller's grains contain various enzymes, organic acids and a large amount of purine, pyrimidine and lipid compounds generated by autolysis of thallus. Research shows that the vinasse is utilized to produce the bio-organic fertilizer, and by the unique advantages of the bio-organic fertilizer, a bridge can be built for agricultural waste and crop production, and a sustainable development road with vinasse-bio-organic fertilizer-crops as a circulation mode is developed. However, due to the high acidity of the vinasse, crude vinasse fibers are difficult to degrade, so that the problems of long decomposition period, incomplete decomposition and the like of the vinasse are caused, and the utilization rate of the vinasse is low. This is not only a great waste of resources, but also a serious environmental pollution, and a sustainable pollution-free utilization of resources cannot be realized.
The biological organic fertilizer is a fertilizer which is prepared by compounding organic materials which are derived from microorganisms with specific functions, animal and plant residues, crop straws and the like and are subjected to innocent treatment and decomposition and has the effects of microbial fertilizer and organic fertilizer. The raw materials of the biological organic fertilizer and the feed mainly come from straws, bean pulp, cottonseed meal, rapeseed cakes, vinasse, vinegar residue, cassava residue, sugar residue, furfural residue, sawdust, kitchen waste, vegetable market tail and the like, and the raw materials all contain rich cellulose. Because the cellulose is not easy to degrade and utilize, the positivity of applying the bio-organic fertilizer in China at the present stage is not high, and the utilization rate is relatively low.
The traditional microbial inoculum is mostly a wild strain of a single strain, the wild strain has the problems of long growth cycle, low cellulase activity and the like, the vinasse contains a large amount of crude fibers, and the degradation of cellulose is a result of the combined action of multiple enzyme systems. The vinasse can experience a temperature rise period, a high temperature period and a rotting period in the decomposing process, the dominant strains in different stages are different, the traditional single microbial inoculum cannot meet the strain requirements in different stages, and the factors have great influence on the vinasse decomposing, so that the vinasse decomposing fermentation time is long, the decomposing degree is not enough, the prepared organic fertilizer is easy to cause secondary fermentation after application, and the phenomenon of burning roots of crops occurs. The acidity of the vinasse is high, crude fibers in the vinasse are difficult to degrade, and few composite bacteria agents specially applied to vinasse decomposition are used at present, so that the problems of long vinasse decomposition period, incomplete decomposition and the like are caused, and the utilization rate of the vinasse is low.
The vinasse belongs to a special acid environment, and poor acid resistance is a bottleneck of the current strain in the aspects of vinasse degradation and application, so that a complex microbial inoculum with high thermal stability and good acid resistance is required to be developed and applied to the aspects of vinasse degradation and decomposition, the problems of long decomposition period, incomplete decomposition and the like of the vinasse are solved, and the utilization rate of the vinasse is improved.
Disclosure of Invention
The invention aims to solve the technical problems and provides a special liquid compound bacterium agent for decomposing vinasse. The composite microbial inoculum belongs to a viable bacteria preparation, improves the activity of cellulase when the microbial inoculum is used for vinasse, improves the acid resistance of the composite microbial inoculum, has high substrate conversion efficiency, improves the substrate utilization rate under the combined action of bacteria and fungi in the composite microbial inoculum at different stages of vinasse decomposition, shortens the vinasse decomposition fermentation period, and improves the product quality.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
the special vinasse decomposing bacterium is Bacillus beiLeisi UN-5, is preserved in China center for type culture Collection in 6 months and 28 days in 2020, and has the preservation number of CCTCC NO: m2020236, the deposit address is: china, wuhan university, zip code: 430072, named Bacillus velezensis UN-5.
The Bacillus belgii UN-5 strain can be used for preparing a composite microbial inoculum for degrading vinasse.
The method for preparing the composite microbial inoculum by utilizing the Bacillus belgii UN-5 strain comprises the following specific steps:
s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei;
s2, primary seed culture: respectively inoculating Bacillus belgii UN-5 and Trichoderma reesei which are activated in S1 and have good growth vigor and large single colony to sodium carboxymethylcellulose liquid enrichment medium and beef extract protein peptone solutionIn a liquid medium, the density OD of the liquid culture medium540nmStopping culturing when the value reaches 0.6-0.8, and respectively obtaining a first-stage seed solution of Bacillus beiLeisi UN-5 and a first-stage seed solution of Trichoderma reesei;
s3, secondary seed culture: inoculating the primary seed liquid of the Bacillus belgii UN-5 and the primary seed liquid of the Trichoderma reesei obtained in the step S2 into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium respectively, and culturing for 24h at 37 ℃ and 150r/min to obtain a secondary seed liquid of the Bacillus belgii UN-5 and a secondary seed liquid of the Trichoderma reesei respectively;
s4, respectively inoculating the secondary seed liquid of the Bacillus belgii UN-5 and the secondary seed liquid of the Trichoderma reesei obtained in the step S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 180r/min for 24h, wherein when the effective viable count in each bacterial liquid exceeds 5 multiplied by 108Completing the culture in/mL to respectively obtain a Bacillus beiLeisi UN-5 microbial inoculum and a Trichoderma reesei microbial inoculum;
s5, mixing the Bacillus beiLeisi UN-5 microbial inoculum and the trichoderma reesei microbial inoculum according to a proportion, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
Preferably, in the step S5, the bacillus beijerinckii UN-5 microbial inoculum and the trichoderma reesei microbial inoculum are mixed in a volume ratio of 2:1 to 3:1, and more preferably in a volume ratio of 2.5: 1.
The culture medium adopted in the preparation method is as follows:
PDA culture medium: 20 wt% of potato, 2 wt% of glucose, 1.5-2 wt% of agar, natural pH, and sterilizing at 121 ℃ for 20 mi;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g，MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone, 0.5 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
The effective viable count in the composite microbial inoculum exceeds 109/ml。
The method for preparing the organic fertilizer by adopting the compound microbial inoculum comprises the following steps:
weighing a certain amount of dried distiller's grains or wet distiller's grains, adding auxiliary materials into the distiller's grains, and uniformly stirring; and spraying the liquid compound microbial inoculum into the mixture, adding urea and water, stirring again, and fermenting for 14d-16d at room temperature to obtain the organic fertilizer. Controlling the water content of the distiller's grains to be 46-56 wt% in the process, and naturally airing the distiller's grains if the water content is high; if the water content is low, water is added for wetting.
The auxiliary material is pulverized peanut shell (particle size is 60-100 meshes), the addition amount of pulverized peanut shell is 25% of the mass of distiller's grains, the addition amount of urea is 1.0g/kg in terms of distiller's grains, and the addition amount of water is preferably 40-50% of the moisture content of the fermented product.
The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing vinasse, the adding amount of the composite microbial inoculum is 2-4 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 109(ii)/g, organic matter content not less than 50%.
The maximum yield of the composite microbial inoculum prepared by the method is 150.4U.
The positive effects of the invention are as follows:
the liquid composite microbial inoculum belongs to a viable bacteria preparation, so that the cellulase activity of the microbial inoculum when the microbial inoculum is used for vinasse is improved, the acid resistance of the composite microbial inoculum is improved, the substrate conversion efficiency is high, the bacteria and fungi in the composite microbial inoculum jointly act at different stages of vinasse decomposition, the substrate utilization rate is improved, the vinasse decomposition fermentation period is shortened, and the product quality is improved.
The composite microbial inoculum prepared by the method has strong acid resistance, is suitable for the acid environment of the vinasse, greatly improves the enzymatic activity of the cellulase, shortens the degradation time of the vinasse, is beneficial to the decomposition of the vinasse, achieves the sustainable development of resources, and solves the problem of environmental pollution caused by the vinasse.
The liquid composite microbial inoculum special for vinasse decomposition is prepared by compounding the mutagenized vinasse degradation dominant bacteria, namely bacillus beili UN-5 and trichoderma reesei, so that the activity and the expression quantity of cellulase when the liquid composite microbial inoculum is applied to vinasse decomposition are improved, the composite microbial inoculum is better applied to degradation of acidic vinasse, and the utilization rate of the vinasse is improved.
Fourthly, compounding the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei in the composite microbial inoculum to provide dominant strains for different stages in the vinasse decomposing process; the prepared composite microbial inoculum is suitable for recycling vinasse in the aspects of acidity and medium-high temperature environment.
Description of the drawings:
FIG. 1 is a bar graph showing the results of acid resistance test
FIG. 2 is a histogram of effective viable count results of single strains and complex microbial agents
FIG. 3 is a histogram of single strain and complex microbial inoculum cellulase activity results
FIG. 4 is a histogram of the results of effective viable count of organic fertilizer
FIG. 5 shows the liquid complex microbial inoculum prepared in example 1
Detailed Description
The present invention will be described in further detail with reference to specific embodiments for making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples.
The special strain for decomposing the vinasse is Bacillus beilaisi UN-5 (UN-5 for short), is preserved in China Center for Type Culture Collection (CCTCC) in 6 months and 28 days in 2020, and has a preservation number of CCTCC NO: m2020236, accession number: china, wuhan university, zip code: 430072, named Bacillus velezensis UN-5.
The Bacillus belgii UN-5 strain can be used for preparing a composite microbial inoculum for degrading vinasse.
The fungus Trichoderma reesei is preferably the fungus Trichoderma reesei with the deposit number CICC 41027.
The method for preparing the composite microbial inoculum by utilizing the Bacillus belgii UN-5 strain comprises the following specific steps:
s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei;
s2, primary seed culture: respectively inoculating Bacillus belgii UN-5 and Trichoderma reesei which are activated in S1 and have good growth vigor and large single colony in sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD540nmStopping culturing when the value reaches 0.6-0.8, and respectively obtaining a first-stage seed solution of Bacillus beiLeisi UN-5 and a first-stage seed solution of Trichoderma reesei;
s3, secondary seed culture: inoculating the primary seed liquid of the Bacillus belgii UN-5 and the primary seed liquid of the Trichoderma reesei obtained in the step S2 into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium respectively, and culturing for 24h at 37 ℃ and 150r/min to obtain a secondary seed liquid of the Bacillus belgii UN-5 and a secondary seed liquid of the Trichoderma reesei respectively;
s4, respectively inoculating the secondary seed liquid of the Bacillus belgii UN-5 and the secondary seed liquid of the Trichoderma reesei obtained in the step S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 180r/min for 24h, wherein when the effective viable count in each bacterial liquid exceeds 5 multiplied by 108Completing the culture in/mL to respectively obtain a Bacillus beiLeisi UN-5 microbial inoculum and a Trichoderma reesei microbial inoculum;
s5, mixing the Bacillus beiLeisi UN-5 microbial inoculum and the trichoderma reesei microbial inoculum according to a proportion, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
Preferably, in the step S5, the bacillus beijerinckii UN-5 microbial inoculum and the trichoderma reesei microbial inoculum are mixed in a volume ratio of 2:1 to 3:1, and more preferably in a volume ratio of 2.5: 1.
The culture medium adopted in the preparation method is as follows:
PDA culture medium: 20 wt% of potato, 2 wt% of glucose, 1.5-2 wt% of agar, natural pH, and sterilizing at 121 ℃ for 20 mi;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g， MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone, 0.5 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
The effective viable count in the composite microbial inoculum exceeds 109And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing vinasse, the adding amount of the composite microbial inoculum is 2-4 wt%, and the number of effective viable bacteria of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 109The organic matter content is not less than 50 percent per gram.
Example 1:
the method for preparing the liquid composite microbial inoculum special for vinasse decomposition by using the special bacterium Bacillus belgii UN-5 for vinasse decomposition in the specific embodiment comprises the following specific steps:
s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei;
s2, primary seed culture: respectively inoculating Bacillus belgii UN-5 and Trichoderma reesei which are activated in S1 and have good growth vigor and large single colony in sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD540nmStopping culturing when the value reaches 0.6 to respectively obtain BelisBacillus UN-5 primary seed liquid and trichoderma reesei primary seed liquid;
s3, secondary seed culture: inoculating the primary seed liquid of the Bacillus belgii UN-5 and the primary seed liquid of the Trichoderma reesei obtained in the step S2 into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium respectively, and culturing for 24h at 37 ℃ and 150r/min to obtain a secondary seed liquid of the Bacillus belgii UN-5 and a secondary seed liquid of the Trichoderma reesei respectively;
s4, respectively inoculating the secondary seed solution of the Bacillus belgii UN-5 and the secondary seed solution of the trichoderma reesei obtained in the step S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 150r/min for 24h to respectively obtain a Bacillus belgii UN-5 microbial inoculum and a trichoderma reesei microbial inoculum;
s5, mixing the Bacillus beiLeisi UN-5 microbial inoculum and the Trichoderma reesei microbial inoculum according to the volume ratio of 2.5:1, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
PDA culture medium: 20 wt% of potato, 2 wt% of glucose and 1.5 wt% of agar, and sterilizing at 121 ℃ for 20mi under natural pH;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g， MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone, 0.5 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
The effective viable count in the composite microbial inoculum exceeds 109And/ml. The composite microbial inoculum can be used for wineIn the step of preparing the organic fertilizer by vinasse rotten cooking, the specific steps are as follows: weighing 50kg of waste wet distiller's grains (controlling the water content of the wet distiller's grains to be 50 +/-2 wt%, and if the water content exceeds 50 wt%, naturally drying the wet distiller's grains to control the water content within the range, wherein the original pH value of the wet distiller's grains is 5.0), adding 12.5kg of crushed peanut shells into the distiller's grains, and uniformly stirring. And spraying the prepared liquid composite microbial inoculum to a mixture of vinasse and peanut shells, then adding urea, stirring again, and fermenting for 15d to obtain the organic fertilizer. The inoculation amount of the compound bactericide is 2.5 wt% (based on the mass of vinasse), the addition amount of urea is 1.0g/kg, the C/N is controlled to be 26, and the organic matter content of the prepared organic fertilizer is 59.35%. The determination method of the organic matter is carried out according to 5.2 in the national standard NY 525-2012.
Example 2:
the method for preparing the compound bactericide by using the special bacterium Bacillus beiLeisi UN-5 for decomposing the vinasse in the specific embodiment comprises the following specific steps:
s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei;
s2, primary seed culture: respectively inoculating Bacillus belgii UN-5 and Trichoderma reesei which are activated in S1 and have good growth vigor and large single colony in sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD540nmStopping culturing when the value reaches 0.8, and respectively obtaining a primary seed solution of Bacillus beiLensis UN-5 and a primary seed solution of Trichoderma reesei;
s3, secondary seed culture: inoculating the primary seed liquid of the Bacillus belgii UN-5 and the primary seed liquid of the Trichoderma reesei obtained in the step S2 into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium respectively, and culturing for 24h at 37 ℃ and 150r/min to obtain a secondary seed liquid of the Bacillus belgii UN-5 and a secondary seed liquid of the Trichoderma reesei respectively;
s4, respectively inoculating the secondary seed solution of the Bacillus belgii UN-5 and the secondary seed solution of the trichoderma reesei obtained in the step S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 180r/min for 24 hours to respectively obtain a Bacillus belgii UN-5 microbial inoculum and a trichoderma reesei microbial inoculum;
s5, mixing the Bacillus beiLeisi UN-5 microbial inoculum and the Trichoderma reesei microbial inoculum according to the volume ratio of 2.5:1, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
PDA culture medium: 20 wt% of potato, 2 wt% of glucose and 2 wt% of agar, and sterilizing at the natural pH of 121 ℃ for 20 mi;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g， MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone, 0.5 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
The effective viable count in the composite microbial inoculum exceeds 109And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing vinasse (the preparation step is the same as the example 1), the inoculation amount of the composite microbial inoculum is 3 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 109The organic matter content is not less than 50 percent per gram.
Example 3:
the method for preparing the compound bactericide by using the special bacterium Bacillus beiLeisi UN-5 for decomposing the vinasse in the specific embodiment comprises the following specific steps:
s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei;
s2, primary seed culture: activated in S1 and grows wellThe single colony large Bacillus beleisi UN-5 and Trichoderma reesei are respectively inoculated in a sodium carboxymethylcellulose liquid enrichment medium and a beef extract peptone liquid medium, and when the liquid culture density OD540nmStopping culturing when the value reaches 0.7, and respectively obtaining a primary seed solution of Bacillus beiLensis UN-5 and a primary seed solution of Trichoderma reesei;
s3, secondary seed culture: inoculating the primary seed liquid of the Bacillus belgii UN-5 and the primary seed liquid of the Trichoderma reesei obtained in the step S2 into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium respectively, and culturing for 24h at 37 ℃ and 150r/min to obtain a secondary seed liquid of the Bacillus belgii UN-5 and a secondary seed liquid of the Trichoderma reesei respectively;
s4, respectively inoculating the secondary seed solution of the Bacillus belgii UN-5 and the secondary seed solution of the trichoderma reesei obtained in the step S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 160r/min for 24 hours to respectively obtain a Bacillus belgii UN-5 microbial inoculum and a trichoderma reesei microbial inoculum;
s5, mixing the Bacillus beiLeisi UN-5 microbial inoculum and the Trichoderma reesei microbial inoculum according to the volume ratio of 2.5:1, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
PDA culture medium: 20 wt% of potato, 2 wt% of glucose and 2 wt% of agar, and sterilizing at the natural pH of 121 ℃ for 20 mi;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g， MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone, 0.5 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
The effective viable count in the composite microbial inoculum exceeds 109And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing vinasse (the preparation step is the same as the example 1), the inoculation amount of the composite microbial inoculum is 4 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 109The organic matter content is not less than 50 percent per gram.
If the composite microbial inoculum in the embodiment is used in the step of preparing the organic fertilizer by decomposing in the distiller's dried grains (the preparation steps are the same as those in embodiment 1), only water is needed to be added into the distiller's dried grains for wetting until the specified water content is reached.
Comparative example 1:
a composite microbial preparation was prepared in the same manner as in example 1 except that Bacillus subtilis UN-5 (Bacillus licheniformis UN-78) was used in example 1 instead of Bacillus subtilis CICC 10829.
The effective viable count of the prepared composite microbial inoculum is 108-0.8×109And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing the distillers' grains, the inoculation amount of the composite microbial inoculum is 2 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is less than 109The organic matter content is 40-42 percent per gram.
Comparative example 2:
the preparation of the complex microbial inoculum was carried out in the same manner as in example 1 except that the culture conditions in the step S5 were changed.
S5, mixing the bacillus agent and the trichoderma reesei agent according to the volume ratio of 3:1, and culturing for 24h under the conditions of pH 7.0 and 150r/min to prepare the composite microbial agent.
The effective viable count of the prepared composite microbial inoculum is 108-0.8×109And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing the distillers' grains, the inoculation amount of the composite microbial inoculum is 2 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is less than 109The organic matter content is 40-42 percent per gram.
Comparative example 3:
the preparation of the complex microbial inoculum was carried out in the same manner as in example 1 except that the medium was changed.
PDA culture medium: 20 wt% of potato, 2 wt% of glucose and 1.5 wt% of agar, and sterilizing at 121 ℃ for 20mi under natural pH;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: CMC-Na 0.5 wt%, peptone 0.2 wt%, KH2PO4 4g，MgSO4·7H2O0.05 wt%, natural pH, 121 deg.C sterilizing for 20 min;
beef extract peptone liquid medium: 0.2 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, and sterilizing at 121 ℃ for 20min at natural pH;
sodium carboxymethylcellulose fermentation medium: 0.5 wt% of CMC-Na, 1.5 wt% of peptone, 0.2 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, 121 deg.C sterilizing for 20 min.
The prepared composite microbial inoculum has effective viable count less than 109And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by vinasse rotting, the adding amount of the composite microbial inoculum is 2 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is less than 109(iv)/g, organic matter content less than 42%.
Comparative example 4:
the preparation of the complex microbial inoculum was carried out in the same manner as in example 1, and only one additional strain was added, namely the complex microbial inoculum was prepared by changing Bacillus belgii UN-5 and Trichoderma reesei into Bacillus belgii UN-5, Bacillus (CICC 10829) and Trichoderma reesei.
The effective viable count in the prepared composite microbial inoculum exceeds 108And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by vinasse rotting, the adding amount of the composite microbial inoculum is 3 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 108The organic matter content is about 40 percent per gram.
Experiment 1:
1. determination of antagonism
And adopting a plate confronting method to cross and streak the suspension of the UN-5 and the Trichoderma reesei on a sodium carboxymethylcellulose culture medium pairwise, culturing at the constant temperature of 37 ℃ for 2 days, wherein the cross joint of the two strains on the culture medium does not appear the phenomenon of bacterial colony reduction or disappearance, and the result shows that the two strains do not have antagonistic action.
2. Determination of acid resistance
Respectively preparing sodium carboxymethylcellulose culture media with different pH values (2.0, 3.0, 4.0, 5.0 and 6.0), respectively inoculating 5mL of bacterial suspension to be detected on the sodium carboxymethylcellulose culture media, and culturing at constant temperature of 37 ℃ for 24 hours. OD measurement before and after incubation at 540nm with an ultraviolet spectrophotometer540nmValue of OD before and after culture540nmThe difference indicates the acid resistance of the strain (the larger the difference is, the stronger the acid resistance is), and the result indicates that the two strains have better acid resistance and are suitable for decomposing vinasse.
4. Determination of effective viable count
Diluting the bacterial suspension to 10 degrees by 10 times of gradient dilution method-10Take 10-9、10-8And 10-7Gradient 0.1mL is respectively coated on sodium carboxymethylcellulose culture media, each concentration is made into 3 parallels, and is compared with a flat plate coated with sterile water (the effective viable count of the composite microbial inoculum is expressed by the maximum order of magnitude). The effective viable count of the composite microbial inoculum reaches 10.3-12.4 multiplied by 108/ml。
Experiment 2:
preparation conditions of composite microbial inoculum
Suspensions of the strains UN-5 and Trichoderma reesei were prepared separately.
1. Influence of strain ratio on microbial inoculum enzyme activity:
preparing single-strain bacterial liquid, respectively taking 120ml and 120ml, 80ml and 160ml, 60ml and 180ml, 160ml and 80ml, 180ml and 60ml of UN-5 and trichoderma reesei bacterial liquid, mixing according to the proportion of 1:1, 1:2, 1:3, 2:1 and 3:1, culturing for 2d under the condition of 37 ℃ and 150r/min, and determining the activity of cellulase of the composite bacterial agent by using a DNS method, wherein the optimal proportion is 2: 1-3: 1.
2. Influence of the pH of the culture medium on the enzyme activity of the microbial inoculum:
adjusting the pH of the fermentation medium to be 2.0, 3.0, 4.0, 5.0 and 6.0 respectively, mixing 160ml of UN-5 bacterial liquid and 80ml of Trichoderma reesei bacterial liquid according to a volume ratio of 2:1, culturing for 2d at 37 ℃ and 150r/min, taking the mixed bacterial liquid, and determining the cellulase activity of the composite microbial inoculum by using a DNS method to obtain the optimal culture pH of 4.0-5.0.
3. Influence of incubation time on enzyme activity:
preparing single strain suspension, mixing 160ml of UN-5 strain liquid and 80ml of trichoderma reesei strain liquid according to a ratio of 2:1, culturing for 12h, 24h, 36h, 48h and 60h respectively at 37 ℃ and 150r/min, and determining the activity of cellulase by using a DNS method to obtain the optimal culture time range of 24-32 h.
Thirdly, optimizing preparation conditions of the compound microbial inoculum
According to a single-factor test result and related documents, the strain ratio, the pH value of a culture medium and the culture time have large influence on the cellulase activity of the composite microbial inoculum, according to an orthogonal test principle, the strain ratio, the pH value of the culture medium and the culture time are selected as independent variables on the basis of the single-factor test, the cellulase activity of the composite microbial inoculum is used as an index, and an SPSS19.0 data processing system is adopted to perform general linear model analysis on orthogonal test data to obtain an optimal preparation condition: the ratio is 2.5:1, the culture pH is 5.0 and the culture time is 28 h.
Preparation of liquid composite bacterial agent
A special liquid compound microbial agent for decomposing vinasse comprises UN-5 and trichoderma reesei.
The UN-5 is Bacillus beiLeisi bred by mutagenesis in a laboratory; trichoderma reesei is CICC 41027.
The preparation method of the special liquid compound microbial agent for decomposing the vinasse comprises the following steps:
(1) solid slant culture: selecting bacteria UN-5 and fungus Trichoderma reesei with high cellulase yield, respectively inoculating the UN-5 and the Trichoderma reesei into a PDA culture medium and a beef extract peptone culture medium, and culturing at 37 ℃ for 2d to fully activate the strains;
(2) first-order seed culture: the UN-5 strain which grows well and has large single colony after being activated in the step (1) is mixed withRespectively inoculating Trichoderma reesei in sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid culture medium, culturing at 37 deg.C and 150r/min, measuring thallus concentration every 4 hr at wavelength of 540nm, and determining liquid culture density OD540nmStopping culturing when the value reaches 0.6-0.8, and respectively obtaining first-grade seed solutions;
(3) secondary seed culture: taking 12.5-25ml of the primary seed liquid of UN-5 and trichoderma reesei obtained in the step (2), respectively inoculating the primary seed liquid and the primary seed liquid into 250ml of sodium carboxymethylcellulose liquid culture medium and 250ml of beef extract peptone liquid culture medium according to the inoculation amount of 5-10%, and culturing for 24h under the conditions of 37 ℃ and 150r/min to respectively obtain secondary seed liquid;
(4) taking 25ml of the UN-5 obtained in the step (3) and the secondary seed solution of the trichoderma reesei according to the inoculation amount of 10%, respectively inoculating the 25ml of the UN-5 and the seed solution into 250ml of a sodium carboxymethylcellulose fermentation culture medium, and carrying out high-density fermentation culture at 37 ℃ and 180r/min for 24h to obtain microbial agents A and B; wherein the microbial inoculum A is UN-5 microbial inoculum, and the microbial inoculum B is Trichoderma reesei microbial inoculum;
(5) 250ml of microbial inoculum A and 100ml of microbial inoculum B are mixed and cultured according to the proportion of 2.5:1, cultured for 28h under the conditions of pH 5.0 and 150r/min, and compounded to prepare the special decomposing agent for decomposing vinasse suitable for the vinasse environment.
(6) Storage conditions are as follows: the prepared special decomposing agent for decomposing the vinasse is stored for 7-12 days at the temperature of 2-8 ℃.
(7) Protocol the media used:
PDA culture medium: 20 wt% of potato, 2 wt% of glucose, 1.5-2 wt% of agar, natural pH, and sterilizing at 121 ℃ for 20 mi;
beef extract peptone medium: 0.3 wt% of beef extract, 1 wt% of peptone, 0.5 wt% of NaCl and 2 wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g， MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone, 0.5 wt% of beef extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
(8) The effective viable count of the composite microbial inoculum exceeds 109The composite microbial inoculum is applied to the preparation of organic fertilizer by vinasse rotting and cooking, the adding amount of the composite microbial inoculum is 2-4%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 109The organic matter content is not less than 50 percent per gram.
(9) The concentration of the bacteria in the bacterial liquid is measured by adopting a photoelectric turbidimetry method: 2mL of the bacterial solution was aspirated, and OD was measured at a wavelength of 540nm540nmThe value is obtained.
(10) The cellulase activity was determined by the DNS method: sucking 2mL of bacterial liquid, centrifuging at 12000 r/min for 5min, taking supernatant, namely taking 0.5mL of crude enzyme liquid as the crude enzyme liquid, adding 1.5mL of 1% CMC buffer solution, carrying out water bath at 50 ℃ for 30min, adding 3mL of DNS solution, carrying out boiling water bath for 10min, rapidly cooling, and then carrying out volume fixing to 10 mL. Adding 0.5mL of inactivated crude enzyme solution to control group instead of crude enzyme solution, and measuring OD at 540nm wavelength under the same conditions540nmThe value is obtained. Enzyme activity calculation formula H ═ D.V1·C/(T·V2) Calculating the cellulase activity of the strain, wherein the cellulase activity is expressed in U, namely IU/mL.
Experiment 2:
1. composition comparison of fresh distillers grains with dried distillers grains
The fresh distillers grains had an initial pH of 3.52, and as shown in Table 1, the fresh distillers grains contained mainly water, nitrogen-free extract, crude fiber, crude protein, crude starch, and the like, and the dried distillers grains contained mainly nitrogen-free extract, crude fiber, crude protein, crude starch, and the like.
TABLE 1 comparison of the composition of fresh distillers grains with dried distillers grains
Figure BDA0002651506080000171
(2) Comparison of acid resistance
The two strains used in the composite microbial inoculum prepared by the method can still grow under the condition of pH 3.0, the strains in the traditional microbial inoculum can normally grow in the environment with pH 4.0 and the growth vigor is weaker than that of UN-5 and Trichoderma reesei, and the acid resistance of the two strains used in the composite microbial inoculum is stronger than that of the strains used in the traditional microbial inoculum. See in particular fig. 1.
As can be seen from FIG. 1, the UN-5 strain is more acid-resistant than Trichoderma reesei and can grow at pH 2.0, whereas Trichoderma reesei is more sensitive to strong acids and cannot grow normally at pH 2.0. Although the two strains can still grow under low pH, the growth of the two strains is obviously inhibited compared with the environment with higher pH and weaker acidity. The strains in the traditional live bacteria preparation start to grow slowly under the condition that the pH value is 4.0, and the strains do not grow well in the composite bacteria agent under the acidic condition. The pH value of the original vinasse is about 3.0-4.0, and acid resistance tests show that UN-5 and Trichoderma reesei have stronger acid resistance and are suitable for being applied to the decomposition fermentation of the vinasse.
(3) Comparison of effective viable count
UN-5 and trichoderma reesei liquid are mixed according to the proportion of 2.5:1(UN-5: trichoderma reesei), and the mixture is cultured for 28 hours under the condition of pH 5.0 to obtain the composite microbial agent, namely the effective viable count of the special liquid composite microbial agent for decomposing the vinasse is greatly improved, and the result is shown in figure 2.
As shown in figure 2, the effective viable count of UN-5 is 12.5 hundred million/g, because Trichoderma reesei is larger than a single fungus body of mould, and the effective viable count in the same volume is less than that of UN-5, the effective viable count in the prepared composite microbial agent is only 10.3 hundred million/g, the effective viable count of the traditional single microbial agent is 3.1 hundred million/g, and the effective viable count in the composite microbial agent is 4.03 times that in the traditional single microbial agent
(4) Comparison of cellulase Activity of Single Strain and Complex microbial inoculum
The cellulase activity of the composite microbial inoculum prepared by optimizing the preparation conditions is greatly improved compared with that of the traditional microbial inoculum, and the result is shown in figure 3.
As shown in figure 3, the cellulase activity of the prepared composite microbial inoculum reaches up to 150.4U, which is higher than that of single-strain cellulase of UN-5 and Trichoderma reesei, the cellulase activity of the traditional microbial inoculum is only 34.6U, and the enzyme activity of the composite microbial inoculum is improved by 3.35 times compared with that of the traditional microbial inoculum, which indicates that the cellulase activity of the composite microbial inoculum is greatly improved.
(5) Comparison of effective viable count of vinasse bio-organic fertilizer prepared by using composite microbial inoculum
The effective viable bacteria number of the organic fertilizer after the vinasse is decomposed and fermented by the liquid compound microbial inoculum is increased by 5.94 times compared with the organic fertilizer by applying the traditional single microbial inoculum. The results are shown in FIG. 4
As shown in figure 4, the effective viable count of the organic fertilizer prepared by vinasse decomposed and composted after the compound microbial inoculum is applied reaches 12.5 hundred million/g, which is far more than 6.1 and 2.6 hundred million/g of single strains UN-5 and trichoderma reesei, while the effective viable count of the vinasse decomposed by the traditional microbial inoculum is only 1.8 hundred million/g. The compound microbial inoculum is higher in effective viable count in the biological organic fertilizer prepared by decomposing the distiller's grains, and has an effect of improving the fertilizer efficiency of the organic fertilizer prepared by decomposing and fermenting the distiller's grains.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A special strain for decomposing vinasse is characterized in that: the strain is Bacillus licheniformis UN-5 and Bacillus velezensis UN-5, is preserved in China center for type culture Collection in 6 months and 28 days in 2020, and has a preservation number of CCTCC NO: m2020236.
2. The application of the special bacteria for decomposing the vinasse as claimed in claim 1, wherein the special bacteria comprise: the strain is used for preparing a composite microbial inoculum for degrading vinasse.
3. The method for preparing the complex microbial inoculum by using the strain as claimed in claim 1 is characterized by comprising the following steps:
s1, respectively carrying out activation culture on the Bacillus beiLeisi UN-5 and the fungus Trichoderma reesei;
s2, primary seed culture: respectively inoculating the Bacillus belgii UN-5 and Trichoderma reesei with good growth and single colony after activation in S1In a sodium carboxymethylcellulose liquid enrichment medium and a beef extract peptone liquid medium, when the liquid culture density OD540nmStopping culturing when the value reaches 0.6-0.8, and respectively obtaining a primary seed solution of Bacillus beiLeisi UN-5 and a primary seed solution of Trichoderma reesei;
s3, secondary seed culture: inoculating the primary seed solution of the Bacillus belgii UN-5 and the primary seed solution of the trichoderma reesei obtained in the step S2 into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium respectively, and culturing for 24 hours at the temperature of 37 ℃ and at the speed of 150r/min to obtain a secondary seed solution of the Bacillus belgii UN-5 and a secondary seed solution of the trichoderma reesei respectively;
s4, respectively inoculating the secondary seed solution of the Bacillus belgii UN-5 and the secondary seed solution of the Trichoderma reesei obtained in the step S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 180r/min for 24h to respectively obtain a Bacillus belgii UN-5 microbial inoculum and a Trichoderma reesei microbial inoculum;
s5, mixing the Bacillus beiLeisi UN-5 microbial inoculum and the trichoderma reesei microbial inoculum according to a proportion, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
4. The method for preparing a complex bacterial agent according to claim 3,
PDA culture medium: 20 wt% of potato, 2 wt% of glucose, 1.5-2 wt% of agar, natural pH, and sterilizing at 121 ℃ for 20 mi;
beef extract peptone medium: beef extract 0.3 wt%, peptone 1 wt%, NaCl 0.5 wt%, agar 2 wt%, pH 7.0, sterilizing at 121 deg.C for 20 min;
sodium carboxymethylcellulose liquid enrichment medium: 1 wt% of CMC-Na, 0.3 wt% of peptone and KH2PO4 4g，MgSO4·7H2O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20 min;
beef extract peptone liquid medium: 0.3 wt% of beef extract, 1 wt% of peptone and 0.5 wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20 min;
sodium carboxymethylcellulose fermentation medium: 1 wt% of CMC-Na, 1 wt% of peptone and 1 wt% of cattle0.5 wt% of meat extract and KH2PO4 2g，MgSO4·7H2O 0.05wt％，(NH4)2SO40.2 wt%, natural pH, sterilizing at 121 deg.C for 20 min.
5. The method for preparing a complex microbial inoculum according to claim 3, which comprises the following steps: in the step S5, the Bacillus belgii UN-5 microbial inoculum and the Trichoderma reesei microbial inoculum are mixed according to the volume ratio of 2.5: 1.
6. The method for preparing a complex microbial inoculum according to claim 3, which comprises the following steps: the effective viable count in the prepared composite microbial inoculum exceeds 109/ml。
7. The complex microbial inoculum prepared by the method of claim 3, which is characterized in that: the composite microbial inoculum is used in the step of preparing the organic fertilizer by decomposing vinasse, the access amount of the composite microbial inoculum is 2-4 wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 109The organic matter content is not less than 50 percent per gram.
8. The method for preparing the organic fertilizer by adopting the composite microbial inoculum as claimed in claim 7 is characterized by comprising the following steps: weighing a certain amount of dried distiller's grains or wet distiller's grains, adding auxiliary materials into the distiller's grains, and uniformly stirring; spraying the liquid composite microbial inoculum into the mixture, adding urea and water, stirring again, and fermenting at room temperature for 14d-16d to obtain the organic fertilizer.
9. The method of claim 8, wherein: the auxiliary material is crushed peanut shells, the addition amount of the crushed peanut shells is 25% of the mass of the vinasse, the addition amount of the urea is 1.0g/kg, and the addition amount of the water is such that the moisture content of the fermentation product is 40-50%.
10. The method of claim 8, wherein: the water content of the dried distiller's grains or the wet distiller's grains is controlled to be 46-56 wt%.
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CN115777443A (en) * 2022-12-05 2023-03-14 安徽大学 Method for cultivating oyster mushroom by using distiller's grains

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