CN112029681B - 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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CN112029681B
CN112029681B CN202010872338.1A CN202010872338A CN112029681B CN 112029681 B CN112029681 B CN 112029681B CN 202010872338 A CN202010872338 A CN 202010872338A CN 112029681 B CN112029681 B CN 112029681B
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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 Bacillus beiLeisi UN-5 and trichoderma reesei; s2, first-stage seed culture; s3, secondary seed culture; s4, respectively inoculating the secondary seed solution of the Bacillus beiLeisi 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 vinasse decomposition
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 output 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 besides, the distiller's grains contain various enzymes, organic acids and a large amount of purine, pyrimidine and lipid compounds generated by thallus autolysis. 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 obtained by taking specific functional microorganisms, animal and plant residues, crop straws and the like as sources and are subjected to harmless treatment and decomposition and has the effects of both the microbial fertilizer and the 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 uses a plurality of wild strains of a single strain, the wild strains have the problems of long growth period, 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 a plurality of enzyme systems. The vinasse can experience a heating period, a high-temperature period and a decomposition period in the decomposition 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 decomposition, so that the vinasse decomposition fermentation time is longer, the decomposition 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 at present, few composite microbial inoculums specially applied to vinasse decomposition are provided, 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 provide a special liquid composite microbial inoculum for decomposing vinasse aiming at the technical problems. 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 strain for decomposing the vinasse is Bacillus beleisi UN-5, is preserved in China center for type culture Collection in 6 months and 28 days of 2020, and has the 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 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 Bacillus beiLeisi UN-5 and trichoderma reesei;
s2, primary seed culture: respectively inoculating the activated Bacillus beijerinckii UN-5 and Trichoderma reesei in S1 into sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD 540nm Stopping 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: taking the primary seed solution of the Bacillus belgii UN-5 and the primary seed solution of the trichoderma reesei obtained in the step (2), respectively inoculating the primary seed solution of the Bacillus belgii UN-5 and the primary seed solution of the trichoderma reesei into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium again, and culturing for 24 hours at 37 ℃ and 150r/min to respectively obtain a secondary seed solution of the Bacillus belgii UN-5 and a secondary seed solution of the trichoderma reesei;
s4, respectively inoculating the secondary seed liquid of the Bacillus beiLeisi UN-5 and the secondary seed liquid of the trichoderma reesei obtained in the S3 into a sodium carboxymethylcellulose fermentation culture medium, performing high-density fermentation culture at 37 ℃ and 150-180r/min for 24h, and when the number of effective viable bacteria in each bacterial liquid exceeds 5 multiplied by 10 8 Completing 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 volume ratio of the bacillus beijerinckii UN-5 microbial inoculum to the trichoderma reesei microbial inoculum is 2 to 1, and further preferably, the mixing is performed according to the volume ratio of 2.5.
The culture medium adopted in the preparation method is as follows:
PDA culture medium: 20wt% of potato, 2wt% of glucose, 1.5-2 wt% of agar, natural pH, and sterilizing at 121 ℃ for 20mi;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
The effective viable count in the composite microbial inoculum exceeds 10 9 /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; 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 46-56wt% 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 access amount of the composite microbial inoculum is 2-4wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 10 9 The organic matter content is not less than 50 percent per gram.
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, the cellulase activity of the microbial inoculum when being 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 vinasse, greatly improves the enzymatic activity of 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 Trichoderma reesei fungus is preferably Trichoderma reesei fungus with a deposit number of 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 Bacillus beiLeisi UN-5 and trichoderma reesei;
s2, first-class seedsCulturing: respectively inoculating the Bacillus belief UN-5 and Trichoderma reesei in S1 which are activated and have good growth vigor and large single colony into a sodium carboxymethylcellulose liquid enrichment medium and a beef extract peptone liquid medium, and when the liquid culture density OD is obtained 540nm Stopping 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: taking the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei obtained in the step S2, respectively inoculating the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium, and culturing for 24h at 37 ℃ and 150r/min to respectively obtain the secondary seed solution of the Bacillus beiLeisi UN-5 and the secondary seed solution of the trichoderma reesei;
s4, respectively inoculating the secondary seed liquid of the Bacillus beiLeisi UN-5 and the secondary seed liquid of the trichoderma reesei obtained in the S3 into a sodium carboxymethylcellulose fermentation culture medium, performing high-density fermentation culture at 37 ℃ and 150-180r/min for 24h, and when the number of effective viable bacteria in each bacterial liquid exceeds 5 multiplied by 10 8 Completing the culture per 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 volume ratio of the bacillus beijerinckii UN-5 microbial inoculum to the trichoderma reesei microbial inoculum is 2 to 1, and further preferably, the mixing is performed according to the volume ratio of 2.5.
The culture medium adopted in the preparation method is as follows:
PDA culture medium: 20wt% of potato, 2wt% of glucose, 1.5-2 wt% of agar, natural pH, and 20mi of sterilization at 121 ℃;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
The effective viable count in the composite microbial inoculum exceeds 10 9 And/ml. The composite microbial inoculum is applied to the step of preparing the organic fertilizer by decomposing vinasse, the access amount of the composite microbial inoculum is 2-4wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 10 9 The organic matter content is not less than 50 percent per gram.
Example 1:
the method for preparing the special liquid composite microbial inoculum for decomposing the vinasse by utilizing the special bacterium Bacillus belief UN-5 for decomposing the vinasse in the specific embodiment comprises the following specific steps of:
s1, respectively carrying out activation culture on Bacillus beiLeisi UN-5 and trichoderma reesei;
s2, first-level seed culture: respectively inoculating the activated Bacillus beijerinckii UN-5 and Trichoderma reesei in S1 into sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD 540nm Stopping culturing when the value reaches 0.6, and respectively obtaining a primary seed solution of Bacillus beiLensis UN-5 and a primary seed solution of Trichoderma reesei;
s3, secondary seed culture: taking the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei obtained in the step S2, respectively inoculating the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium, and culturing for 24h at 37 ℃ and 150r/min to respectively obtain the secondary seed solution of the Bacillus beiLeisi UN-5 and the secondary seed solution of the trichoderma reesei;
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 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 a volume ratio of 2.5 to 1, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
PDA culture medium: 20wt% of potato, 2wt% of glucose and 1.5wt% of agar, and sterilizing at 121 ℃ for 20mi under natural pH;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, with the pH of 7.0, and sterilizing at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
The effective viable count in the composite microbial inoculum exceeds 10 9 And/ml. The composite microbial inoculum is used in the step of preparing the organic fertilizer by decomposing vinasse, and the specific steps are as follows: weighing 50kg of 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 50wt%, naturally drying the wet distiller's grains to control the water content within the range, and controlling the original pH value of the wet distiller's grains to be 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 complex microbial inoculum is 2.5wt% (based on vinasse)By mass), the adding amount of the 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 composite microbial inoculum by using the special bacterium Bacillus belgii UN-5 for decomposing the vinasse in the specific embodiment comprises the following specific steps:
s1, respectively carrying out activation culture on Bacillus beiLeisi UN-5 and trichoderma reesei;
s2, primary seed culture: respectively inoculating the activated Bacillus beijerinckii UN-5 and Trichoderma reesei in S1 into sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD 540nm Stopping 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: taking the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei obtained in the step S2, respectively inoculating the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium, and culturing for 24h at 37 ℃ and 150r/min to respectively obtain the secondary seed solution of the Bacillus beiLeisi UN-5 and the secondary seed solution of the trichoderma reesei;
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 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 volume ratio of 2.5 to 1, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
PDA culture medium: 20wt% of potato, 2wt% of glucose and 2wt% of agar, and sterilizing at 121 ℃ for 20mi under natural pH;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
The effective viable count in the composite microbial inoculum exceeds 10 9 And/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 that of the example 1), the inoculation amount of the composite microbial inoculum is 3wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 10 9 The organic matter content is not less than 50 percent per gram.
Example 3:
the method for preparing the composite microbial inoculum by using the special bacterium Bacillus belgii UN-5 for decomposing the vinasse in the specific embodiment comprises the following specific steps:
s1, respectively carrying out activation culture on Bacillus beiLeisi UN-5 and trichoderma reesei;
s2, primary seed culture: respectively inoculating the Bacillus belief UN-5 and Trichoderma reesei in S1 which are activated and have good growth vigor and large single colony into a sodium carboxymethylcellulose liquid enrichment medium and a beef extract peptone liquid medium, and when the liquid culture density OD is obtained 540nm Stopping 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: taking the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei obtained in the step S2, respectively inoculating the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium, and culturing for 24h at 37 ℃ and 150r/min to respectively obtain the secondary seed solution of the Bacillus beiLeisi UN-5 and the secondary seed solution of the trichoderma reesei;
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 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 to 1, and culturing for 28h under the conditions of pH 5.0 and 150r/min to prepare the composite microbial inoculum.
PDA culture medium: 20wt% of potato, 2wt% of glucose and 2wt% of agar, and sterilizing at 121 ℃ for 20mi under natural pH;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
The effective viable count in the composite microbial inoculum exceeds 10 9 And (4) the concentration is/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 4wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 10 9 Per g, the organic content is not less than 50 percent.
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 (CICC 10829) was used instead of Bacillus beleisi UN-5 in example 1.
The effective viable count of the prepared composite microbial inoculum is 10 8 -0.8×10 9 And/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 2wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is less than 10 9 Per gram, the organic content is 40-42 percent.
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.
The effective viable count of the prepared composite microbial inoculum is 10 8 -0.8×10 9 And/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 2wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is less than 10 9 The organic matter content is 40-42 percent per gram.
Comparative example 3:
the complex microbial inoculum was prepared in the same manner as in example 1, except that the medium was changed.
PDA culture medium: 20wt% of potato, 2wt% of glucose and 1.5wt% of agar, and sterilizing at 121 ℃ for 20mi under natural pH;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: CMC-Na 0.5wt%, peptone 0.2wt%, KH 2 PO 4 4g，MgSO 4 ·7H 2 0.05wt% of O, natural pH, sterilizing at 121 ℃ for 20min;
beef extract peptone liquid medium: 0.2wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, sterilizing at 121 ℃ for 20min at natural pH;
sodium carboxymethylcellulose fermentation medium: 0.5wt% of CMC-Na, 1.5wt% of peptone, 0.2wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH,121 deg.C sterilizing for 20min.
The prepared composite microbial inoculum has effective viable count less than 10 9 And/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 2wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is less than 10 9 (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 10 8 And/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 3wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 10 8 The 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
Preparing sodium carboxymethylcellulose culture medium with different pH values (2.0, 3.0, 4.0, 5.0, 6.0), respectively, inoculating 5mL of bacterial suspension to be tested on carboxymethyl celluloseCulturing on sodium acetate medium at 37 deg.C for 24 hr. OD measurement before and after incubation at 540nm with an ultraviolet spectrophotometer 540nm Value, OD before and after culture 540nm The 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 times by using a 10-fold gradient dilution method -10 Take 10 out -9 、10 -8 And 10 -7 Gradient 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 10 8 /ml。
Experiment 2:
1. preparation conditions of complex microbial inoculum
Suspensions of strains UN-5 and Trichoderma reesei were prepared, respectively.
1. The influence of the strain ratio on the enzyme activity of the microbial inoculum is as follows:
preparing single-strain bacterial liquid, mixing 120ml and 120ml, 80ml and 160ml, 60ml and 180ml, 160ml and 80ml, 180ml and 60ml of UN-5 and Trichoderma reesei bacterial liquid according to the following ratio of 1, 2, 1, 3, 1 and 3, culturing for 2d at 37 ℃ under 150r/min, and determining the cellulase activity of the composite bacterial agent by using a DNS method, wherein the optimal ratio is 2.
2. Influence of the pH of the culture medium on the enzyme activity of the microbial inoculum:
adjusting the pH of a 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, culturing for 2d at 37 ℃ under the condition of 150r/min, taking the mixed bacterial liquid, and measuring 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 bacterial suspension, mixing 160ml of UN-5 bacterial liquid and 80ml of trichoderma reesei bacterial liquid according to a ratio of 2 to 1, culturing for 12h, 24h, 36h, 48h and 60h under the conditions of 37 ℃ and 150r/min, respectively, and measuring the activity of cellulase by using a DNS method to obtain the optimal culture time range of 24-32 h.
3. Preparation condition optimization of complex 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 mixture ratio is 2.5.
4. 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 in 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: respectively inoculating the UN-5 strain and the Trichoderma reesei strain which are activated in the step (1) and have good growth vigor and large single colony in a sodium carboxymethylcellulose liquid enrichment medium and a beef extract peptone liquid medium, culturing at 37 ℃ and 150r/min, measuring the concentration of the strain every 4 hours at the wavelength of 540nm, and determining the OD (OD) when the density of the liquid culture strain is 540nm Stopping culturing when the value reaches 0.6-0.8, and respectively obtaining first-stage 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 UN-5 obtained in the step (3) and the secondary seed solution of trichoderma reesei according to the inoculation amount of 10%, respectively inoculating the 25ml of UN-5 and the seed solution into 250ml of sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 150-180r/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 to 1, and are cultured for 28 hours under the conditions of pH 5.0 and 150r/min, and the special decomposing agent for decomposing vinasse suitable for the vinasse environment is prepared by compounding.
(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 media used:
PDA culture medium: 20wt% of potato, 2wt% of glucose, 1.5-2 wt% of agar, natural pH, and sterilizing at 121 ℃ for 20mi;
beef extract peptone medium: beef extract 0.3wt%, peptone 1wt%, naCl 0.5wt%, agar 2wt%, pH 7.0, sterilizing at 121 deg.C for 20min;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
(8) The effective viable count of the composite microbial inoculum exceeds 10 9 The composite microbial inoculum is applied to the preparation of organic fertilizer by decomposing vinasse, the inoculation 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 10 9 The organic matter content is not less than 50 percent per gram.
(9) Bacterial liquidThe concentration of the medium thallus is measured by adopting a photoelectric turbidimetry method: 2mL of the bacterial solution was aspirated, and OD was measured at a wavelength of 540nm 540nm The value is obtained.
(10) The cellulase activity was determined by the DNS method: sucking 2mL of bacterial liquid, centrifuging for 5min at 12 000r/min, taking the supernatant, namely taking 0.5mL of crude enzyme liquid as the crude enzyme liquid, adding 1.5mL of 1-percent 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 diluting to 10mL. 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 conditions 540nm The value is obtained. Enzyme activity calculation formula H = D.V 1 ·C/(T·V 2 ) The cellulase activity of the strain is calculated and expressed in U, i.e. 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 GDA0002752459180000171
(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 viable bacteria preparation start to grow slowly only under the condition that the pH value is 4.0, and the strains in the traditional viable bacteria preparation do not grow well 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 strong acid resistance and are suitable for being applied to the decomposition fermentation of the vinasse.
(3) Comparison of effective viable count
The UN-5 and the trichoderma reesei liquid are mixed according to the proportion of 2.5 (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 inoculum is only 10.3 hundred million/g, the effective viable count of the traditional single microbial inoculum is 3.1 hundred million/g, and the effective viable count in the composite microbial inoculum is 4.03 times of that in the traditional single microbial inoculum
(4) Comparison of cellulase Activity of Single Strain and Complex microbial inoculum
The cellulase activity of the complex 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, the enzyme activity of the composite microbial inoculum is improved by 3.35 times compared with that of the traditional microbial inoculum, and the cellulase activity of the composite microbial inoculum is improved greatly.
(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 using the liquid compound microbial inoculum is increased by 5.94 times compared with the organic fertilizer using 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 decomposing and composting the vinasse by applying the composite microbial inoculum 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 prepared by decomposing and composting the vinasse by applying the traditional microbial inoculum is only 1.8 hundred million/g. The composite microbial inoculum is higher in effective viable count in the biological organic fertilizer prepared by decomposing the vinasse, and has an effect of improving the fertilizer efficiency of the organic fertilizer prepared by decomposing and fermenting the vinasse.
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 liquid composite microbial inoculum for decomposing vinasse is characterized in that: the liquid composite microbial agent comprises a Bacillus subtilis UN-5 microbial agent and a Trichoderma reesei microbial agent, wherein the Bacillus subtilis UN-5 is classified and named as Bacillus velezensis UN-5 and is preserved in a China Center for Type Culture Collection (CCTCC) within 6-28 months in 2020, and the preservation number is CCTCC NO: m2020236.
2. The use of the liquid complex microbial inoculum according to claim 1, wherein: the microbial inoculum is used for degrading vinasse.
3. The method for preparing the complex microbial inoculum by the strain as claimed in claim 1, which is characterized by comprising the following steps:
s1, respectively carrying out activation culture on Bacillus beiLeisi UN-5 and trichoderma reesei;
s2, primary seed culture: respectively inoculating the activated Bacillus beijerinckii UN-5 and Trichoderma reesei in S1 into sodium carboxymethylcellulose liquid enrichment medium and beef extract peptone liquid medium, and when the liquid culture density OD 540nm Stopping 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: taking the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei obtained in the step S2, respectively inoculating the primary seed solution of the Bacillus beiLeisi UN-5 and the primary seed solution of the trichoderma reesei into a sodium carboxymethylcellulose liquid culture medium and a beef extract peptone liquid culture medium, and culturing for 24h at 37 ℃ and 150r/min to respectively obtain the secondary seed solution of the Bacillus beiLeisi UN-5 and the secondary seed solution of the trichoderma reesei;
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 S3 into a sodium carboxymethylcellulose fermentation culture medium, and performing high-density fermentation culture at 37 ℃ and 150-180r/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,
beef extract peptone medium: 0.3wt% of beef extract, 1wt% of peptone, 0.5wt% of NaCl and 2wt% of agar, wherein the pH is 7.0, and the beef extract is sterilized for 20min at 121 ℃;
sodium carboxymethylcellulose liquid enrichment medium: 1wt% of CMC-Na, 0.3wt% of peptone and KH 2 PO 4 4g，MgSO 4 ·7H 2 O0.03 wt%, pH 6.0, sterilizing at 121 deg.C for 20min;
beef extract peptone liquid medium: 0.3wt% of beef extract, 1wt% of peptone and 0.5wt% of NaCl, wherein the pH is 7.0, and the beef extract is sterilized at 121 ℃ for 20min;
sodium carboxymethylcellulose fermentation medium: 1wt% of CMC-Na, 1wt% of peptone, 0.5wt% of beef extract and KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.05wt％，(NH 4 ) 2 SO 4 0.2wt%, natural pH, sterilizing at 121 deg.C for 20min.
5. The method for preparing the complex microbial inoculum according to claim 3, which is characterized in that: 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.
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 10 9 /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 2-4wt%, and the effective viable count of the organic fertilizer prepared by adding the composite microbial inoculum is not less than 10 9 The 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 according to claim 7 is characterized by comprising the following steps of: 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 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.
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 46-56wt%.
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