CN113729110B - High-efficiency low-cost pretreatment combined solid state fermentation method for biomass material and application of biomass material in single-cell protein feed production - Google Patents
High-efficiency low-cost pretreatment combined solid state fermentation method for biomass material and application of biomass material in single-cell protein feed production Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a biomass material treatment method combining high-efficiency low-cost pretreatment and solid state fermentation, wherein continuous steam explosion with variable temperature stage can realize effective separation of cellulose, hemicellulose and lignin in biomass, and the solution is neutral without detoxification and direct fermentation, so that the energy consumption is greatly reduced; in the subsequent solid state fermentation process of biological materials, the bred Trichoderma reesei strain and Aspergillus niger strain capable of efficiently utilizing lignocellulose are added to secrete cellulase and beta-glucosidase outwards respectively in the growth process of utilizing biomass materials, and the two enzyme systems are mutually cooperated to efficiently degrade cellulose in lignocellulose agricultural wastes into fermentable sugar which can be utilized by thalli. The concentration of fermentable sugar in the culture medium can be reduced effectively by adding yeast, and the feedback inhibition effect of glucose on Aspergillus niger/Trichoderma reesei cellulase can be reduced effectively, so that the secretion of Aspergillus niger/Trichoderma reesei cellulase can be improved, and the enzymolysis efficiency of straw can be accelerated.
Description
Technical Field
The invention relates to the field of biomass treatment and feed, in particular to a high-efficiency low-cost pretreatment method for biomass materials and application of the pretreatment method in single-cell protein feed production.
Background
It is estimated that the global production of agricultural waste is about 22 million tons (kishaore, v., bhandari, p.m., p.gupta, p. (2004) Biomass Energy technologies for rural infrastructure andvillage power-opportunities and challenges in the context of global climate change concepts, energy Policy, 32,801-810.doi:10.1016/S0301-4215 (03) 00002-8), and that the total amount of agricultural straw in the largest agricultural world in China is 7.8 million tons, which contains about 4.5 million tons of standard coal, which is 60% of the total amount of organic waste resources in China, is not effectively utilized, resulting in great waste and environmental problems (Wu, z., zhang, m., wang, l. (2013) Biomass digestibility is predominantly afected bythree factors of wall polymer features distinctive in wheat accessions and rice polypeptides, biotechnology for Biofuels, 6 (1), 183; qu, y.b., fang, x. (2018) Fungal cellulolytic enzymes: microbial production and application, spring angle). According to the statistical data of China, at present, crop straws in China are taken as feed and industrial raw materials, most of the crop straws are taken as primary fuel in rural areas, the utilization efficiency is extremely low, and serious environmental pollution is caused. The high-value utilization of agricultural wastes becomes an important direction of sustainable development of economy and society in China.
The production of corn stalks, which are agricultural wastes, is up to 3.6 hundred million tons per year, the annual yield of corncobs is more than 1 hundred million tons, and the yield of bagasse is nearly 1 hundred million tons. Corn stalks contain substantially the same total energy as ordinary grains and also contain a number of nutrients beneficial to the growth and development of livestock and poultry. Is the largest carbohydrate resource which is not fully utilized and is a precious biological resource. However, with the rapid development of corn and sugarcane planting in recent years, the treatment of agricultural wastes with two features of one north and one south has become a great difficulty. On the other hand, the shortage of traditional agricultural protein raw material resources is a primary problem restricting the development of the feed industry and animal husbandry. Soybeans are important traditional protein raw materials, the import amount of the soybeans is about 9000 ten thousand tons each year, and two thirds of the import amount is used for a feed part (Yin Jie, liu Gongna, li Tiejun (2019) of protein feed resource shortage state and solution in China, proc. Natl. Acad. Sci. Of China, 34 (01), 97-101). The phenomenon of human and animal contending for food is further aggravated due to the shortage of food in China, and the food safety is seriously threatened. The method is significant for development and utilization of straw resources in the face of food shortage and waste of potential agricultural waste feed resources. In recent years, corn stalks are widely applied to the field of silage and direct field fertilizer. The defect of silage straw feed is that the limit of low protein content of corn straw cannot be changed, and the protein requirement of animals cannot be met by single use. The straw fermented feed still stays in a laboratory or a small production scale, and the crude protein content in the straw fermented feed can be improved to 16-25%, but the amino acid content is only about 12% generally. Corn cob and bagasse resources are developed into primary feed, and the digestibility is very low. The agricultural wastes are utilized to produce feed protein by a biosolid fermentation method, the nutrition quality of the straw feed is effectively improved by the characteristics of high protein content and excellent amino acid ratio, and the digestion utilization rate of the livestock and poultry to the protein feed from the agricultural wastes is improved, so that the immunity of the livestock and poultry is improved. The large-scale biosynthesis of feed proteins from agricultural wastes is the best way for low-cost healthy cultivation, and the large-scale production of feed proteins from agricultural wastes is an ideal way for solving the problem of comprehensive utilization of agricultural wastes. Therefore, the single-cell product developed by utilizing agricultural wastes in the prior art has low true protein content and low cost, and the single-cell protein fermented by the agricultural wastes still has no large-scale industrialized production, so that the substitution of the traditional agricultural protein can not be realized.
The main reasons are two points, namely, most of agricultural wastes are lignocellulose materials, the anti-degradation barrier is relatively intractable, the anti-nutritional factors are still in existence, and how to develop a high-efficiency low-cost pretreatment process and equipment to deal with large-scale treatment of straws;
the agricultural waste raw material straw has complex components, nonuniform structure, tight combination among the components and strong degradation resistance. At present, the straws are converted into high-value products, and pretreatment is an indispensable process. Hemicellulose and lignin components can be partially removed through pretreatment, the crystallinity of cellulose is reduced, the contact area of cellulase and a substrate is increased, and the accessibility of the cellulase is improved, so that the efficiency of degrading cellulose by enzyme is improved.
In general, pretreatment is classified into biological pretreatment, physical pretreatment, and chemical pretreatment, or a combination of these methods. The physical pretreatment mainly changes biomass into smaller particles through mechanical means such as crushing, grinding and the like of the biomass, increases the contact area of enzyme on cellulose, and can also partially break the crystalline structure of the cellulose. However, the mechanical pulverization method has a disadvantage of high energy consumption. The chemical pretreatment is to remove hemicellulose and lignin in the lignocellulose raw material by using an added catalyst under high temperature and high pressure, or to change the compact structure of the raw material so as to be beneficial to enzymolysis of cellulase.
Acid-base pretreatment tends to be highly corrosive, toxic and dangerous, requires high corrosion resistance of the reaction vessel, and is also recovered after use for economical reasons, so that practical use thereof is small. The pretreatment of the organic solvent is to break the internal nodes of hemicellulose and lignin under the catalysis of inorganic acid by means of the organic solvent or the aqueous solution of the organic solvent so as to realize the separation of cellulose, lignin and hemicellulose; in order to be economically feasible and not to inhibit the next enzymatic hydrolysis and fermentation process, the organic solvent must be evaporated, concentrated and recovered after the reaction is completed, and the operation steps are relatively complicated. The biological pretreatment method has the advantages of low energy consumption and environmental friendliness, but has the defect of low degradation speed. Currently, the only pretreatment technology that can be industrially applied is the steam explosion pretreatment technology. The steam explosion is to steam the plant material in high temperature and high pressure steam to produce some acid matters to degrade hemicellulose into soluble sugar, and to soften lignin and degrade lignin partially to weaken the adhesion between fiber, and to make the material separate into single fiber cell via the adiabatic expansion process to form work. The prior commercial pretreatment processes often damage lignocellulose deconstruction by increasing pretreatment intensity and reduce enzyme consumption. However, these pretreatment techniques and processes generally have the problems of high energy consumption and cost, and simultaneously generate a large amount of enzymolysis inhibitors and three wastes, so that most pretreatment processes are difficult to realize industrialized popularization and application (Yue nationality Jun.2014. Cellulose ethanol engineering theory, chemical industry Press).
Meanwhile, the mixed bacteria fermentation process cannot be regulated and controlled under the industrialized rough condition, the existing production strains of the microbial mycoprotein of the old generation in China have the problems of low straw conversion efficiency, small accumulation of essential amino acids, insufficient protein synthesis capacity and the like, the quality of the feed produced by solid state fermentation of agricultural wastes is relatively low, the laboratory result cannot be reproduced, and the method is also a factor for restricting the large-scale production of the straw fermented feed. The establishment of an optimizable and reproducible solid state fermentation technology, the definition of a dynamic correlation mode between the activity of thalli and other factors, and the monitoring of the process characteristic change when the fodder strain is used for solid state fermentation of straws are key to realizing industrialization of straw fodder proteins.
Disclosure of Invention
Based on the above requirements, the invention develops an efficient low-cost pretreatment method for biomass materials and application of the pretreatment method in single-cell protein feed production.
The invention provides a biomass material treatment method combining efficient low-cost pretreatment and solid state fermentation, which is characterized by comprising the following steps:
1) A biomass material digestion stage; one or more of corn stalks, corn cobs and bagasse which are biomass materials of agricultural wastes are soaked in sulfuric acid solution, and the temperature and the pressure are raised;
2) And (3) a biomass material blasting stage: depressurizing and blasting;
3) Respectively taking the biomass material pretreated by the method, adding water, and adding trichoderma reesei and aspergillus niger spore liquid for solid state fermentation;
wherein, the collection number of the aspergillus niger is: CGMCC No.22465; the preservation number of the trichoderma reesei is as follows: CGMCC No.21470.
In the preferred embodiment, the temperature and pressure rise in the step 1) are carried out by heating to 140-180 ℃ and preserving the heat for 4-10min, and the pressure is 0.8-1.0Mpa.
In another preferred embodiment, the pressure is reduced to 0.4-0.7Mpa by pressure-reducing blasting in step 2), and the same temperature is maintained for 4-8min.
In a further preferred embodiment, the solid state fermentation in step 3) is carried out at 25-35℃for 24-36 hours.
Further preferably, after the solid state fermentation of 3), the candida utilis seed solution is added and the cultivation is continued at 25-35 ℃ for 100-150 hours.
In other embodiments, in step 1), the biomass material is prepared according to 1: and (4) after the solid-liquid ratio of 4-10, heating and pressurizing.
In other embodiments, in step 3), the pretreated biomass material is added with water in a ratio of 1:2 to 3 as a medium for solid state fermentation.
Preferably, in step 3), the spore liquids of trichoderma reesei and aspergillus niger are preferably mixed according to a ratio of 1:1, wherein the spore concentration of Aspergillus niger and Trichoderma reesei is 1×10 7 From one/mL to 2X 10 9 And each mL.
The invention also provides application of the biomass material treatment method in single-cell protein feed production. Further preferably, after the fermentation of the 3 rd step is completed, a step of separating crude protein is further included.
The invention develops a biomass material treatment method combining efficient low-cost pretreatment and solid state fermentation, which comprises two aspects of a variable-temperature steam explosion pretreatment method and solid state fermentation of agricultural wastes. The continuous steam explosion method with variable temperature can realize the effective separation of cellulose, hemicellulose and lignin in biomass, the xylose removal rate reaches more than 90%, and the xylose solution is neutral and can be used for downstream direct fermentation without detoxification; and the energy consumption is reduced to 40% of the current blasting technology, the inhibitor is reduced to below 10% of the current steam explosion pretreatment technology, and the pretreatment cost is less than 200 yuan/ton of raw materials. In the fermentation process of biological materials, trichoderma reesei and Aspergillus niger strains which are bred by the inventor and can efficiently utilize straw are added to secrete cellulase and beta-glucosidase outwards respectively in the growth process of utilizing biomass materials, and the two enzyme systems are mutually cooperated to efficiently degrade cellulose of corn straw and convert the cellulose into fermentable sugar which can be utilized by thalli. After the yeast seed liquid is added, the yeast can efficiently utilize the fermentable sugar to be converted into mycoprotein; the addition of the yeast can effectively reduce the concentration of fermentable sugar in the culture medium, effectively reduce the feedback inhibition effect of glucose on the Aspergillus niger/Trichoderma reesei cellulase, thereby being beneficial to improving the secretion of the Aspergillus niger/Trichoderma reesei cellulase and accelerating the enzymolysis efficiency of the straw.
Detailed Description
The invention is illustrated by the following detailed description in order to provide a better understanding of the invention. But do not constitute a limitation of the invention.
Example 1: establishment of the Process
A first stage, a cooking stage; the agricultural waste biomass material corn stalk, corn cob and bagasse are cut into small pieces of about 10cm by a pulverizer, and the small pieces are respectively processed according to the following steps: 8 (solid-liquid ratio), soaking in 0.5% dilute sulfuric acid solution, maintaining at 160deg.C for 6min, and under 0.9Mpa;
and a second stage, namely a blasting stage: after cooking, the pressure is reduced to 0.6Mpa, and the temperature is kept at 160 ℃ for 6min.
And comparing the composition change of the biomass material before and after the steam explosion pretreatment.
TABLE 1 composition variation after steam explosion pretreatment of various agricultural waste biomass materials
Example 2: production of single cell proteins
Biomass materials (including bagasse, corn stover and corn cob) subjected to steam explosion pretreatment are used for producing single-cell proteins.
Trichoderma reesei/Aspergillus niger spore liquid: taking Aspergillus niger 60B-3DW and Trichoderma reesei A2H, respectively streaking PDA plate, culturing at 30deg.C for 5d, eluting spores with sterile water, filtering out mycelium with cell filter, counting with blood cell counting plate, and regulating spore concentration of Aspergillus niger and Trichoderma reeseiThe degrees are all 1.2×10 8 And each mL.
Wherein, the strain Aspergillus niger Aspergillus niger B-3DW is preserved in China general microbiological culture Collection center with the accession number: CGMCC No.22465, the preservation time is: 2021, 07, 05, deposit unit address: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute. The strain is a mutant strain with greatly improved beta-glucosidase activity, which is bred by taking Aspergillus niger 3.316 (preserved in China national academy of sciences microbiological culture Collection center, CGMCC No. 3.316) as an original strain and adopting an ARTP mutagenesis and liquid drop microfluidic high-throughput screening technology.
Trichoderma reesei Trichoderma reesei A H, deposited in China general microbiological culture Collection center, accession number: CGMCC No.21470, the preservation time is: 2021, 03, 17, deposit unit address: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.
Steam explosion pretreatment of corn stover, bagasse and corn cob according to the method of example 1 was performed separately, and water was added at a water-to-water ratio of 1:2.5 as a solid state fermentation medium. Trichoderma reesei and Aspergillus niger spore liquid are added to the surface of a culture medium for solid state fermentation of a steam explosion pretreatment biomass material according to the inoculation amount of 5% (i.e. the ratio of 1:1), and the culture is carried out for 24-36 hours at the temperature of 30 ℃.
After the above-mentioned Aspergillus niger and Trichoderma reesei hydrolyzed biomass material for 24 hours, candida utilis seed solution was added thereto in an amount of 10% added thereto, and solid-state fermentation was continued at 30℃for 120 hours.
The corn stover, bagasse, and corn cob pretreated by steam explosion according to example 1 were subjected to solid state fermentation for 7 days, respectively, and after drying the stover, the total nitrogen content and the amino acid content of the single cell protein product were determined. The crude protein content of the straw after fermentation was calculated as crude protein = solid total nitrogen content 6.25. The result shows that the crude protein content of the steam exploded straw after 7 days of solid state fermentation is about 28.25%; the crude protein content of the steam exploded bagasse is about 30.25%, and the crude protein content of the steam exploded corncob is about 29.88%.
Crude protein GB/T6432-1994 method for measuring crude protein in feed. The amino acid content is determined by adopting an A200 amino Nova amino acid analyzer and referring to national standard GB/T18246-2000 of the people's republic of China. As can be seen from the following table, the amino acid content of the steam exploded straw after 7 days of solid state fermentation is about 21.21%; the amino acid content of the steam exploded bagasse is about 23.45%, and the amino acid content of the steam exploded corncob is about 22.76%.
TABLE 2 analysis of single cell products produced by solid state fermentation of post-steam explosion biomass materials
| Material | Corn stalk | Bagasse | Corn cob |
| Total nitrogen content (%) | 4.52 | 4.84 | 4.78 |
| Crude protein content (%) | 28.25 | 30.25 | 29.88 |
| Amino acid content (%) | 21.21 | 23.45 | 22.76 |
The invention starts from agricultural wastes, synthesizes novel single-cell protein feed with high protein, full amino acid and compound probiotics functions by using a bioconversion method through key technologies of integrated optimization biomass refining, such as a variable-temperature steam explosion pretreatment technology, a mixed bacteria solid state fermentation technology and the like. The steam explosion pretreatment method and the solid state fermentation method can be duplicated and amplified, can be applied in industrialization, and have good industrialization prospect. After the bagasse, the corn stalks and the corn cobs which are subjected to steam explosion pretreatment are used as substrates, and the specific trichoderma reesei, the specific aspergillus niger and the candida utilis are subjected to mixed fermentation, the crude protein content in single cell proteins is more than 28%, and the amino acid content is more than 21%, so that the agricultural waste resources are changed into valuables, the traditional agricultural protein production mode is subverted, the self-sufficiency of protein raw materials in China is promoted, and the important way of the sustainable development of the recycling economy and agriculture in China is realized. Meanwhile, the two problems of low utilization rate of agricultural wastes and shortage of traditional agricultural proteins in China are solved accurately, the comprehensive productivity and competitiveness of agriculture in China are improved, and the method has important strategic significance.
Claims (10)
1. The biomass material treatment method combining high-efficiency low-cost pretreatment and solid state fermentation is characterized by comprising the following steps of:
1) A biomass material digestion stage; soaking one or more of corn stalks, corn cobs and bagasse which are agricultural waste biomass materials in sulfuric acid solution, and heating and pressurizing, wherein the heating and pressurizing are carried out by heating to 140-180 ℃, the heat preservation time is 4-10min, and the pressure is 0.8-1.0Mpa;
2) And (3) a biomass material blasting stage: depressurizing and blasting, wherein the pressure is reduced to 0.4-0.7Mpa by the depressurizing and blasting, and the same temperature is maintained for 4-8min;
3) Respectively taking the biomass material pretreated by the method, adding water, and adding trichoderma reesei and aspergillus niger spore liquid for solid state fermentation;
wherein, the collection number of the aspergillus niger is: CGMCC No.22465; the preservation number of the trichoderma reesei is as follows: CGMCC No.21470.
2. The method according to claim 1, wherein the temperature and pressure increase in step 1) is performed by heating to 160℃for 6min at a pressure of 0.9MPa.
3. The method for treating biomass material according to claim 1, wherein in step 2), the pressure is reduced by pressure-reducing blasting to 0.6Mpa and the same temperature is maintained for 6min.
4. The method for processing biomass material according to claim 1, wherein in step 3), the solid state fermentation is performed at 25 to 35 ℃ for 24 to 36 hours.
5. A method of biomass material treatment according to any one of claims 1 to 3, wherein after solid state fermentation of 3) candida utilis seed solution is added and cultivation is continued for 100-150 hours at 25-35 ℃.
6. The method of biomass material treatment according to claim 1, wherein in step 1), biomass material is produced according to 1: and (4) after the solid-liquid ratio of 4-10, heating and pressurizing.
7. The method for treating biomass material according to claim 1, wherein in step 3), the pretreated biomass material is added with water in a ratio of 1:2 to 3 as a medium for solid state fermentation.
8. The method of biomass material treatment according to claim 1, wherein in step 3), trichoderma reesei and aspergillus niger spore liquid are mixed according to a ratio of 1:1, wherein the spore concentration of Aspergillus niger and Trichoderma reesei is 1×10 7 From one/mL to 2X 10 9 And each mL.
9. Use of the biomass material treatment method according to any one of claims 1 to 8 in single cell protein feed production.
10. The use according to claim 9, further comprising the step of isolating the crude protein after the fermentation of step 3) is completed.
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