CN113729110A - Efficient low-cost pretreatment and solid-state fermentation combined method for biomass material and application of method in production of single-cell protein feed - Google Patents
Efficient low-cost pretreatment and solid-state fermentation combined method for biomass material and application of method in production of single-cell protein feed Download PDFInfo
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- 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
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- 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
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- 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
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Abstract
The invention discloses a biomass material treatment method combining high-efficiency low-cost pretreatment with solid state fermentation, wherein the biomass material treatment method can realize effective separation of cellulose, hemicellulose and lignin in biomass by continuous steam explosion with step temperature change, 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 the biological material, the selected Trichoderma reesei and Aspergillus niger strains capable of efficiently utilizing lignocellulose are added to secrete cellulase and beta-glucosidase to the outside of cells respectively in the growth process of utilizing the biological material, and the two enzyme systems are mutually cooperated to efficiently degrade cellulose in agricultural wastes of the lignocellulose and convert the cellulose into fermentable sugar which can be utilized by thalli. And the subsequent addition of the yeast can effectively reduce the concentration of fermentable sugar in the culture medium, and 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.
Description
Technical Field
The invention relates to the field of biomass treatment and feed, in particular to a high-efficiency low-cost pretreatment method of a biomass material and application of the method in single-cell protein feed production.
Background
It is estimated that about 22 million tons of agricultural wastes (Kishore, V., Bhand, P. M., P. guide, P. (2004). Biomass Energy technologies for road in front and bottom of plants and grains in the context of biological chemistry, 32,801 well 810. doi:10.1016/S0301-4215(03) 00002-8) are produced globally every year, and our country is the largest agricultural straw total of 7.8 tons, which contains about 4.5 million tons of standard coal, which accounts for 60% of the total organic waste resources, and most of them are not effectively utilized, causing huge waste and environmental problems (Wu, Z., Zhang, M., Wang, L. 3. biological wastes, 2013. biological wastes and biological wastes, 6(1), 183; qu, Y.B., Fang, X. (2018). Fungal cellulosic enzymes, Microbial production and application, Springer nature). According to the statistical data of China, the crop straws in China are mostly used as primary fuel in rural areas except for being used as feed and industrial raw materials, so that the utilization efficiency is extremely low, and the serious environmental pollution is caused. The high-value utilization of agricultural wastes becomes an important direction for the sustainable development of economy and society in China.
The agricultural wastes, namely corn straws, can be produced by 3.6 million tons every year, the annual output of corn cobs is more than 1 million tons, and the annual output of bagasse is nearly 1 million tons. The corn stalks contain the total energy basically equivalent to that of common grains and also contain a plurality of nutrient substances beneficial to the growth and development of livestock and poultry. Is the maximum amount of carbohydrate resources which are not fully developed and utilized, and is a very precious biological resource. However, with the rapid development of corn and sugarcane planting in recent years, the treatment of the agricultural wastes with two characteristics of one south and one north becomes a big problem. On the other hand, the shortage of the conventional agricultural protein raw material resources is a primary problem restricting the development of the feed industry and the animal husbandry. Soybeans are important traditional protein raw materials, the imported quantity of the soybeans is about 9000 ten thousand tons every year, and two thirds of the imported quantity of the soybeans are used for feed parts (yijie, liuhong nan, li iron army. (2019) the current situation and the solution of the shortage of protein feed resources in China, the periodical of the academy of sciences of China, 34(01), 97-101.). Due to the shortage of feed in China, the phenomenon of food fight between people and livestock is further aggravated, and the food safety is seriously threatened. The method has great significance for developing and utilizing straw resources in the face of food shortage and potential waste of agricultural waste feed resources. In recent years, corn straws are widely applied to the fields of silage and direct returning to field fertilizers. The silage straw feed has the defects that the limit of low protein content of the maize straw cannot be changed, and the requirement of animals on protein cannot be met by using the silage straw feed singly. The straw fermented feed still stays in a laboratory or a small production scale, the content of crude protein in the straw fermented feed can be improved to 16-25%, but the content of amino acid is only about 12% generally. The corn cob and bagasse resources are developed into primary feed, and the digestibility is low. The feed protein is produced by utilizing the agricultural wastes through a biological solid state fermentation method, the nutritional quality of the straw feed is effectively improved through the characteristics of high protein content and excellent amino acid proportion, and the digestion utilization rate of the protein feed of livestock and poultry to the agricultural wastes is improved, so that the immunity of the livestock and poultry is improved. The scale biosynthesis of the feed protein by the agricultural wastes is the best way for low-cost healthy culture, and the scale production of the feed protein by the agricultural wastes is an ideal way for solving the problem of comprehensive utilization of the 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 and can not be replaced by the traditional agricultural protein.
The main reasons are two, firstly, most of agricultural wastes are lignocellulose materials, the degradation-resistant barrier is relatively stubborn, the anti-nutritional factors are still in existence, and how to develop a high-efficiency low-cost pretreatment process and equipment is to deal with the large-scale treatment of straws;
the agricultural waste raw material straw has complex components, uneven structure, tight combination among the components and strong degradation resistance. At present, the straw is converted into a high-valued product, and the pretreatment is an essential process. The pretreatment can partially remove hemicellulose and lignin components, reduce the crystallinity of cellulose, increase the contact area of cellulase and a substrate, and improve the accessibility of the cellulase, thereby improving the efficiency of degrading the cellulose by the enzyme.
Generally, the pretreatment is classified into a biological pretreatment method, a physical pretreatment method, and a chemical pretreatment method, or a combination thereof. The physical pretreatment is mainly to change the biomass into smaller particles through mechanical arm sections such as crushing, grinding and the like of the biomass, increase the contact area of enzyme to cellulose, and simultaneously possibly partially destroy the crystalline structure of the cellulose. But the mechanical crushing treatment mode has the defect of high energy consumption. The chemical pretreatment is to remove hemicellulose and lignin in the wood fiber raw material by using an added catalyst at 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, has high requirements for corrosion resistance of reaction vessels, and is also recycled after use for economic reasons, so that it has few practical applications. The organic solvent pretreatment is to realize the separation of cellulose, lignin and hemicellulose by means of the organic solvent or the aqueous solution of the organic solvent to destroy the inner nodes of the hemicellulose and the lignin under the catalytic action of inorganic acid; in order to be economically feasible and not to inhibit the next enzyme hydrolysis and fermentation process, the organic solvent must be evaporated, concentrated and recycled after the reaction is finished, and the operation steps are relatively complex. The biological pretreatment method has the advantages of low energy consumption and environmental friendliness, but has the defect of slow degradation speed. Currently, only the steam explosion pretreatment technology is industrially applicable and amplified. Steam explosion refers to the process of steaming plant raw materials in high-temperature and high-pressure steam to generate some acidic substances, so that hemicellulose is degraded into soluble sugar, lignin in a composite intercellular layer of a cell wall is softened and partially degraded, so that bonding among fibers is weakened, and then the material is dissociated into single fiber cells from the intercellular layer through an adiabatic expansion process instantaneously completed by the steam explosion to do work externally (Chen H, Lan W.2017. Chapter 3-advanced Strategies for Biochemical Conversion of Biomass. technol. biochem. converters. Biomass, Elsevier, 21-64.). These current commercial pretreatment processes tend to destroy the lignocellulosic destructuring and reduce the enzyme dosage by increasing the pretreatment intensity. However, these pretreatment technologies and processes generally have the problems of high energy consumption and high cost, and are accompanied by the generation of a large amount of enzymatic hydrolysis inhibitors and three wastes, and most of the pretreatment technologies are difficult to realize industrial popularization and application (Yue Guojun. 2014. cellulose ethanol engineering general theory, chemical industry publishers).
Meanwhile, the mixed fermentation process cannot be regulated and controlled under the extensive industrialized condition, the existing domestic production strains of the microbial mycoprotein of the old generation have the problems of low straw conversion efficiency, small accumulation of essential amino acid, insufficient protein synthesis capacity and the like, the quality of the feed produced by solid fermentation of agricultural wastes is relatively low, the laboratory result cannot be reproduced, and the mixed fermentation process is also a factor for restricting the large-scale production of the straw fermented feed. Establishing an optimized and reproducible solid state fermentation technology, determining the dynamic correlation mode between the thallus activity and other factors, and monitoring the process characteristic change of the straw during solid state fermentation of the straw by the feeding strain, which is the key for realizing the industrialization of the straw feeding protein.
Disclosure of Invention
Based on the requirements, the invention develops a high-efficiency 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 high-efficiency and low-cost pretreatment with solid state fermentation, which is characterized by comprising the following steps of:
1) a biomass material cooking stage; soaking one or more of agricultural waste biomass materials, namely corn stalks, corn cobs and bagasse in a sulfuric acid solution, and heating and pressurizing;
2) and (3) biomass material blasting stage: carrying out pressure reduction blasting;
3) adding water into the biomass material pretreated by the method respectively, and adding trichoderma reesei and aspergillus niger spore liquid for solid state fermentation;
wherein the preservation number of the Aspergillus niger is as follows: CGMCC No. 22465; the preservation number of the trichoderma reesei is as follows: CGMCC number 21470.
In a preferred embodiment, the temperature and pressure increase in the step 1) is carried out at 140-.
In another preferred embodiment, the pressure is reduced to 0.4-0.7MPa by pressure reduction blasting in step 2), and the same temperature is maintained for 4-8 min.
In still another 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 in step 3), the Candida utilis seed solution is added and the culture is continued at 25-35 ℃ for 100-150 hours.
In other embodiments, in step 1), the biomass material is mixed according to a ratio of 1: 4-10 solid-liquid ratio, and then heating and pressurizing.
In other embodiments, in step 3), water is added to the pretreated biomass material as a culture medium for solid state fermentation at a feed-water ratio of 1:2 to 3.
Preferably, in step 3), the ratio of the spore liquid of trichoderma reesei and aspergillus niger is preferably 1: 1 proportion, the spore concentration of the aspergillus niger and the trichoderma reesei is 1 multiplied by 107one/mL to 2X 109one/mL.
The invention also provides the application of the biomass material treatment method in the production of single-cell protein feed. Further preferably, the step 3) further comprises a step of separating crude protein after the fermentation is completed.
The invention discloses a biomass material treatment method combining efficient low-cost pretreatment with solid state fermentation, which comprises two aspects of a staged temperature-changing steam explosion pretreatment method and solid state fermentation of agricultural wastes. The continuous steam explosion method capable of changing temperature in stages can realize effective separation of cellulose, hemicellulose and lignin in biomass, the xylose removal rate reaches over 90 percent, and the xylose solution is neutral, can be used for downstream direct fermentation, and does not need detoxification; and the energy consumption is reduced to 40 percent of the prior blasting technology, the inhibitor is reduced to less than 10 percent of the prior steam explosion pretreatment technology, and the pretreatment cost is less than 200 yuan per ton of raw materials. During the fermentation process of the biological material, Trichoderma reesei and Aspergillus niger strains which are selected by the inventor and can efficiently utilize the straws are added to secrete cellulose and beta-glucosidase to the outside of cells respectively during the growth process of utilizing the biological material, and the two enzyme systems are mutually cooperated to efficiently degrade cellulose of the corn straws and convert the cellulose into fermentable sugar which can be utilized by the bacteria. After yeast seed liquid is added, the yeast can efficiently utilize fermentable sugar to convert the fermentable sugar into mycoprotein; the addition of the yeast can efficiently reduce the concentration of fermentable sugar in the culture medium, and 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 specific embodiments in order that the invention may be better understood. But are not to be construed as limiting the invention.
Example 1: establishment of the Process
A first stage, a cooking stage; cutting agricultural waste biomass materials such as corn stalks, corn cobs and bagasse into small blocks of about 10cm by a crusher, wherein the grain size is 1: 8 (solid-to-liquid ratio), soaking in 0.5% dilute sulfuric acid solution, keeping the temperature at 160 ℃, keeping the temperature for 6min, and keeping the pressure at 0.9 Mpa;
the second stage, the blasting stage: after cooking, the pressure is reduced to 0.6Mpa, and the temperature is kept at 160 ℃ for 6 min.
Comparing the component change of the biomass material before and after the steam explosion pretreatment.
TABLE 1 compositional changes after steam explosion pretreatment of various agricultural waste biomass materials
Example 2: production of single cell proteins
Single cell protein is produced by using biomass material (including bagasse, corn stalk and corn cob) after steam explosion pretreatment.
Trichoderma reesei/aspergillus niger spore liquid: respectively streaking PDA plate with Aspergillus niger 60B-3DW and Trichoderma reesei A2H, culturing at 30 deg.C for 5d, eluting spore with sterile water, filtering to remove mycelium with cell filter, counting with blood counting plate, and adjusting spore concentration of Aspergillus niger and Trichoderma reesei to 1.2 × 108one/mL.
Wherein the strain Aspergillus niger 60B-3DW is preserved in China general microbiological culture Collection center with the preservation number: CGMCC No.22465, the preservation time is as follows: on 2021, 07/05, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The strain is a mutant strain which takes Aspergillus niger 3.316 (preserved in CGMCC 3.316, a common strain preservation center of institute of microbiology of Chinese academy of sciences) as an initial strain and has greatly improved beta-glucosidase activity and is bred by ARTP mutagenesis and droplet microfluidic high-throughput screening technology.
Trichoderma reesei A2H, deposited in the China general microbiological culture Collection center, with the following numbers: CGMCC No.21470, the preservation time is as follows: on the day of 2021, 03 and 17, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
The steam-exploded pretreated corn stalks, bagasse and corn cobs obtained by the method described in example 1 were taken respectively, and water was added in a feed-water ratio of 1:2.5 to serve as a culture medium for solid state fermentation. The trichoderma reesei and aspergillus niger spore liquid are added on the surface of a culture medium for the solid state fermentation of the biomass material by steam explosion pretreatment according to the inoculum size of 5 percent (namely the ratio of 1: 1), and cultured for 24 to 36 hours at the temperature of 30 ℃.
After the Aspergillus niger and Trichoderma reesei hydrolyze the biomass material for 24 hours, Candida utilis seed solution is added according to the addition amount of 10 percent, and solid state fermentation is continued for 120 hours at 30 ℃.
Corn stalks, bagasse and corncobs pretreated by steam explosion in example 1 were taken after solid-state fermentation for 7 days, respectively, and the fermented stalks were dried, and then the total nitrogen content and amino acid content of the single-cell protein product were measured. The crude protein content of the fermented straw was calculated as crude protein = total nitrogen content of solid 6.25. The results show 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 corn cob is about 29.88%.
Crude protein GB/T6432 1994 method for determining crude protein in feed. The amino acid content is measured by an A200 amino Nova amino acid analyzer according to national standard GB/T18246-. As can be seen from the following table, the amino acid content of the steam exploded straw after 7 days of solid state fermentation was 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 corn cob is about 22.76%.
TABLE 2 analysis of single-cell products produced by solid state fermentation of biomass material after steam explosion
| 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 high-protein, full-amino-acid and composite probiotic function single-cell protein feed by utilizing a biotransformation method through integrating and optimizing key technologies of biomass refining, such as a step-variable temperature steam explosion pretreatment technology, a mixed-strain solid-state fermentation technology and the like. The steam explosion pretreatment method and the solid state fermentation method can be copied and amplified, can be applied industrially and have better industrialization prospect. The bagasse, the corn straw and the corncob after steam explosion pretreatment are used as substrates, and after specific Trichoderma reesei, specific Aspergillus niger and Candida utilis are mixed and fermented, the content of crude protein in single-cell protein exceeds 28 percent, and the content of amino acid exceeds 21 percent, so that the waste agricultural waste resources are changed into valuable, the traditional agricultural protein production mode is overturned, the self-sufficiency of protein raw materials in China is promoted, and the important way of the circular economy and agricultural sustainable development in China is realized. Meanwhile, two problems of low resource utilization rate of agricultural wastes in China and shortage of traditional agricultural proteins are accurately solved, the comprehensive productivity and competitiveness of agriculture in China are improved, and the method has important strategic significance.
Claims (10)
1. A biomass material treatment method combining high-efficiency and low-cost pretreatment with solid state fermentation is characterized by comprising the following steps:
1) a biomass material cooking stage; soaking one or more of agricultural waste biomass materials, namely corn stalks, corn cobs and bagasse in a sulfuric acid solution, and heating and pressurizing;
2) and (3) biomass material blasting stage: carrying out pressure reduction blasting;
3) adding water into the biomass material pretreated by the method respectively, and adding trichoderma reesei and aspergillus niger spore liquid for solid state fermentation;
wherein the preservation number of the Aspergillus niger is as follows: CGMCC No. 22465; the preservation number of the trichoderma reesei is as follows: CGMCC number 21470.
2. The method for treating biomass material according to claim 1, wherein the temperature rise and pressure increase in step 1) is carried out at 140-180 ℃, the temperature preservation time is 4-10min, and the pressure is 0.8-1.0 MPa.
3. The biomass material treatment method according to claim 1, wherein in the step 2), the pressure is reduced to 0.4-0.7Mpa by pressure reduction blasting, and the same temperature is maintained for 4-8 min.
4. The biomass material treatment method according to claim 1, wherein in the step 3), the solid state fermentation is carried out at 25 to 35 ℃ for 24 to 36 hours.
5. The method for treating a biomass material according to any one of claims 1 to 3, wherein after the solid state fermentation of the step 3), the Candida utilis seed solution is added and the cultivation is continued at 25 to 35 ℃ for 100-150 hours.
6. The biomass material processing method according to claim 1, wherein in the step 1), the biomass material is treated in a ratio of 1: 4-10 solid-liquid ratio, and then heating and pressurizing.
7. The biomass material treatment method according to claim 1, wherein in the step 3), water is added to the pretreated biomass material as a culture medium for solid state fermentation at a ratio of 1:2 to 3.
8. The biomass material treatment process according to claim 1, wherein in step 3), the spore liquors of trichoderma reesei and aspergillus niger are preferably mixed in a ratio of 1: 1 proportion, the spore concentration of the aspergillus niger and the trichoderma reesei is 1 multiplied by 107one/mL to 2X 109one/mL.
9. Use of a biomass material processing method according to any one of claims 1 to 8 in single cell protein feed production.
10. The use of claim 9, further comprising the step of isolating the crude protein after the fermentation of step 3).
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