Method for coupling agricultural and forestry waste pretreatment with low organic acid under assistance of low-concentration auxiliary agent and in-situ enzyme hydrolysis

Abstract

The invention discloses a coupling in-situ enzyme hydrolysis method for low-organic-acid pretreatment of agricultural and forestry wastes under the assistance of a low-concentration auxiliary agent. The method comprises the following steps: by taking crushed agricultural and forestry waste as a raw material, adding an aqueous solution containing a low-concentration auxiliary agent and organic acid into the raw material, reacting at 140-185 DEG C for 0.5-4.0 hours for pretreatment, cooling to 45-55 DEG C after the reaction is finished, adding cellulase accounting for 5-25 mg / g of the raw material into the raw material, hydrolyzing at 50 DEG C for 72 hours, filtering, washing, and drying to obtain the cellulose. And after enzyme hydrolysis is finished, carrying out solid-liquid separation to obtain solid residues and enzyme hydrolysate, adjusting the pH value of the enzyme hydrolysate, and carrying out oil-producing fermentation. According to the method, organic acid pretreatment is assisted by the aid of the auxiliaries, so that pollution or fermentation inhibition caused by alkali, solvents or inorganic acid is avoided, meanwhile, the enzyme hydrolysis efficiency is improved by the aid, and fermentable sugar with higher concentration is obtained.

Description

Technical Field

[0001] This invention relates to the field of biomass energy conversion technology, and in particular to a method for pretreating agricultural and forestry waste with low concentration of adjuvants and low organic acid content, coupled with in-situ enzymatic hydrolysis. Background Technology

[0002] A crucial step in the process of converting agricultural and forestry waste into energy and chemical products is to hydrolyze the cellulose and hemicellulose in the raw materials into fermentable sugars such as pentoses and hexoses, which are then subjected to bio-fermentation to produce liquid fuels such as microbial oils, ethanol, and butanol.

[0003] Conventional pretreatment methods for lignocellulosic biomass are as follows: 1) Hemicellulose priority: After acid pretreatment to remove hemicellulose, solid-liquid separation is performed. The hemicellulose hydrolysis products, xylose, arabinose, or xylooligosaccharides, are further utilized in the pretreatment liquid. The pretreatment residue is further hydrolyzed by cellulase to obtain glucose, which is present in the enzyme hydrolysate. The remaining unhydrolyzed cellulose and lignin are in the hydrolysate. The liquid after solid-liquid separation is used for subsequent fermentation. Pentose and hexose are present in the pretreatment liquid and enzyme hydrolysate, respectively. The solid content of enzyme hydrolysis is generally no more than 15% (Xiao et al., 2004a, Kristensen et al., 2009). Usually, the sugar concentration in both the pretreatment liquid and the enzyme hydrolysate does not exceed 30 g / L. Subsequent fermentation requires concentration; otherwise, the sugar concentration will be too low, reducing production efficiency and increasing equipment investment. 2) Lignin priority: After alkali / solvent pretreatment to remove lignin, enzyme hydrolysis is performed to obtain fermentable sugars. The inorganic acids and alkalis in methods 1) and 2) above can easily cause environmental pollution, and the solvents in method 2) can easily remain in the pretreatment residue, affecting the enzyme activity of enzymatic hydrolysis and subsequent fermentation activity. In recent years, more and more researchers have considered using hydrothermal treatment, low organic acid, and auxiliaries to synergistically pretreat lignocellulosic biomass before enzymatic hydrolysis. The enzymatic hydrolysate is then used for downstream fermentation to produce liquid fuels such as ethanol, oils, and butanol. However, during the separation of solid products, due to the high water absorption of lignocellulose (usually with a moisture content of over 50%), whether hemicellulose or lignin is removed first during pretreatment, the separated solid products will carry away a large amount of hydrolysate (pentose sugars or oligosaccharides obtained from hydrolyzing hemicellulose) or solvent, requiring additional washing steps to recover more by-products. This process is lengthy and involves significant investment.

[0004] To address the aforementioned issues, some researchers have employed solvent-coupled acid or alkali pretreatment with enzymes. For example, ZL202211532425.8 discloses a method for pretreating biomass using glycerol formaldehyde-catalyst-aqueous solution, where the glycerol formaldehyde content is 50-100%, and the catalyst is a soluble acid (sulfuric acid, hydrochloric acid, formic acid, or p-toluenesulfonic acid), alkali (sodium hydroxide, potassium hydroxide, ethylenediamine, or acetamide), or salt (FeCl3, Al2(SO4)3Cl3, Na3PO4, or CH3COONa). This method can separate hemicellulose, cellulose, and lignin. Although the subsequent enzymatic hydrolysis is highly efficient, the solvent and water have a certain degree of miscibility, making complete removal difficult during distillation. When the hemicellulose hydrolysate is used as a fermentation substrate, residual glycerol formaldehyde or catalyst can easily inhibit subsequent fermentation. The process of recovering hemicellulose and lignin requires multiple steps of solid-liquid separation, pH adjustment, and solvent washing of hydrolyzed residue, making it a lengthy process. In addition, a large amount of organic solvents such as 50-100% glycerol formaldehyde, tetrahydrofurfuryl alcohol, and γ-valerol are used, and the high saturated vapor pressure of the solvents leads to high reaction pressure, resulting in high reactor costs. Furthermore, the inorganic acids, alkalis, and salts used cause severe corrosion to the equipment.

[0005] US20220298531A1 discloses a method in which biomass and water are mixed in a continuous countercurrent extrusion / reactor, and after treatment at a certain time and temperature, solid-liquid separation is achieved to obtain a solid phase rich in cellulose and a liquid phase rich in lignin and hemicellulose-derived xylose. The solid phase is further fermented to produce cellulase using microorganisms. These enzymes are then used to enzymatically hydrolyze the cellulose or hemicellulose in the solid phase, and the resulting monosaccharides are fermented into other useful chemicals or fuels.

[0006] CN103774478A discloses the use of oxalic acid (5-10% concentration) for pre-hydrolysis to remove hemicellulose in papermaking pulp production, which has a certain effect. However, after pretreatment, solid-liquid separation and washing are still required to obtain pulp. The catalyst has a high concentration of oxalic acid, which may inhibit subsequent fermentation.

[0007] CN 116426586 A discloses a method for mildly degrading agricultural and forestry biomass raw materials to improve hydrolysis sugar production. The method involves mixing biomass raw materials with glycerol at a mass ratio of 1:5 to 1:10, adding 2-8% (by weight of the raw materials) of nonionic surfactants such as PEG-series, Tween-series, Triton-X-series, and AEO-series, pretreating at 130-200℃ for 15-60 min, cooling, adding boiling water and stirring, and then filtering to separate the mixture. The resulting solid matrix is ​​added to citrate buffer at a material-to-liquid ratio of 2g:100mL to 30g:100mL, while simultaneously adding 1-6% (by weight of the solid matrix) of nonionic surfactant for enzymatic hydrolysis to obtain a sugar-containing hydrolysate. This patent achieves good results by using high-concentration glycerol combined with surfactants to modify the raw material components during the pretreatment process. However, the glycerol consumption is 5-10 times that of the raw materials, and the nonionic surfactant is 2-8% of the raw materials, resulting in very large reagent consumption. Furthermore, the subsequent utilization of sugar in the enzymatic hydrolysate faces challenges such as the removal of glycerol and nonionic surfactants.

[0008] CN121137093A discloses a method for preparing fermentable sugars from lignocellulose by pretreating it with recyclable low-concentration p-toluenesulfonic acid. First, lignocellulose is pretreated with a low-concentration p-toluenesulfonic acid aqueous solution. After solid-liquid separation, a hemicellulose hydrolysate and a pretreated solid component are obtained. Then, the pretreated solid component is saccharified with cellulase to obtain a glucose-rich hydrolysate. The hemicellulose hydrolysate is adsorbed using a highly cross-linked adsorption resin, and after solid-liquid separation, a xylose-rich hydrolysate is obtained. The resin is desorbed using industrial ethanol, and the desorbed liquid is rotary evaporated to remove the industrial ethanol, yielding p-toluenesulfonic acid. This method efficiently degrades hemicellulose to obtain its derived sugars while improving cellulase saccharification efficiency. The use of a highly cross-linked adsorption resin for p-toluenesulfonic acid recovery offers advantages such as high fermentable sugar yield, recyclable acidic catalyst, low cost, and environmental friendliness. However, a lengthy process still exists.

[0009] With the increasing demand for sustainable aviation fuels (oils) in the aviation industry, the production of microbial oils through fermentation of lignocellulosic hydrolysates using oil-producing microorganisms has become one of the most promising methods for sustainable aviation fuel production. Therefore, the economic efficiency of the pretreatment and fermentation process is crucial, and the aforementioned issues urgently need to be addressed. Summary of the Invention

[0010] The purpose of this invention is to provide a method for pretreating agricultural and forestry waste with low concentration of additives and low organic acid, coupled with in-situ enzymatic hydrolysis. This method does not require solid-liquid separation after pretreatment and can directly carry out subsequent enzymatic hydrolysis. It has the advantages of short process and no need to wash pretreatment residue. At the same time, the additives and organic acids used can be obtained through biomass hydrolysis, fermentation and other means, and can be directly fermented for oil production without detoxification, realizing a green and low-carbon cycle throughout the entire process.

[0011] This invention is achieved through the following technical solutions:

[0012] This invention protects a method for pretreating agricultural and forestry waste with low concentration of adjuvants and low organic acid, coupled with in-situ enzymatic hydrolysis. The method includes the following steps: using pulverized agricultural and forestry waste as raw material, adding an aqueous solution containing low concentrations of adjuvants and organic acids to the raw material. The adjuvants are selected from one or more of propylene glycol, glycerol, Tween, ethylene glycol, and polyethylene glycol. The mass fraction of the adjuvants in the aqueous solution containing the adjuvants and organic acids is 0-1.5%, and the mass fraction of the organic acids is 0.5%-2.0%. Pretreatment is carried out at 140℃-185℃ for 0.5-4.0 h. After the reaction, the temperature is lowered to 45℃-55℃, and 5-25 mg / g of cellulase from the raw material is added. Hydrolysis is carried out at 45℃-55℃ for 65-75 h. After enzymatic hydrolysis, solid-liquid separation is performed to obtain solid residue and in-situ enzymatic hydrolysate. The pH of the in-situ enzymatic hydrolysate is adjusted before oil production fermentation.

[0013] To fully utilize the three components of biomass, reduce physical losses, and shorten the process flow, this invention utilizes mild, low-concentration organic acids to hydrolyze part of the hemicellulose and in-situ enzymes to hydrolyze the remaining xylan and cellulose. Simultaneously, it employs adjuvants with strong permeability or surface activity to control the condensation of lignin and the deep hydrolysis of soluble sugars under high-temperature, low-concentration organic acid pretreatment conditions, reducing the formation of soluble lignin and humin. This yields a hydrolysate with a high concentration of fermentable sugars. The organic acids and adjuvants used can be utilized by oil-producing microorganisms or are non-toxic and recyclable. Ultimately, this achieves efficient one-step sugar production and microbial oil production from lignocellulosic biomass. Through the design of each unit and the seamless integration of processes, it solves the problems of long processes and significant sugar losses in actual production caused by multiple solid-liquid separations and washing processes required in existing technologies. Furthermore, it applies this technology to oil-producing fermentation, achieving green preparation of liquid fuels.

[0014] Preferably, the agricultural and forestry waste is selected from one or more of sugarcane bagasse, wheat stalks, and corn stalks.

[0015] Preferably, the mass ratio of the agricultural and forestry waste to the aqueous solution containing auxiliaries and organic acids is 1:6 to 1:12.

[0016] Further preferred, the agricultural and forestry waste is sugarcane bagasse, and the pretreatment conditions are a reaction at 160℃-170℃ for 1.0-1.5 h. The aqueous solution containing the auxiliaries and organic acids has a mass fraction of 1.0%-1.5% for organic acids and 0.5% for auxiliaries. The aqueous solution is prepared by diluting 1.0-1.5 g of organic acid and 0.5 g of auxiliaries with water to a quantitative amount of 100 g.

[0017] Preferably, the organic acid is selected from one of oxalic acid, acetic acid, levulinic acid, and succinic acid. All organic acids can be prepared from agricultural and forestry waste, and the raw and auxiliary materials possess fully bio-based, green, and carbon-reducing properties.

[0018] Further preferably, the organic acid is acetylpropionic acid or succinic acid, and no additives are added to the aqueous solution.

[0019] More preferably, the auxiliary agent and organic acid are a combination of acetic acid and glycerol.

[0020] Preferably, after the reaction is completed, the temperature is lowered to 50°C, and 5-25 mg / g of cellulase from the raw material is added. The mixture is then hydrolyzed at 50°C for 24-72 h.

[0021] Preferably, the specific steps for oil-producing fermentation after adjusting the pH of the in-situ enzyme hydrolysate are as follows: the oil-producing yeast strain is transferred from the slant to the seed culture medium and activated for 24-36 h. The in-situ enzyme hydrolysate is adjusted to pH 5.0-7.0 to serve as the fermentation medium. The activated strain is inoculated into the fermentation medium at an inoculation rate of 5%-10%. After fermentation at 25℃-35℃ for 84-120 h, the cells are collected by centrifugation or filtration. The microbial oil is then extracted by acid-heat method or direct oil extraction. The seed culture medium consists of the following raw materials in the following mass percentages: glucose 2%, yeast powder 1%, peptone 1%, and the remainder is deionized water.

[0022] Further preferably, the lipophilic yeast strain is selected from one or more of the following: R. glutinis, Trichosporoncutaneum, Cryptococcus albidus, Yarrowia lipolytica, Rhodosporidium torulodides, Lipomyces starkeyi, Cutaneotrichosporon dermatis, and Trichosporon mucoides.

[0023] Further optimization involves adjusting the pH of the in-situ enzyme hydrolysate for oil production fermentation as follows: the oil-producing yeast strain is transferred from the slant culture to the seed culture medium and activated for 24 h. The in-situ enzyme hydrolysate is then adjusted to pH 5.0-7.0 to serve as the fermentation medium. The activated strain is inoculated into the fermentation medium at a 10% inoculation rate. After fermentation at 28℃ for 120 h, the cells are collected by centrifugation. The microbial oil is then extracted using the acid-heat method or by direct oil extraction. The seed culture medium consists of the following raw materials in the following mass percentages: glucose 2%, yeast powder 1%, peptone 1%, and the remainder is deionized water.

[0024] The specific steps of the acid-heat method are as follows: Add 5–100 mL of 1–7 mol / L HCl per gram of bacterial cells, let stand for 20 min, reflux in a boiling water bath for 0.2–0.5 h, intermittently shaking during this period to ensure sufficient acid-heat treatment and cell disruption, then quickly place in an ice-water bath; add a methanol-chloroform or methanol-cyclohexane mixture with a volume ratio of 1:2, shake for 60 min to fully extract the oil, centrifuge at 8000–10000 r / min for 5–10 min, the upper layer of the system is aqueous phase + methanol, and the lower layer is oil phase (containing oil), collect the lower oil phase, evaporate the solvent to dryness in a rotary evaporator (40℃–50℃) to obtain crude microbial oil. The advantage of the method for extracting microbial oil proposed in this invention is that the process is short, requiring only one solid-liquid separation step, without the need for a detoxification step.

[0025] Preferably, the pH of the enzyme hydrolysate is adjusted to 5.0-7.0 by adding NaOH, Ca(OH)2, or CaO. If the in-situ enzyme hydrolysate is used for oil-producing fermentation, the organic acid is one of acetic acid, levulinic acid, or succinic acid, and the adjuvant is glycerol, ethylene glycol, or polyethylene glycol, or none is added.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1) This invention avoids pollution or fermentation inhibition caused by alkali, solvent or inorganic acid by using auxiliary agents to assist in organic acid pretreatment. At the same time, the auxiliary agents improve the efficiency of enzyme hydrolysis and obtain a higher concentration of fermentable sugar.

[0028] 2) This invention reduces one solid-liquid separation step and washing steps by in-situ enzymatic hydrolysis, thereby reducing equipment investment and increasing the total sugar yield.

[0029] 3) The enzyme hydrolysate proposed in this invention can be used directly for oil production fermentation without detoxification, reducing the consumption of microbial oil auxiliary materials and improving economic efficiency.

[0030] 4) The additives and organic acids used in this invention can be obtained through biomass hydrolysis, fermentation and other methods, achieving a green, low-carbon cycle throughout the entire process. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the process flow of the method for coupling in-situ enzyme hydrolysis of agricultural and forestry waste according to the present invention.

[0032] Figure 2 This is a schematic diagram of a conventional process flow. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are considered to be commercially available materials and reagents that can be obtained through conventional markets and other commercial channels.

[0034] In the following examples or comparative examples, the preferred steps for oil-producing fermentation after adjusting the pH of the in-situ enzyme hydrolysate are as follows: the oil-producing yeast strain is transferred from the slant culture to the seed culture medium and activated for 24-36 h. The in-situ enzyme hydrolysate is adjusted to pH 5.0-7.0 to serve as the fermentation medium. The activated strain is inoculated into the fermentation medium at an inoculation rate of 5%-10%. After fermentation at 25℃-35℃ for 84-120 h, the cells are collected by centrifugation or filtration. The microbial oil is then extracted by acid-heat method or direct oil extraction. The seed culture medium consists of the following raw materials in the following mass percentages: glucose 2%, yeast powder 1.0%, peptone 1%, and the remainder is deionized water.

[0035] The lipophilic yeast strain is selected from one or more of the following: *R. glutinis*, *Trichosporon cutaneum*, *Cryptococcus albidus*, *Yarrowia lipolytica*, *Rhodosporidium torudides*, *Lipomyces starkeyi*, *Cutaneotrichosporon dermatis*, and *Trichosporon mucoides*. Preferably, in the following examples or comparative examples, the lipophilic yeast strain is *Trichosporon cutaneum*.

[0036] Further optimization involves adjusting the pH of the in-situ enzyme hydrolysate for oil production fermentation as follows: The oil-producing yeast strain is transferred from the slant culture medium to the seed culture medium and activated for 24 hours. The in-situ enzyme hydrolysate is then adjusted to pH 5.0-7.0 to serve as the fermentation medium. The activated strain is inoculated into the fermentation medium at a 10% inoculum size. After fermentation at 28°C for 120 hours, the cells are collected by centrifugation. The microbial oil is then extracted using an acid-heat method or by direct oil extraction. The seed culture medium consists of the following raw materials in the following mass percentages: glucose 2%, yeast powder 1.0%, peptone 1%, and the remainder is deionized water.

[0037] The specific steps of the acid-heat method are as follows: Add 5–100 mL of 1–7 mol / L HCl per gram of bacterial cells, let stand for 20 min, reflux in a boiling water bath for 0.2–0.5 h, and intermittent shaking can be performed during this period to ensure sufficient acid-heat treatment for cell disruption. Then, quickly place the mixture in an ice-water bath. Next, add a methanol-chloroform or methanol-cyclohexane mixture with a volume ratio of 1:2, shake for 60 min to fully extract the oil, and centrifuge at 8000–10000 r / min for 5–10 min. The upper layer of the system is an aqueous phase + methanol, and the lower layer is an oil phase (containing oil). Collect the lower oil phase and evaporate the solvent in a rotary evaporator (40℃–50℃) to obtain crude microbial oil.

[0038] Further optimization involves the following steps for the acid-heat method: Add 40 mL of 4 mol / L HCl per gram of dry bacterial cells, let stand for 20 min, then reflux in a boiling water bath for 10 min; quickly place in a -20 ºC refrigerator, then add 50 mL of methanol and 100 mL of chloroform respectively, shake on a shaker at 200 r / min for 1 h, centrifuge at 10,000 r / min for 10 min, separate the layers using a separatory funnel, collect the lower layer solution in a pre-weighed flask, remove the chloroform using a rotary evaporator, dry in a 60 ºC oven for 12 h, and weigh. Calculate the oil content and oil yield based on this.

[0039] Example 1

[0040] like Figure 1 As shown, wheat straw was crushed and added to an aqueous solution containing 0.5 wt% acetic acid and 0.5 wt% ethylene glycol at a mass ratio of wheat straw to aqueous solution of 1:8. The reaction was carried out at 160℃ for 2.5 h. After the reaction was completed, the temperature was lowered to 50℃, and 25 mg / g of cellulase from the raw material was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the in-situ enzyme hydrolysate was 30.56 g / L. CaO was added to adjust the pH of the enzyme hydrolysate to 5.0 before oil production fermentation was carried out. The organic matter-biomass yield was 20%, and the sugar-oil yield was 10%.

[0041] like Figure 2 As shown, the conventional process involves solid-liquid separation after pretreatment, washing or drying the pretreatment residue, and then enzymatic hydrolysis using the pretreatment residue. Example 1 involves in-situ enzymatic hydrolysis. Compared with the conventional process, Example 1 reduces the solid-liquid separation steps and washing steps through in-situ enzymatic hydrolysis, thereby reducing equipment investment and increasing the total sugar yield.

[0042] Example 2

[0043] Sugarcane bagasse was crushed and added to an aqueous solution containing 0.5 wt% acetic acid and 0.5% glycerol at a mass ratio of 1:8. The reaction was carried out at 160℃ for 2.5 h. After the reaction was completed, the temperature was lowered to 50℃, and 10 mg / g of cellulase was added. The mixture was then hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the in-situ enzyme hydrolysate was 39.09 g / L. CaO was added to adjust the pH of the enzyme hydrolysate to 6.0 before oil production fermentation was carried out. The organic matter-to-biomass yield was 25%, and the sugar-to-oil yield was 13%.

[0044] Comparative Example 1

[0045] Sugarcane bagasse was crushed and added to an aqueous solution containing 0.5 wt% acetic acid at a mass ratio of 1:8. The reaction was carried out at 160℃ for 2.5 h. After the reaction was completed, the temperature was lowered to 50℃, and 10 mg / g of cellulase was added. The mixture was then hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the in-situ enzyme hydrolysate was 24.74 g / L. CaO was added to adjust the pH of the enzyme hydrolysate to 6.0 before oil production fermentation was carried out. The organic matter-to-biomass yield was 21%, and the sugar-to-oil yield was 11%.

[0046] Example 3

[0047] Sugarcane bagasse was crushed and added to an aqueous solution containing 1 wt% acetic acid and 0.5 wt% glycerol at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. After the reaction, the temperature was lowered, and 25 mg / g of cellulase was added. The enzymatic hydrolysis conditions were 50℃ for 72 h. After enzymatic hydrolysis, solid-liquid separation was performed. The fermentable sugar concentration in the in-situ enzymatic hydrolysate was 60.14 g / L. The pH of the enzymatic hydrolysate was adjusted to 6.5 with CaO before oil-producing fermentation. The organic matter-to-biomass yield was 30%, and the sugar-to-oil yield was 16%.

[0048] Example 4

[0049] Sugarcane bagasse was crushed and added to an aqueous solution containing 0.6 wt% succinic acid, 0.65 wt% acetic acid, and 0.5 wt% glycerol at a mass ratio of 1:12. The mixture was pretreated at 185℃ for 0.5 h. After the reaction was completed, the temperature was lowered, and 20 mg / g of cellulase was added. The enzymatic hydrolysis conditions were 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the enzymatic hydrolysate was 35.6 g / L. After adjusting the pH to 6.5 with CaO, oil production fermentation was carried out. The organic matter-to-biomass yield was 23%, and the sugar-to-oil yield was 12%.

[0050] Comparative Example 2

[0051] Sugarcane bagasse was crushed and added to an aqueous solution containing 1.1 wt% succinic acid and 0.65 wt% acetic acid at a mass ratio of 1:12. The mixture was pretreated at 185℃ for 0.5 h. After the reaction was completed, the temperature was lowered, and 20 mg / g of cellulase was added. The enzymatic hydrolysis conditions were 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the enzymatic hydrolysate was 30.7 g / L. After adjusting the pH to 7.0 with CaO, oil-producing fermentation was carried out. The organic matter-to-biomass yield was 22%, and the sugar-to-oil yield was 11.5%.

[0052] Comparative Example 3

[0053] Sugarcane bagasse was crushed and added to an aqueous solution containing 0.65 wt% acetic acid and 1.1 wt% glycerol at a mass ratio of 1:12. The mixture was pretreated at 185℃ for 0.5 h. After the reaction was completed, the temperature was lowered, and 20 mg / g of cellulase was added. The enzymatic hydrolysis conditions were 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the enzymatic hydrolysate was 28.86 g / L. After adjusting the pH to 7.0 with CaO, oil-producing fermentation was carried out. The organic matter-to-biomass yield was 18.6%, and the sugar-to-oil yield was 8.6%.

[0054] Example 5

[0055] Sugarcane bagasse was crushed and added to an aqueous solution containing 1.25 wt% acetic acid and 0.5 wt% glycerol at a mass ratio of 1:12. The mixture was pretreated at 185℃ for 0.5 h. After the reaction was completed, the temperature was lowered, and 20 mg / g of cellulase was added. The enzymatic hydrolysis conditions were 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the enzymatic hydrolysate was 29.42 g / L. After adjusting the pH to 7.0 with CaO, oil-producing fermentation was carried out. The organic matter-to-biomass yield was 25%, and the sugar-to-oil yield was 12%.

[0056] Example 6

[0057] Corn stalks were crushed and added to an aqueous solution containing 1 wt% oxalic acid and 0.5 wt% propylene glycol at a mass ratio of 1:6. The mixture was pretreated at 170℃ for 1 h. After the reaction was completed, the temperature was lowered, and 5 mg / g of cellulase was added. The mixture was then hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the hydrolysate was 35.3 g / L. After adjusting the pH to 7.0 with CaO, oil-producing fermentation was carried out. The organic matter-to-biomass yield was 15%, and the sugar-to-oil yield was 7.5%.

[0058] Example 7

[0059] Sugarcane bagasse was crushed and added to an aqueous solution containing 0.5 wt% acetic acid and 0.5 wt% glycerol at a mass ratio of 1:8. The mixture was pretreated at 160℃ for 4 h. After the reaction was completed, the temperature was lowered and 15 mg / g of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzymatic hydrolysate was 34.34 g / L.

[0060] Example 8

[0061] Sugarcane bagasse was crushed and added to an aqueous solution containing 2 wt% acetic acid and 0.1% glycerol at a mass ratio of 1:8. The mixture was pretreated at 155℃ for 4 h. After the reaction was completed, the temperature was lowered and 20 mg / g of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzymatic hydrolysate was 33.18 g / L.

[0062] Example 9

[0063] Corn stalks were crushed and added to an aqueous solution containing 1 wt% acetic acid and 1.5 wt% glycerol at a mass ratio of 1:10. The mixture was pretreated at 180℃ for 0.5 h. After the reaction was completed, the temperature was lowered and 25 mg / L of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzymatic hydrolysate was 53.7 g / L.

[0064] Example 10

[0065] Sugarcane bagasse was crushed and added to an aqueous solution containing 1 wt% levulinic acid at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. After the reaction was completed, the temperature was lowered and 20 mg / g of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzyme hydrolysate was 55.05 g / L.

[0066] Example 11

[0067] Sugarcane bagasse was crushed and added to an aqueous solution containing 1 wt% levulinic acid and 0.5 wt% glycerol at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. After the reaction was completed, the temperature was lowered and 20 mg / g of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzymatic hydrolysate was 51.78 g / L.

[0068] Example 12

[0069] Sugarcane bagasse was crushed and added to an aqueous solution containing 1 wt% acetic acid and 0.5 wt% Tween 60 at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. After the reaction was completed, the temperature was lowered and 20 mg / g of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzymatic hydrolysate was 56.45 g / L.

[0070] Example 13

[0071] Sugarcane bagasse was crushed and added to an aqueous solution containing 1 wt% acetic acid and 0.5 wt% Tween 80 at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. After the reaction was completed, the temperature was lowered and 20 mg / g of cellulase was added. The mixture was hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzyme hydrolysate was 54.70 g / L.

[0072] Example 14

[0073] Sugarcane bagasse was crushed and added to an aqueous solution containing 1 wt% acetic acid and 0.5 wt% polyethylene glycol at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. Cellulase at 10 mg / g was added and hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugars in the enzymatic hydrolysate was 54.94 g / L.

[0074] Example 15

[0075] Sugarcane bagasse was crushed and added to an aqueous solution of 1.25% acetic acid and 0.5% glycerol at a solid-liquid mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h, then cooled to 50℃, and 20 mg / g of cellulase was added. Enzymatic hydrolysis was performed at 50℃ for 72 h. After hydrolysis, solid-liquid separation was performed, and the concentration of fermentable sugars in the hydrolysate was 45.53 g / L.

[0076] Comparative Example 4

[0077] Wheat straw was crushed and added to an aqueous solution containing 0.5 wt% acetic acid at a mass ratio of 1:8. The reaction was carried out at 160℃ for 2.5 h. After the reaction was completed, the temperature was lowered to 50℃, and 25 mg / g of cellulase from the raw material was added. The mixture was then hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the in-situ enzyme hydrolysate was 24.80 g / L. CaO was added to adjust the pH of the enzyme hydrolysate to 5.0 before oil production fermentation was carried out. The organic matter-to-biomass yield was 18%, and the sugar-to-oil yield was 8.8%.

[0078] Comparative Example 5

[0079] Sugarcane bagasse was crushed and added to an aqueous solution containing 1.5 wt% acetic acid at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. Cellulase at 10 mg / g was added and hydrolyzed at 50℃ for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the in-situ enzymatic hydrolysate was 32.55 g / L.

[0080] Comparative Example 6

[0081] Sugarcane bagasse was crushed and added to an aqueous solution containing 1.5 wt% polyethylene glycol at a mass ratio of 1:8. The mixture was pretreated at 170°C for 1 h. Cellulase at 10 mg / g was added and hydrolyzed at 50°C for 72 h. After the enzymatic hydrolysis was completed, solid-liquid separation was performed. The concentration of fermentable sugar in the in-situ enzymatic hydrolysate was 13.4 g / L.

[0082] Comparative Example 7

[0083] Sugarcane bagasse was crushed and added to water at a mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 h. Then, 20 mg / g of cellulase was added, and the enzymatic hydrolysis was performed at 50℃ for 72 h. After enzymatic hydrolysis, solid-liquid separation was performed, and the concentration of fermentable sugars in the in-situ enzymatic hydrolysate was 28.50 g / L.

[0084] Comparative Example 8

[0085] Sugarcane bagasse was crushed and added to water at a mass ratio of 1:8. The mixture was pretreated at 160℃ for 2.5 h, and then 10 mg / g cellulase was added. Enzymatic hydrolysis was performed at 50℃ for 72 h. After hydrolysis, solid-liquid separation was performed, and the concentration of fermentable sugars in the in-situ enzyme hydrolysate was 19.73 g / L.

[0086] Comparative Example 9

[0087] Sugarcane bagasse was crushed and added to a 1.25% sulfuric acid aqueous solution at a solid-liquid mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 hour, then cooled to 50℃, and 20 mg / g cellulase was added. Enzymatic hydrolysis was performed at 50℃ for 72 hours. After hydrolysis, solid-liquid separation was performed, and the concentration of fermentable sugars in the in-situ enzyme hydrolysate was 21.60 g / L.

[0088] Comparative Example 10

[0089] Sugarcane bagasse was crushed and added to a 1.25% acetic acid aqueous solution at a solid-liquid mass ratio of 1:8. The mixture was pretreated at 170℃ for 1 hour, then cooled to 50℃, and 20 mg / g cellulase was added. Enzymatic hydrolysis was performed at 50℃ for 72 hours. After hydrolysis, solid-liquid separation was performed, and the concentration of fermentable sugars in the in-situ enzyme hydrolysate was 40.53 g / L.

[0090] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for pretreating agricultural and forestry waste with low concentration of adjuvants and low organic acid content, coupled with in-situ enzymatic hydrolysis, characterized in that, The process includes the following steps: using pulverized agricultural and forestry waste as raw material, adding an aqueous solution containing low concentrations of adjuvants and organic acids to the raw material, wherein the adjuvants are selected from one or more of propylene glycol, glycerol, Tween, ethylene glycol, and polyethylene glycol, the mass fraction of the adjuvants in the aqueous solution containing the adjuvants and organic acids is 0-1.5%, and the mass fraction of the organic acids in the aqueous solution containing the adjuvants and organic acids is 0.5%-2.0%, pretreatment by reacting at 140℃-185℃ for 0.5-4.0 h, after which the temperature is lowered to 45℃-55℃, and 5-25 mg / g of cellulase of the raw material is added, hydrolyzing at 45℃-55℃ for 65-75 h, after which solid-liquid separation is performed to obtain solid residue and in-situ enzyme hydrolysate, and the in-situ enzyme hydrolysate is adjusted to pH and then fermented for oil production.

2. The method according to claim 1, characterized in that, The agricultural and forestry waste mentioned above is selected from one or more of sugarcane bagasse, wheat straw, and corn stalks.

3. The method according to claim 1 or 2, characterized in that, The mass ratio of the agricultural and forestry waste to the aqueous solution containing auxiliaries and organic acids is 1:6 to 1:

12.

4. The method according to claim 2, characterized in that, The agricultural and forestry waste is sugarcane bagasse. The pretreatment conditions are 160℃-170℃ reaction for 1.0-1.5 h. The aqueous solution containing auxiliaries and organic acids has an organic acid mass fraction of 1.0%-1.5% and an auxiliaries mass fraction of 0.5%.

5. The method according to claim 1 or 2, characterized in that, The organic acid is selected from one of oxalic acid, acetic acid, levulinic acid, and succinic acid.

6. The method according to claim 5, characterized in that, The organic acid is acetylpropionic acid or succinic acid, and no additives are added to the aqueous solution.

7. The method according to claim 1, characterized in that, After the reaction is complete, the temperature is lowered to 50°C, and 5-25 mg / g of cellulase from the raw material is added. The mixture is then hydrolyzed at 50°C for 24-72 hours.

8. The method according to claim 1, characterized in that, The specific steps for oil-producing fermentation after adjusting the pH of the in-situ enzyme hydrolysate are as follows: the oil-producing yeast strain is transferred from the slant to the seed culture medium and activated for 24-36 h. The in-situ enzyme hydrolysate is adjusted to pH 5.0-7.0 to serve as the fermentation medium. The activated strain is inoculated into the fermentation medium at an inoculation rate of 5%-10%. After fermentation at 25℃-35℃ for 84-120 h, the cells are collected by centrifugation or filtration. The microbial oil is then extracted by acid-heat method or direct oil pressing. The seed culture medium consists of the following raw materials in the following mass percentages: glucose 2%, yeast powder 1%, peptone 1%, and the remainder is deionized water.

9. The method according to claim 8, characterized in that, The oily yeast strains mentioned are selected from one or more of the following: R. glutinis, Trichosporon cutaneum, Cryptococcus albidus, Yarrowialipolytica, Rhodosporidium torulodides, Lipomyces starkeyi, Cutaneotrichosporon dermatis, and Trichosporon mucoides.

10. The method according to claim 8, characterized in that, The pH of the enzyme hydrolysate can be adjusted to 5.0-7.0 by adding NaOH, Ca(OH)2 or CaO, without the need for other detoxification steps.

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