Method for producing lactic acid by efficiently utilizing lignocellulose pretreatment liquid

Abstract

The invention discloses a method for producing lactic acid by efficiently utilizing lignocellulose pretreatment liquid through fermentation. The method comprises the steps of preparing lignocellulose detoxification saccharification liquid and producing lactic acid through fermentation. The preparation process of the lignocellulose detoxified saccharified liquid mainly comprises the following steps: adding hydrogen peroxide under an acidic condition to react for a period of time, then adding manganese dioxide to remove excessive hydrogen peroxide, and then adding cellulase to obtain the lignocellulose saccharified liquid; the process for producing lactic acid by fermentation comprises the following steps: activating seeds, preparing a seed solution, introducing gas for a period of time after inoculation, and then keeping anaerobic fermentation to produce lactic acid. According to the method, most of aldehydes such as furfural, 5-hydroxymethylfurfural and vanillin and phenolic inhibitors such as acid-soluble lignin in the pretreatment liquid are removed. Divalent manganese ions generated in the detoxification step and a proper amount of ventilation operation in the initial stage of fermentation are beneficial to growth metabolism and fermentation production of lactic acid bacteria, and the sugar-acid conversion rate reaches up to 96% or above.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and particularly relates to a method for efficiently utilizing lignocellulose hydrolyzate to ferment and produce lactic acid. Background Art

[0002] Lactic acid is one of the three major organic acids and one of the most commonly used organic acids in human daily life. Lactic acid is an important biochemical product and is widely used in food, beverages, daily necessities, medicine, petrochemicals, tobacco, textiles and other fields; it can also be used as an acidulant, flavoring agent, antibacterial agent and preservative. Lactic acid polymers are biodegradable and biocompatible polymer materials that can be used to make disposable tableware, shopping bags, plastic bags, agricultural films, sanitary products, etc., thereby alleviating the environmental pressure caused by white pollution. It can also be used to prepare orthopedic fixation materials, drug sustained-release materials, surgical sutures and other modern medical materials. Therefore, lactic acid has broad market applications and demand.

[0003] At present, the industrial production of lactic acid is mainly based on biofermentation, with starch-based raw materials such as corn, cassava, and potatoes, and sugar-based raw materials such as sugarcane and beets. This production method not only has high raw material prices, but also has the problem of "competing with people for food and competing with food for land". Cheap and readily available agricultural and forestry wastes, such as corn straw, sugarcane bagasse, wheat straw and other lignocellulose, are good alternative raw materials. Lignocellulose is a heterogeneous polymer mainly composed of cellulose, hemicellulose, and lignin. Lignin is directly cross-linked with cellulose and hemicellulose, and esterified with carbohydrates by ferulic acid cross-linking to form a carbohydrate-ferulic acid lignin structure, and is also attached to the cellulose surface through van der Waals forces and hydrogen bonds. This further exacerbates the complexity and degradation resistance of the lignocellulose cell wall, seriously hindering its bioconversion.

[0004] Pretreatment is the first and key step in the utilization of lignocellulose, which is mainly divided into physical, chemical, biological and physicochemical methods. In the process of pretreatment of lignocellulose by different methods, it is inevitable to produce inhibitors, and there are often more than one inhibitor. The presence of multiple inhibitors also has a synergistic inhibitory effect on fermentation. Acid pretreatment, alkali pretreatment and steam explosion pretreatment are the most widely used pretreatment methods. During the acid pretreatment process, hemicellulose produces pentose and hexose under the action of acid, and pentose and hexose are dehydrated to produce furfural and 5-hydroxymethylfurfural. Alkaline pretreatment is mainly caused by alkali attacking lignin to produce phenolic inhibitors. According to the type of R branched functional group on the benzene ring, phenolic substances are divided into phenolic aldehydes represented by coniferyl aldehyde and p-hydroxybenzaldehyde; phenolic alcohols represented by coniferyl alcohol, mustard alcohol, and p-hydroxybenzyl alcohol. The steam explosion pretreatment process is accompanied by the degradation of hemicellulose and lignin to produce some phenolic and furfural substances, which makes the utilization rate of pretreatment low.

[0005] Furan compounds such as furfural and 5-hydroxymethylfurfural inhibit bacterial growth mainly by destroying the permeability of cell membranes and inhibiting the activity of key enzymes involved in microbial metabolism in cells; they attenuate a large amount of translation activities in microorganisms, thereby degenerating the physiological state of microorganisms; furfural can also induce the accumulation of reactive oxygen in microbial cells, leading to damage to mitochondria, vacuole membranes and nuclear chromatin, and even causing cell death. The inhibitory mechanism of furans involves aspects such as the integrity, synthesis and metabolic processes of microbial cell structure, which seriously affects the bioconversion rate. The inhibitory effect of phenolic substances is mainly reflected in the ability of the hydroxyl groups on the phenolic side chains to adsorb proteins, bind to enzymes to precipitate and inactivate them; cause gene mutations, destroy the integrity of cells; and destroy the antioxidant defense system of cells.

[0006] Lignocellulose pretreatment inhibitors have a significant inhibitory effect on microorganisms and are one of the main bottlenecks in lactic acid fermentation production. In order to reduce the negative impact of inhibitors, researchers have conducted some research on the improvement of fermentation processes in recent years, such as adding a detoxification step. Adsorption detoxification is a common detoxification method, which mainly uses adsorption resins and cross-linked polyethyleneimine to selectively adsorb furans and phenols in the pretreatment liquid. Deng (Ind Crops Prod, 2018, 112: 681-690) et al. used PEI and polydiallyldimethylammonium chloride to treat sugarcane bagasse hydrolyzate after dilute ammonia pretreatment, which can remove 43% of organic acids and 73% of total phenolic compounds. López-Linares et al. (Enzyme Microb Technol, 2020, 135:109490) used ion exchange resins to adsorb inhibitors in sulfuric acid hydrolysis of rapeseed straw, and phenolic and furanaldehyde compounds were reduced to a relatively low level. Although physical adsorption is effective in removing inhibitors, the activation and regeneration of adsorbents such as resins require a large amount of water and chemicals, which puts great pressure on the environment and is costly. In addition, the adsorption method often fills the resin column to facilitate recycling, which increases equipment and site investment.

[0007] Persson et al. (J Agric Food Chem, 2002, 50(19):5318-5325.) studied the detoxification effects of four alkalis: sodium hydroxide, calcium hydroxide, potassium hydroxide, and ammonia. The results showed that the removal rate of the inhibitor was about 40%. Haq et al. (J Chem Technol Biotechnol, 2018, 93(10): 3011-3017) used calcium hydroxide for detoxification and increased the removal rate of phenolic substances to 60% by increasing the reaction temperature to 80 °C. Xie et al. (Bioresour Technol, 2015, 186: 106-113.) used 20 amino acids to treat lignocellulosic pretreatment liquid and found that cysteine ​​had the best effect, consuming 96% of 5-hydroxymethylfurfural, but the removal effect of other inhibitors was not obvious. Reducing agents such as dithionite, sulfite, and thiothioate have also been used in the detoxification process of lignocellulosic pretreatment liquid. Although reducing agents can be used for in situ detoxification under mild conditions, the effect is poor and needs to be combined with other detoxification methods such as alkaline detoxification.

[0008] In the current lignocellulose-based lactic acid fermentation process, some of them directly use hydrolyzate containing a large amount of inhibitors for fermentation without detoxification, resulting in low lactic acid yield; and the fermentation process using physical adsorption detoxification method has low production efficiency due to the investment of additional equipment and cumbersome operation procedures; the fermentation process using chemical detoxification method often introduces new inhibitors due to incomplete detoxification or residual detoxification agent. Therefore, it is still necessary to develop a fermentation process that includes an in-situ detoxification method that does not require additional equipment investment to facilitate the efficient fermentation production of lactic acid. Summary of the invention

[0009] The purpose of the present invention is to provide a method for producing lactic acid by efficiently utilizing lignocellulose pretreatment liquid in view of the deficiencies of the prior art.

[0010] In order to achieve the above purpose, the following technical solutions are adopted: A method for efficiently utilizing wood fiber pretreatment liquid to produce lactic acid comprises the following steps: (1) The lignocellulose pretreatment liquid is placed in a fermentation tank, the pH is adjusted, a detoxifying agent is added, and the mixture is stirred evenly, and the mixture is heated and kept warm for a specified time; (2) Add oxidant in batches until no bubbles are generated in the system; (3) heating to a certain temperature, adjusting the pH, adding cellulase for hydrolysis and saccharification, and adjusting the pH with alkali during the hydrolysis and saccharification process to obtain a detoxified lignocellulose hydrolyzate; (4) inoculating Bacillus coagulans on a slant medium to obtain a single colony, and inoculating the single colony into a seed medium to obtain a seed solution; (5) Inoculate the seed liquid into the fermentation medium, maintain a certain temperature, and allow air to ferment; during the fermentation process, add alkaline solution to maintain the pH of the fermentation system at 6.

[0011] Furthermore, the raw material of the lignocellulose pretreatment liquid in step (1) is any one of corn straw, sugarcane bagasse, rice straw, tobacco straw, and bamboo shoot husk.

[0012] Furthermore, the detoxifying agent in step (1) is hydrogen peroxide with a concentration of 0.5% to 1.5%, and the amount of the detoxifying agent is 0.1% to 1% of the mass of the lignocellulose pretreatment liquid raw material; the pH is adjusted to 1 to 4, the heating temperature is 30 to 40°C, and the rated insulation time is 0.5 to 2 h.

[0013] Furthermore, the oxidant in step (2) is manganese dioxide, and the amount used is 0.01% to 0.2% of the mass of the lignocellulose pretreatment liquid raw material.

[0014] Furthermore, in step (3), the heating temperature is 45-55°C, the pH is 4.5-6; the amount of cellulase added is 5-20 FPU per gram of lignocellulose raw material, and the hydrolysis saccharification time is 36-48 h.

[0015] Furthermore, the alkali solution in step (3) and step (5) is any one of ammonia water, potassium hydroxide, calcium hydroxide, and sodium hydroxide.

[0016] Furthermore, the slant culture medium in step (4) comprises 5 g / L tryptone, 15 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 2 g / L glucose, 1 g / L xylose, and 20 g / L agar, and the pH is adjusted to 7-8.

[0017] Furthermore, the components of the seed culture medium in step (5) are yeast extract powder 20 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, glucose 45 g / L, xylose 15 g / L, manganese sulfate monohydrate 0.015 g / L, and calcium carbonate 20 g / L.

[0018] Furthermore, the fermentation medium in step (5) comprises detoxified lignocellulose hydrolyzate and yeast extract, and the amount of yeast extract is 10 g of yeast extract per liter of lignin pretreatment liquid.

[0019] Furthermore, in step (5), the fermentation temperature is 45-55°C, the ventilation time is 1-3 h, and the ventilation volume is 1-3 vvm.

[0020] The beneficial effects of the present invention are: The present invention uses hydrogen peroxide as the primary detoxification agent and has the following advantages: (1) The detoxification step and saccharification step can be carried out in the same reactor, which is easy to operate.

[0021] (2) Hydrogen peroxide oxidizes furfural, 5-hydroxymethylfurfural, vanillin and other aldehydes in lignocellulose into acids, greatly reducing the toxicity of inhibitors; and oxidizes phenolic substances in acid-soluble lignin into quinones, reducing their cytotoxicity to microorganisms. At the same time, the reduction product of hydrogen peroxide is water, which is clean and pollution-free.

[0022] (3) The oxidant manganese dioxide can quickly remove hydrogen peroxide from the solution, preventing the residual hydrogen peroxide from having adverse effects on the subsequent saccharification and fermentation steps; at the same time, manganese dioxide itself is reduced to divalent manganese ions, which are cofactors of many enzymes and participate in catalyzing various biochemical reactions. It can promote the growth and metabolism of lactic acid bacteria and improve production efficiency.

[0023] (4) Hydrogen peroxide is oxidized to release oxygen, which can be used as dissolved oxygen by facultative anaerobic lactic acid bacteria. After the oxygen is dissolved to saturation, the remaining oxygen is discharged through the bubbler, which can be used as a criterion for judging whether the hydrogen peroxide has been completely decomposed.

[0024] (5) This process can be detoxified in situ, is easy to operate, and has mild conditions. The detoxifying agent has no adverse effects on the subsequent saccharification and fermentation steps, and the manganese ions produced by reduction also contribute to lactic acid fermentation production.

[0025] The lignocellulose lactic acid fermentation process has the following advantages: (1) Adding xylose to the slant culture medium and seed culture medium is beneficial to quickly activate the xylose metabolic pathway of lactic acid bacteria and improve fermentation efficiency.

[0026] (2) The ratio of carbon sources glucose and xylose in the seed liquid is 3:1, which is close to the carbon source ratio in the lignocellulose hydrolysate, which helps lactic acid bacteria adapt to the fermentation liquid environment faster.

[0027] (3) The fermentation medium does not require the addition of additional inorganic salts and contains trace amounts of divalent manganese ions obtained in the detoxification step, which is beneficial to the growth, metabolism and fermentation production of lactic acid bacteria.

[0028] (4) The fermentation temperature is high and other bacteria cannot survive, so open fermentation can be performed.

[0029] (5) Introducing an appropriate amount of air at the initial stage of seed liquid inoculation is conducive to the rapid growth of facultative anaerobic lactic acid bacteria.

[0030] In summary, the present invention solves the problem of incomplete detoxification and residual detoxification agent in the production process of lignocellulose-based lactic acid. Manganese dioxide is used as an oxidant to oxidize hydrogen peroxide under acidic conditions, and it is itself reduced to divalent manganese ions that are beneficial to the growth of lactic acid bacteria; glucose and xylose with a carbon source ratio close to that in the saccharification liquid are added to the slant and seed culture medium, which is beneficial for lactic acid bacteria to adapt to the fermentation environment as soon as possible. After inoculation, an appropriate amount of air is introduced, which is beneficial to the rapid growth of facultative anaerobic lactic acid bacteria and improves the fermentation production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Example 1 The content of inhibitors in corn straw pretreatment liquid before and after detoxification.

[0032] Figure 2 : Example 2 The content of inhibitors in bagasse pretreatment liquid before and after detoxification.

[0033] Figure 3 : Example 3 Content of inhibitors in rice straw pretreatment liquid before and after detoxification.

[0034] Figure 4 : Comparative Example 2: Content of inhibitors in corn straw pretreatment liquid before and after detoxification.

[0035] Figure 5 : Comparative Example 3: Content of inhibitors in corn straw pretreatment liquid before and after detoxification.

[0036] Figure 6 : Comparative Example 4: Content of inhibitors in corn straw pretreatment liquid before and after detoxification. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The exemplary implementation modes of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0038] The formula of the following slant medium is: tryptone 5 g / L, yeast extract 15 g / L, potassium dihydrogen phosphate 3 g / L, glucose 2 g / L, xylose 1 g / L, agar 20 g / L, potassium hydroxide to adjust pH 7-8.

[0039] The formula of the seed culture medium is: yeast extract powder 20 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, glucose 45 g / L, xylose 15 g / L, manganese sulfate monohydrate 0.015 g / L, and calcium carbonate 20 g / L.

[0040] Bacillus coagulans was provided by Tan Tianwei's laboratory at Beijing University of Chemical Technology.

[0041] Example 1 10 kg corn stalks were soaked in 12 kg 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain corn stalk pretreatment liquid. 2 L of pretreatment liquid was loaded into a fermenter, the pH was adjusted to 2 with potassium hydroxide, 30 mL of 1% hydrogen peroxide was added and stirred evenly, heated to 35 °C and kept warm for 1 h. Manganese dioxide was slowly added in 4 batches until no bubbles were generated in the system (1.86 g of manganese dioxide was used in the end). Heat to 50 °C, adjust the pH to 4.5 with potassium hydroxide, add cellulase 10 FPU / g corn stalks, and hydrolyze and saccharify for 36 h. After saccharification, adjust the pH in the fermenter to 6 with potassium hydroxide, and add 20 g of yeast extract powder for standby use.

[0042] Bacillus coagulans was inoculated on the slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into the seed medium and cultured at 50 °C for 24 h to prepare the seed solution. The seed solution was inoculated into the fermenter at a mass ratio of 3%, and compressed air was introduced at a flow rate of 1vvm for 2 h. The gas valve was then closed and fermented at 50 °C and 300 rpm for 36 h. A 30% potassium hydroxide solution was added to keep the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 128 g / L, and the sugar-acid conversion rate was 96.3%.

[0043] Example 2 10 kg of sugarcane bagasse was soaked in 12 kg of 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain the sugarcane bagasse pretreatment liquid. 2 L of pretreatment liquid was loaded into the fermentation tank, the pH was adjusted to 2 with potassium hydroxide, 30 mL of 1% hydrogen peroxide was added and stirred evenly, and the mixture was heated to 35 °C and kept warm for 1 h. Manganese dioxide was slowly added in 4 batches until no bubbles were generated in the system (1.77 g of manganese dioxide was used in the end). Heat to 50 °C, adjust the pH to 5 with potassium hydroxide, add cellulase 10 FPU / g corn straw, and hydrolyze and saccharify for 36 h. After saccharification, the pH in the fermentation tank was adjusted to 6 with potassium hydroxide, and 20 g of yeast extract powder was added for standby use.

[0044] Bacillus coagulans was inoculated on the slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into the seed medium and cultured at 50 °C for 24 h to prepare the seed solution. The seed solution was inoculated into the fermenter at a mass ratio of 3%, and compressed air was introduced at a flow rate of 1.5 vvm and maintained for 2 h. The gas valve was then closed, and the fermentation was carried out at 50 °C and 300 rpm for 36 h, and 30% potassium hydroxide solution was added to keep the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 136 g / L, and the sugar-acid conversion rate was 96.7%.

[0045] Example 3 10 kg of rice straw was soaked in 12 kg of 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain rice straw pretreatment liquid. 2 L of pretreatment liquid was put into a fermentation tank, the pH was adjusted to 2 with potassium hydroxide, 30 mL of 1% hydrogen peroxide was added and stirred evenly, and the mixture was heated to 35 °C and kept warm for 1 h. Manganese dioxide was slowly added in 4 batches until no bubbles were generated in the system (1.90 g of manganese dioxide was used in the end). Heat to 50 °C, adjust the pH to 5.5 with potassium hydroxide, add cellulase 15 FPU / g corn straw, and hydrolyze and saccharify for 36 h. After saccharification, the pH in the fermentation tank was adjusted to 6 with potassium hydroxide, and 20 g of yeast extract powder was added for standby use.

[0046] Bacillus coagulans was inoculated on a slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into the seed medium and cultured at 50 °C for 24 h to prepare the seed solution. The seed solution was inoculated into the fermenter at a mass ratio of 3%, and compressed air was introduced at a flow rate of 1 vvm and maintained for 2 h. The gas valve was then closed, and the fermentation was carried out at 50 °C and 300 rpm for 36 h, and 30% potassium hydroxide solution was added to maintain the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 118 g / L, and the sugar-acid conversion rate was 95.2%.

[0047] Comparative Example 1 (without detoxification step) 10 kg corn stalks were soaked in 12 kg 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain corn stalk pretreatment liquid. 2 L of pretreatment liquid was put into a fermentation tank, heated to 50 °C, adjusted to pH 4.5 with potassium hydroxide, added with cellulase 10 FPU / g corn stalks, and hydrolyzed for 36 h. After saccharification, potassium hydroxide was used to adjust the pH in the fermentation tank to 6, and 20 g of yeast extract powder was added for standby use.

[0048] Bacillus coagulans was inoculated on a slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into the seed medium and cultured at 50 °C for 24 h to prepare the seed solution. The seed solution was inoculated into the fermenter at a mass ratio of 3%, and compressed air was introduced at a flow rate of 1vvm and maintained for 2 h. The gas valve was then closed, and the fermentation was carried out at 50 °C and 300 rpm for 36 h, and 30% potassium hydroxide solution was added to maintain the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 78 g / L, and the sugar-acid conversion rate was 58.7%.

[0049] Comparative Example 2 (without addition of manganese dioxide) 10 kg corn stalks were soaked in 12 kg 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain corn stalk pretreatment liquid. 2 L of pretreatment liquid was put into a fermentation tank, the pH was adjusted to 2 with potassium hydroxide, 30 mL of 1% hydrogen peroxide was added and stirred evenly, and the mixture was heated to 35 °C and kept warm for 1 h. Heat to 50 °C, adjust the pH to 4.5 with potassium hydroxide, add 10 FPU / g corn stalks of cellulase, and hydrolyze for 36 h. After saccharification, the pH in the fermentation tank was adjusted to 6 with potassium hydroxide, and 20 g of yeast extract powder was added for standby use.

[0050] Bacillus coagulans was inoculated on the slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into the seed medium and cultured at 50 °C for 24 h to prepare the seed solution. The seed solution was inoculated into the fermenter at a mass ratio of 3%, and compressed air was introduced at a flow rate of 1vvm and maintained for 2 h. The gas valve was then closed, and the fermentation was carried out at 50 °C and 300 rpm for 36 h, and 30% potassium hydroxide solution was added to maintain the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 95 g / L, and the sugar-acid conversion rate was 71.5%.

[0051] Comparative Example 3 10 kg corn stalks were soaked in 12 kg 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain corn stalk pretreatment liquid. 2 L of pretreatment liquid was put into a fermentation tank, the pH was adjusted to 2 with potassium hydroxide, 30 mL of 1% hydrogen peroxide was added, stirred evenly, heated to 35 °C, and kept warm for 1 h. Manganese dioxide was slowly added in 4 batches until no bubbles were generated in the system (1.85 g of manganese dioxide was used in the end). Heat to 50 °C, adjust the pH to 4.5 with potassium hydroxide, add cellulase 10 FPU / g corn stalks, and hydrolyze and saccharify for 36 h. After saccharification, the pH in the fermentation tank was adjusted to 6 with potassium hydroxide, and 20 g of yeast extract powder was added for standby use.

[0052] Bacillus coagulans was inoculated on the slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into the seed medium and cultured at 50 °C for 24 h to prepare the seed solution. The seed solution was inoculated into the fermenter at a mass ratio of 3%, and compressed air was introduced at a flow rate of 1vvm and maintained for 2 h. The gas valve was then closed, and the fermentation was carried out at 50 °C and 300 rpm for 36 h, and 30% potassium hydroxide solution was added to maintain the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 101 g / L, and the sugar-acid conversion rate was 76.0%.

[0053] In this embodiment, the slant culture medium is 5 g / L tryptone, 15 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 2 g / L glucose, and 20 g / L agar, and the pH is adjusted to 7-8.

[0054] The seed culture medium comprises components in the following concentrations: 20 g / L yeast extract, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L potassium hydrogen phosphate, 45 g / L glucose, 0.015 g / L manganese sulfate monohydrate, and 20 g / L calcium carbonate.

[0055] Comparative Example 4 10 kg corn stalks were soaked in 12 kg 2% dilute sulfuric acid solution for 8 h, and steam explosion pretreatment was performed at 180 °C and 800 kPa for 10 min to obtain corn stalk pretreatment liquid. 2 L of pretreatment liquid was put into a fermentation tank, the pH was adjusted to 2 with potassium hydroxide, 30 mL of 1% hydrogen peroxide was added, stirred evenly, heated to 35 °C, and kept warm for 1 h. Manganese dioxide was slowly added in 4 batches until no bubbles were generated in the system (1.86 g of manganese dioxide was used in the end). Heat to 50 °C, adjust the pH to 4.5 with potassium hydroxide, add cellulase 10 FPU / g corn stalks, and hydrolyze and saccharify for 36 h. After saccharification, adjust the pH in the fermentation tank to 6 with potassium hydroxide, and add 20 g of yeast extract powder for standby use.

[0056] Bacillus coagulans was inoculated on a slant medium and cultured at 50 °C for 24 h to obtain a single colony. An appropriate amount of single colonies was picked and inoculated into a seed medium and cultured at 50 °C for 24 h to prepare a seed solution. The seed solution was inoculated into a fermenter at a mass ratio of 3%. Fermentation was carried out at 50 °C and 300 rpm for 36 h, and 30% potassium hydroxide solution was added to maintain the pH of the fermentation system at 6. After the fermentation, the lactic acid content was measured by high performance liquid chromatography to be 98 g / L, and the sugar-acid conversion rate was 73.7%.

[0057] The lignocellulose pretreatment liquids of Examples 1-3 and Comparative Examples 2-4 were sampled before and after detoxification to measure the contents of furfural, 5-hydroxymethylfurfural, vanillin and acid-soluble lignin. Figure 1-6 The results showed that detoxification with hydrogen peroxide can significantly reduce inhibitors such as furfural, 5-hydroxymethylfurfural, vanillin, and acid-soluble lignin in lignocellulose.

[0058] The present invention uses lignocellulose pretreatment liquid as raw material, detoxifies the pretreatment liquid under acidic conditions with hydrogen peroxide as a detoxifying agent, and removes excess hydrogen peroxide with manganese dioxide, which is itself reduced to divalent manganese ions that are beneficial to the growth of lactic acid bacteria; glucose and xylose with a carbon source ratio close to that in the saccharification liquid are added to the slant and seed culture medium, which is beneficial for lactic acid bacteria to adapt to the fermentation environment as soon as possible; after inoculation, a proper amount of air is introduced, which is beneficial for the rapid growth of facultative anaerobic lactic acid bacteria, and after fermentation for 36 hours, the lactic acid concentration is as high as 136 g / L, and the sugar-acid conversion rate is 96.7%. In the comparative example, undetoxified lignocellulose pretreatment liquid and detoxified pretreatment liquid that has not been detoxified but has not been detoxified with manganese dioxide to remove excess hydrogen peroxide are used respectively, followed by enzymatic saccharification and inoculation of Bacillus coagulans, and fermentation for 36 hours under the same conditions, and the lactic acid concentration and sugar-acid conversion rate in the fermentation liquid are greatly reduced. Under the conditions of detoxification of the pretreatment liquid under acidic conditions with hydrogen peroxide as an oxidant and removal of excess hydrogen peroxide with manganese dioxide, the use of slant and seed culture medium without xylose addition or the fermentation tank was not ventilated for 2 hours after inoculation, all led to a decrease in lactic acid fermentation efficiency, with a sugar-acid conversion rate of less than 76%. In summary, the process of the present invention has great advantages over the traditional lactic acid fermentation process.

[0059] The specific embodiments described above can further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail, but it should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for efficiently utilizing wood fiber pretreatment liquid to produce lactic acid, characterized in that: The steps include: (1) The lignocellulose pretreatment liquid is placed in a fermentation tank, the pH is adjusted, a detoxifying agent is added, and the mixture is stirred evenly, and the mixture is heated and kept warm for a specified time; (2) Add oxidant in batches until no bubbles are generated in the system; (3) heating to a certain temperature, adjusting the pH, adding cellulase for hydrolysis and saccharification, and then adjusting the pH with alkali to obtain a detoxified lignocellulose hydrolyzate; (4) inoculating Bacillus coagulans on a slant medium to obtain a single colony, and inoculating the single colony into a seed medium to obtain a seed solution; (5) Inoculate the seed liquid into the fermentation medium, maintain a certain temperature, and allow air to flow for fermentation; during the fermentation process, add alkaline solution to maintain the pH of the fermentation system at 6.

2. The method according to claim 1, characterized in that: The raw material of the lignocellulose pretreatment liquid in step (1) is any one of corn straw, sugarcane bagasse, rice straw, tobacco straw and bamboo shoot husk.

3. The method according to claim 1, characterized in that: In step (1), the detoxifying agent is hydrogen peroxide with a concentration of 0.5% to 1.5%, and the amount of the detoxifying agent is 0.1% to 1% of the mass of the lignocellulose pretreatment liquid raw material; the pH is adjusted to 1 to 4, the heating temperature is 30 to 40°C, and the rated insulation time is 0.5 to 2 h.

4. The method according to claim 1, characterized in that: The oxidant in step (2) is manganese dioxide, and the amount used is 0.01% to 0.2% of the mass of the lignocellulose pretreatment liquid raw material.

5. The method according to claim 1, characterized in that: The heating temperature in step (3) is 45-55°C, the pH is 4.5-6; the amount of cellulase added is 5-20 FPU per gram of lignocellulose raw material, and the hydrolysis saccharification time is 36-48 h.

6. The method according to claim 1, characterized in that: The base in step (3) and step (5) is any one of ammonia water, potassium hydroxide, calcium hydroxide and sodium hydroxide.

7. The method according to claim 1, characterized in that: The slant culture medium in step (4) comprises 5 g / L tryptone, 15 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 2 g / L glucose, 1 g / L xylose, and 20 g / L agar, and the pH is adjusted to 7-8.

8. The method according to claim 1, characterized in that: The components of the seed culture medium in step (5) are yeast extract powder 20 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, glucose 45 g / L, xylose 15 g / L, manganese sulfate monohydrate 0.015 g / L, and calcium carbonate 20 g / L.

9. The method according to claim 1, characterized in that: The fermentation medium in step (5) comprises detoxified lignocellulose hydrolyzate and yeast extract, wherein the amount of yeast extract is 10 g of yeast extract per liter of lignin pretreatment solution.

10. The method according to claim 1, characterized in that: The fermentation temperature in step (5) is 45-55°C, the ventilation time is 1-3 h, and the ventilation volume is 1-3 vvm.

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