Method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation. The method comprises the following specific steps: carrying out enzyme hydrolysis on lignin-removed lignocellulose obtained by pretreating lignocellulose with alkali ammonia; l-lactic acid fermentation liquor obtained by fermenting the hydrolysate is subjected to lactic acid removal treatment, and L-lactic acid and lactic acid removal fermentation liquor are obtained; and carrying out xylitol fermentation on the lactic acid-removed fermentation liquor to obtain the xylitol. According to the method, through alkali ammonia pretreatment, the cellulose content is remarkably increased, the lignin removal rate is increased, the pretreated substrate does not need to be washed and dried, and use of chemical reagents is reduced; meanwhile, the hydrolysate is low in toxicity, detoxification treatment is not needed, and first-stage L-lactic acid fermentation can be directly carried out. The lactic acid-removed fermentation liquor can be directly used for xylitol fermentation, and the co-production fermentation index is stable. The method starts with low dosage of chemical reagents and reduction of operation units, and has remarkable economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation. Background Art

[0002] L-lactic acid (L-LA) and xylitol are important industrial raw materials and are widely used in food, medicine, plastics and other fields. These two raw materials can be synthesized by chemical methods, but they still face the disadvantage of requiring expensive catalysts and equipment. At present, after extensive research, biofermentation methods can produce L-LA and xylitol, but they still face many challenges, mainly due to the high prices of raw materials and reagents and complex operating processes.

[0003] Industrial fermentation to produce L-lactic acid generally uses the first generation of biomass raw materials, mainly food crops such as corn and sweet potatoes. These materials have high starch content, high glucose concentration after saccharification, and high fermentation efficiency. These food raw materials are expensive, accounting for about 50% of the total cost, and will cause food problems. The second generation of biomass raw materials uses agricultural and forestry waste to produce L-LA and xylitol. Its rich resource potential and relatively low cost gradually replace the first generation of biomass raw materials.

[0004] The sources of second-generation biomass raw materials are wide, but seeking suitable raw materials is not only directly related to economic benefits, but also plays a vital role in achieving environmental protection goals. Among them, lignocellulose resources are huge, low-priced, and rich in cellulose, hemicellulose and lignin. However, lignin, as an obstacle to biological fermentation, needs to be removed. Then glucose and xylose are produced by hydrolysis, and finally converted into L-lactic acid and xylitol respectively by fermentation. At present, there are few methods for directly fermenting lignocellulose to produce L-lactic acid and xylitol, mainly because of its complex structure, which is not easy to be degraded and utilized, and the high pretreatment cost, long pretreatment time, and low concentration and yield of L-lactic acid and xylitol make it difficult to achieve industrialization. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation in order to solve the problem of low comprehensive utilization rate of carbon in hydrolyzate in the prior art. Figure 1 A flowchart of the method is shown in FIG.

[0006] In order to solve the above technical problems, the present invention discloses a method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation, comprising the following steps:

[0007] (1) pretreating lignocellulose with alkali ammonia to obtain lignocellulose with lignin removed;

[0008] (2) enzymatically hydrolyzing the delignified lignocellulose obtained in step (1) to obtain a hydrolyzate;

[0009] (3) using the hydrolyzate obtained in step (2) to perform L-lactic acid fermentation to obtain L-lactic acid fermentation liquid;

[0010] (4) performing a delactic acid treatment on the L-lactic acid fermentation broth in step (3) to obtain calcium lactate and delactic acid fermentation broth, and purifying the calcium lactate to obtain L-lactic acid;

[0011] (5) using the delactic acid fermentation liquid obtained in step (4) to perform xylitol fermentation to obtain xylitol fermentation liquid, and then separating and purifying the xylitol fermentation liquid to obtain xylitol;

[0012] Wherein, when the delignified lignocellulose is subjected to enzymatic hydrolysis, it is not necessary to wash and dry the delignified lignocellulose;

[0013] Wherein, before the hydrolyzate is subjected to L-lactic acid fermentation, the hydrolyzate does not need to be detoxified.

[0014] Wherein, in step (1), the lignocellulose includes any one or a combination of corn straw, corn cob, wheat straw, rice straw and sugarcane bagasse.

[0015] Preferably, the lignocellulose is corn stalks.

[0016] Wherein, in step (1), the alkali ammonia pretreatment is to add 1 to 5 wt% NaOH and 5 to 10 wt% ammonia water to the lignocellulose, and then add water until the solid content of the lignocellulose is 200 to 300 g / L before pretreatment; the alkali ammonia pretreatment has the following pretreatment conditions: temperature 70 to 90°C, time 10 to 24 hours.

[0017] Preferably, the alkali ammonia pretreatment is to add 3wt% NaOH and 7.5wt% ammonia water to the lignocellulose, and then add water until the solid content of the lignocellulose is 300g / L before pretreatment; the alkali ammonia pretreatment has the following pretreatment conditions: temperature 80°C, time 12h.

[0018] Wherein, in step (2), before the delignined lignocellulose is subjected to enzymatic hydrolysis, the moisture content of the delignined lignocellulose is adjusted to 70-80%.

[0019] Preferably, the moisture content of the delignified lignocellulose is adjusted to 70%.

[0020] The enzymatic hydrolysis is carried out by using cellulase, and the amount of the cellulase used is: 5 to 20 FPU cellulase is added for every 1 g of lignin-removed lignocellulose.

[0021] Preferably, the dosage of the cellulase is: 15 FPU cellulase is added per 1 g of delignified lignocellulose.

[0022] The enzymatic hydrolysis conditions are as follows: pH value 4.8-5.2, temperature 45-55° C., rotation speed 150-250 rpm, and time 48-72 h.

[0023] Preferably, the enzymatic hydrolysis is carried out under the following conditions: pH value 4.8-5.0, temperature 50° C., rotation speed 200 rpm, and time 72 h.

[0024] The enzymatic hydrolysis is carried out in batch feeding mode. In the initial stage, 100-150 g / L of delignified lignocellulose with a solid content is added for enzymatic hydrolysis. After 5-8 hours, the remaining delignified lignocellulose is added to a solid content of 200-300 g / L.

[0025] Preferably, the enzymatic hydrolysis is carried out in a batch feeding manner, wherein 15 wt% of the delignified lignocellulose solid content is first added for enzymatic hydrolysis in the initial stage, and after 6 hours, the remaining delignified lignocellulose is supplemented to a solid content of 300 g / L.

[0026] Wherein, in step (3), the hydrolyzate obtained in step (2) is used for L-lactic acid fermentation, specifically, the bacterial liquid of Lactobacillus rhamnosus is inoculated into the hydrolyzate for L-lactic acid fermentation.

[0027] Specifically, before inoculating the bacterial liquid of Lactobacillus rhamnosus into the hydrolyzate, 0.5-5 g / L yeast extract, 0.5-5 g / L tryptone, 1-3 g / L ammonium sulfate, 0.5-2.0 g / L potassium dihydrogen phosphate, and 0.2-0.8 g / L magnesium sulfate heptahydrate are added to the hydrolyzate, and after adjusting the pH to 5.5-6.5, 25-35 g / L calcium carbonate is added.

[0028] Preferably, before inoculating the Lactobacillus rhamnosus bacterial solution into the hydrolyzate, 2 g / L yeast extract, 2 g / L tryptone, 3 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, and 0.6 g / L magnesium sulfate heptahydrate are added to the hydrolyzate, and after adjusting the pH to 6.0, 35 g / L calcium carbonate is added.

[0029] The Lactobacillus rhamnosus includes but is not limited to the Lactobacillus rhamnosus (NCBI accession number: PV300386.1) described in the embodiments of the present invention. All Lactobacillus rhamnosus capable of L-lactic acid fermentation in the prior art are within the scope of protection of the present invention.

[0030] The inoculation amount is 5-15% v / v; the fermentation conditions of L-lactic acid fermentation are: temperature 35-45°C, rotation speed 150-250 rpm, and time 96-120 hours.

[0031] Preferably, the inoculation amount is 10% v / v; the L-lactic acid fermentation has the following fermentation conditions: temperature 37° C., rotation speed 200 rpm, and time 120 h.

[0032] Wherein, in step (4), the delactic acid treatment includes two steps of decolorization and crystallization.

[0033] Specifically, the decolorization step is as follows: adjusting the pH to 10.0 with a Ca(OH)2 solution, adding 50 g / L activated carbon to the fermentation broth, mixing at 200 rpm and 60°C for 2 hours, and collecting the fermentation broth by suction filtration; the crystallization step is as follows: vacuum distilling and concentrating the decolorized fermentation broth until the concentration of calcium lactate reaches about 150 g / L, crystallizing at 4°C overnight, collecting calcium lactate crystals and concentrated fermentation broth by suction filtration, filling the concentrated fermentation broth with water to the original concentration to obtain a delactic acid fermentation broth, and purifying the calcium lactate crystals to obtain L-lactic acid.

[0034] Wherein, in step (5), xylitol fermentation is carried out using the delactic acid fermentation liquid obtained in step (4), specifically, the bacterial liquid of tropical Candida albicans is inoculated into the delactic acid fermentation liquid for xylitol fermentation;

[0035] Specifically, before the Candida tropicalis bacterial liquid is inoculated into the delactic acid fermentation liquid, 5-10 g / L yeast extract and 15-23 g / L tryptone are added to the delactic acid fermentation liquid to adjust the pH to 4.0-5.0.

[0036] Preferably, before the bacterial liquid of Candida tropicalis is inoculated into the de-lactic acid fermentation liquid, 10 g / L yeast extract and 20 g / L tryptone are added to the de-lactic acid fermentation liquid to adjust the pH to 4.5.

[0037] The tropical Candida includes but is not limited to the tropical Candida (BNCC 187234) described in the embodiments of the present invention. All tropical Candida that can perform xylitol fermentation in the prior art are within the scope of protection of the present invention.

[0038] The inoculation amount is 2-8% v / v. The xylitol fermentation has the following fermentation conditions: temperature 28-32° C., rotation speed 150-200 rpm, and time 96-120 h.

[0039] Preferably, the inoculation amount is 2% v / v; the xylitol fermentation conditions are: temperature 30° C., rotation speed 150-200 rpm, and time 120 h.

[0040] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0041] (1) This method effectively destroys the rigid structure of lignocellulose under mild conditions, and the synergistic effect of low-concentration NaOH and ammonia water significantly increases the cellulose content and improves the lignin removal rate. This method not only improves the conversion rate of raw materials, but also reduces the use of chemicals, which helps to reduce production costs.

[0042] (2) This method proposes a high-solid-phase enzymatic hydrolysis method that does not require water washing and drying, which reduces process steps and resource consumption, while improving hydrolysis efficiency.

[0043] (3) This method adopts a two-stage fermentation method to produce L-lactic acid and xylitol in stages. In the first stage, the hydrolyzate can be directly fermented with L-lactic acid without detoxification, and the ammonia used in the pretreatment provides part of the nitrogen source for the fermentation; the second stage is xylitol fermentation. This method improves the utilization rate of pentose and hexose sugars and improves the economic efficiency of the overall process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0045] Figure 1 The present invention provides a flow chart of a method for co-producing L-lactic acid and xylitol based on lignocellulose.

[0046] Figure 2 This is the effect of whether the hydrolyzate is detoxified on L-lactic acid fermentation in Example 5 of the present invention.

[0047] Figure 3 The concentration changes of xylose and xylitol in the xylitol fermentation of the de-lactic acid fermentation broth in Example 6 of the present invention are shown. DETAILED DESCRIPTION

[0048] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0049] In the following examples, the Lactobacillus rhamnosus has been published in NCBI, and its accession number is: PV300386.1; the Candida tropicalis is Candida tropicalis BNCC187234, purchased from Beina Biotechnology. CTec3 HS, cellulase activity of 240 FPU / mL, was purchased from Novozymes (China) Biotechnology Co., Ltd.

[0050] In the following examples, the detection methods of glucose, xylose, L-lactic acid and xylitol are as follows: high performance liquid chromatography (HPLC) analysis (1260infinity, Agilent) was used, and the HPLC was equipped with a refractive index detector (RID) and an HPX-87H chromatographic column (300mm×7.8mm), the mobile phase was 5mM H2SO4, the flow rate was 0.5mL / min, the temperature was 65°C, and the RID detector temperature was 45°C.

[0051] In the following examples, the calculation formula for the cellulose and hemicellulose conversion rate is:

[0052] Cellulose conversion rate (%) = (C glucose ×86.7×0.9)×100%

[0053] Hemicellulose conversion rate (%) = (C xylose ×86.7×0.88)×100%

[0054] Where: C glucose , C xylose are the concentrations of glucose and xylose in the solution, mg / mL; 86.7 is the volume of the solution, mL; 0.9 is the conversion coefficient of glucose to cellulose, and 0.88 is the conversion coefficient of xylose to hemicellulose.

[0055] In the following examples, the calculation formula for the lignin removal rate is:

[0056] Cellulose conversion rate (%) = ((m unpretreated -m pretreated ) / m unpretreated )×100%

[0057] Where: m unpretreated is the mass of lignin in the untreated raw material, g; m pretreated is the mass of lignin in the raw material after pretreatment, g.

[0058] In the following examples, the calculation formulas for the xylose utilization rate and the xylose yield rate are:

[0059] Xylose utilization rate (%) = ((Cm0 -C m1 ) / C m0 )×100%

[0060] Xylitol yield (g / g xylose) = C xylitol / C xylose

[0061] Where: C m0 , C m1 are the xylose concentrations before and after fermentation, g / L; C xylitol , C xylose are the concentrations of xylitol and xylose, g / L, respectively.

[0062] Example 1: Optimization of different alkali ammonia pretreatment conditions

[0063] After the corn stalks are crushed, they are passed through a 40-mesh round hole sieve and pretreated with alkali ammonia under different conditions. The optimal conditions for alkali ammonia pretreatment are determined by the delignification effect of the corn stalks after pretreatment. The specific method is: 1-5wt% NaOH and 5-10wt% ammonia water are added to every 100g of crushed dry corn stalks, and water is added to a solid content of 30% (w / v, i.e. 300g / L). The pretreatment conditions are: temperature 30-80℃, time 12-48h. After pretreatment, the corn stalks are washed with pure water to neutrality, the water dosage is about 15g / g corn stalks, and dried at 65℃ to constant weight, and the lignin removal rate, cellulose conversion rate and hemicellulose conversion rate are calculated respectively. The specific amount of NaOH and ammonia water added, as well as the pretreatment conditions and corresponding pretreatment results are shown in Table 1.

[0064] Table 1 Alkali ammonia pretreatment results under different conditions

[0065]

[0066] From the results in the table, it can be found that with the increase of NaOH addition, ammonia addition, temperature, and pretreatment time, the lignin removal rate, cellulose and hemicellulose conversion rates all increased. When the NaOH concentration increased from 1% to 5%, the lignin removal rate increased from 35.19% to 43.96%, and the cellulose conversion rate increased from 69.19% to 74.37%. Similarly, when the ammonia concentration increased from 5% to 10%, the lignin removal rate increased from 39.51% to 48.88%, and the cellulose conversion rate increased from 73.66% to 88.55%. The temperature effect during the pretreatment process was more significant. When the temperature increased from 30℃ to 80℃, the lignin removal rate increased from 28.26% to 52.06%, the cellulose conversion rate increased from 59.33% to 88.57%, and the hemicellulose conversion rate increased from 33.45% to 42.33%. The effect of pretreatment time on the pretreatment effect was relatively small. When the pretreatment time was extended from 12 h to 48 h, the lignin removal rate increased slightly from 52.31% to 52.61%, the cellulose conversion rate increased from 88.36% to 89.88%, and the hemicellulose conversion rate increased from 41.77% to 42.15%.

[0067] In summary, the pretreatment conditions of 3 wt % NaOH, 7.5 wt % ammonia water, a pretreatment temperature of 80° C., and a pretreatment time of 12 hours were selected for subsequent experiments.

[0068] Example 2: High solid phase alkali ammonia pretreatment and comparison

[0069] Dry corn stover was pretreated with alkali ammonia, NaOH and ammonia respectively, and a control group without pretreatment was set up. The delignification effect of corn stover after the four treatments was used to evaluate the effect of alkali ammonia pretreatment.

[0070] 1. Alkali ammonia pretreatment method: add 3wt% NaOH and 7.5wt% ammonia water to every 100g dry corn stalks, and add water to a solid content of 30% (w / v, i.e. 300g / L). Mix the stalks, reagents and water evenly and seal them in a blue-mouth bottle.

[0071] 2. NaOH pretreatment method: add 3 wt% NaOH to every 100 g of dry corn stalks, add water to a solid content of 30% (w / v, i.e. 300 g / L), mix well, and seal in a blue-mouth bottle.

[0072] 3. Ammonia pretreatment: add 7.5 wt% ammonia to every 100 g of dry corn stalks, add water to a solid content of 30% (w / v, i.e. 300 g / L), mix well, and seal in a blue-mouth bottle.

[0073] The temperature of the alkaline ammonia pretreatment, NaOH pretreatment and ammonia water pretreatment was 80°C, and the pretreatment time was 12h. After pretreatment, the corn stalks were washed with pure water until neutral, with a water dosage of about 15g / g corn stalks, and dried at 65°C to constant weight, and the lignin removal rate, cellulose conversion rate and hemicellulose conversion rate were calculated respectively. The pretreatment results are shown in Table 2.

[0074] Table 2 Comparison of different pretreatments

[0075] Preprocessing methods Lignin removal rate Cellulose conversion rate Hemicellulose conversion rate Corn stalks (control) - 16.18％ 6.41％ Alkali ammonia pretreatment 53.88％ 88.36％ 40.76％ NaOH pretreatment 27.68％ 46.90％ 20.88％ Ammonia pretreatment 43.11％ 82.19％ 35.13％

[0076] As can be seen from the table, different pretreatments have different effects on lignin removal rate, cellulose and hemicellulose conversion rate. After alkali ammonia pretreatment, the lignin removal rate increased from 27.68% in NaOH pretreatment and 43.11% in ammonia pretreatment to 53.88%. The cellulose conversion rate of the original straw was 16.18%. After alkali ammonia pretreatment, the cellulose conversion rate can be increased to 88.36%, which is 4.46 times higher than that of NaOH pretreatment and ammonia pretreatment alone. In addition, the hemicellulose conversion rate of corn straw after alkali ammonia pretreatment was 40.77%, which is 6.63 times that of the original corn straw. Therefore, the combined action of NaOH and ammonia significantly improved the lignin removal rate, cellulose and hemicellulose conversion rate.

[0077] Example 3: Effects of washing and drying processes on enzymatic hydrolysis

[0078] Based on the pretreatment of 3wt% NaOH, 7.5wt% ammonia water, pretreatment temperature of 80°C, and pretreatment time of 12 hours, the effects of water washing and drying processes of alkaline ammonia pretreated corn straw on enzymatic hydrolysis were further explored.

[0079] The washing process is as follows: using pure water to wash the alkaline ammonia pretreated corn straw to neutrality as a hydrolysis substrate, and the water consumption is about 15g / g corn straw.

[0080] The drying process is as follows: the corn stalks pretreated with alkaline ammonia are dried in an oven at 65°C, with an initial moisture content of 70%. Samples are taken at regular intervals to measure the moisture content, and corn stalks with moisture contents of 70%, 50%, 30%, 10%, and 0% are collected, stored in sealed bags, and used as hydrolysis substrates.

[0081] The non-washing process / non-drying process is: the corn stalks pretreated with alkali ammonia are directly used as the hydrolysis substrate, and the moisture content of the substrate is 70%. The hydrolysis rate of the corn stalks after the washing process and the corn stalks without the washing process is compared to evaluate the effect of the washing process on the enzymatic hydrolysis.

[0082] Enzyme hydrolysis: Take 1g of corn stalks from the above-mentioned washing process, drying process, and non-washing / non-drying process, add water to 20mL, and use 10% H2SO4 to adjust the pH to 4.8-5.0. The hydrolysis temperature is 50°C, the hydrolysis speed is 200rpm, and the hydrolysis time is 72h.

[0083] The effects of the washing process and the drying process on enzymatic hydrolysis were compared, and the results are shown in Table 3. The concentrations of glucose and xylose obtained by enzymatic hydrolysis in the washing process were higher than those in the non-washing process, but there was no significant difference in the cellulose conversion rate and hemicellulose conversion rate. Therefore, the washing process did not further improve the conversion rate, so the washing process can be omitted. In addition, as the moisture content of the corn straw pretreated with alkali ammonia decreased, it was found that the contact efficiency between the enzyme and the substrate decreased, resulting in a significant decrease in the hydrolysis effect. The cellulose conversion rate decreased from 89.97% to 74.56%, and the hemicellulose conversion rate decreased from 38.79% to 32.15%. Alkali ammonia pretreatment of corn straw without drying can keep the substrate moist to a certain extent, which is conducive to the diffusion of the enzyme and the contact of the substrate, thereby improving the conversion rate of cellulose and hemicellulose.

[0084] In summary, corn stover pretreated with alkali ammonia can be directly used as a hydrolysis substrate without the need for washing and drying processes.

[0085] Table 3 Effects of washing and drying processes on enzymatic hydrolysis

[0086]

[0087]

[0088] Example 4: Effect of corn straw feeding method on enzymatic hydrolysis

[0089] The alkali-ammonia pretreated corn stover in Example 3 without washing and drying was directly used as a hydrolysis substrate, and the effect of the feeding method on the enzymatic hydrolysis was further explored by setting the feeding method in batches and in non-batch feeding.

[0090] 1. Batch feeding: The corn stover pretreated with alkaline ammonia is subjected to enzymatic hydrolysis in a batch feeding manner.

[0091] The solid content in the initial stage was 15% (w / v, i.e. 150 g / L), and all the cellulase (15 FPU / g substrate) was added to the system, and the remaining substrate was supplemented at 6 hours to reach a total solid content of 30% (w / v, i.e. 300 g / L), and 10% H2SO4 was used to adjust the pH to 4.8-5.0 after each substrate supplementation. The hydrolysis temperature was 50°C, the hydrolysis speed was 200 rpm, and the hydrolysis time was 72 hours.

[0092] 2. Without batch feeding: the alkaline-ammonia pretreated corn stover is subjected to enzymatic hydrolysis without batch feeding.

[0093] The solid content in the initial stage was 30% (w / v, i.e. 300 g / L), all the cellulase (15 FPU / g substrate) was added to the system, and 10% H2SO4 was used to adjust the pH to 4.8-5.0. The hydrolysis temperature was 50°C, the hydrolysis speed was 200 rpm, and the hydrolysis time was 72 h.

[0094] After HPLC detection, the test results are shown in Table 4. The glucose concentration and xylose concentration after batch feed enzymatic hydrolysis are higher than those of direct hydrolysis. Without batch feed hydrolysis, the glucose and xylose concentrations in the hydrolyzate are 70.00g / L and 29.65g / L, respectively, and the cellulose and hemicellulose conversion rates are 63.12% and 58.71%, respectively. After batch feed hydrolysis, the glucose and xylose concentrations in the hydrolyzate are increased to 78.18g / L and 38.74g / L, respectively, and the cellulose and hemicellulose conversion rates are increased to 73.39% and 67.12%, respectively. The monosaccharide concentrations are increased by about 9g / L, and the conversion rate is increased by about 10%. Therefore, it is better to use batch feed for high solid phase hydrolysis.

[0095] Table 4 Comparison of enzymatic hydrolysis process

[0096]

[0097] Example 5: Effect of hydrolyzate detoxification on L-lactic acid fermentation

[0098] The hydrolyzate obtained by batch feed hydrolysis using cellulase in Example 4 was used to investigate the changes in the main components of the hydrolyzate after detoxification treatment to explore the effect of detoxification on L-lactic acid fermentation.

[0099] 1. Detoxification of hydrolyzate

[0100] The hydrolyzate was detoxified by activated carbon adsorption. 100 g / L activated carbon was added to the hydrolyzate obtained by batch feed hydrolysis, and the detoxification treatment was carried out at 50 ° C and 200 rpm for 4 hours. After the activated carbon detoxification was completed, the activated carbon was removed by a vacuum pump and the detoxified hydrolyzate was collected. The undetoxified hydrolyzate was used as a control.

[0101] The results are shown in Table 5. After the hydrolyzate was detoxified by activated carbon, it was found that the activated carbon had a good adsorption capacity for phenolic substances, and also had a certain adsorption effect on formic acid and acetic acid. A total of 72.96% of total phenolic substances were removed, but it also caused a loss of 3.02% of glucose and 4.28% of xylose.

[0102] Table 5 Changes of main components in hydrolyzate before and after detoxification

[0103]

[0104] 2. L-lactic acid fermentation

[0105] (1) Preparation of Lactobacillus rhamnosus bacterial solution

[0106] Lactobacillus rhamnosus was cultured at 37°C using MRS solid culture medium, and the single colony with the best growth was selected and inoculated into MRS liquid culture medium. The culture temperature was 37°C, the rotation speed was 200 rpm, and the culture time was 24 h to obtain a bacterial solution of Lactobacillus rhamnosus.

[0107] The MRS culture medium formula is: 50 g / L glucose, 5 g / L yeast extract, 5 g / L tryptone, 3 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, the pH is adjusted to 6.0, and 35 g / L calcium carbonate and 20 g / L agar are added (added to MRS solid culture medium, not added to MRS liquid culture medium).

[0108] (2) Fermentation to produce L-lactic acid

[0109] 2g / L yeast extract, 2g / L tryptone, 3g / L ammonium sulfate, 1.50g / L potassium dihydrogen phosphate, and 0.60g / L magnesium sulfate heptahydrate were added to the detoxified and non-detoxified hydrolyzates, and after adjusting the pH to 6.0, 35g / L calcium carbonate was added. Then, the bacterial solution of Lactobacillus rhamnosus was inoculated into the above hydrolyzate at an inoculum amount of 10% (v / v), and cultured at 37°C and 200rpm for 120h to ferment and produce L-lactic acid.

[0110] like Figure 2 As shown, the concentration of L-lactic acid in the non-detoxified group (74.06 g / L) was higher than that in the detoxified group (70.31 g / L). At the same time, the remaining xylose in the non-detoxified group (32.38 g / L) was higher than that in the detoxified group (29.86 g / L), and the glucose in both groups was completely consumed. This result shows that the toxic inhibitory components in the hydrolyzate do not significantly affect the fermentation of L-lactic acid. At the same time, after the hydrolyzate is detoxified, glucose and xylose will be lost, and the concentration of L-lactic acid will not be increased, so the hydrolyzate does not need to be detoxified.

[0111] Example 6: Xylitol fermentation of L-lactic acid-free fermentation broth

[0112] The L-lactic acid fermentation broth obtained without detoxification and fermentation of the hydrolyzate in Example 5 was de-lactic acidized using the calcium lactate precipitation method. Specifically, the pH of the L-lactic acid fermentation broth obtained without detoxification and fermentation of the hydrolyzate in Example 5 was adjusted to 10.0 using a Ca(OH)2 solution, 50 g / L of activated carbon was added to the fermentation broth, mixed at 200 rpm and 60°C for 2 hours for decolorization, and the decolorized fermentation broth was collected by suction filtration. The decolorized fermentation broth was then vacuum distilled and concentrated until the concentration of calcium lactate reached about 150 g / L. Crystallization was carried out overnight at 4°C, and calcium lactate crystals and concentrated fermentation broth were collected by suction filtration. The concentrated fermentation broth was supplemented with water to the original concentration (xylose content was 32.38 g / L) to obtain a de-lactic acidized fermentation broth, and the calcium lactate crystals were purified to obtain L-lactic acid.

[0113] 10 g / L yeast extract and 20 g / L tryptone were added to the de-lactic acid fermentation broth to adjust the pH to 4.5, and then sterilized at 115° C. for 20 min. After cooling to room temperature, a 2% (v / v) inoculation amount of tropical Candida was inoculated therein, and the xylitol fermentation was carried out by culturing at 30° C. and 150-200 rpm for 120 h.

[0114] The preparation method of the bacterial liquid of tropical Candida is as follows: using YM solid culture medium, culturing tropical Candida tropicalis at 30°C, picking the best growing single colony and inoculating it into YM liquid culture medium, the culture temperature is 30°C, the rotation speed is 200rpm, and the culture time is 16 to 24h. The YM culture medium is composed of the following concentration raw materials: 3g / L yeast extract, 3g / L malt extract, 10g / L glucose, 5g / L peptone, and 20g / L agar (added to YM solid culture medium, not added to YM liquid culture medium).

[0115] The results of xylitol fermentation are as follows Figure 3 As shown. In the early stage of fermentation (0-12h), the xylose concentration decreased and the xylitol concentration increased slowly. This stage was mainly due to the growth of tropical Candida. In 12-72h, xylose decreased rapidly and xylose accumulated rapidly. This stage was used for the output of xylitol, and then the xylitol accumulation zone stabilized. The final xylitol concentration was 16.49g / L, the xylose concentration decreased from 32.38g / L to 0.67g / L, the xylose utilization rate was 97.93%, and the xylitol yield was 0.51g / g xylose.

[0116] The present invention provides a method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for co-producing L-lactic acid and xylitol based on lignocellulose fermentation, characterized in that: The steps include: (1) pretreating lignocellulose with alkali ammonia to obtain lignocellulose with lignin removed; (2) enzymatically hydrolyzing the delignified lignocellulose obtained in step (1) to obtain a hydrolyzate; (3) using the hydrolyzate obtained in step (2) to perform L-lactic acid fermentation to obtain L-lactic acid fermentation liquid; (4) performing a delactic acid treatment on the L-lactic acid fermentation broth in step (3) to obtain calcium lactate crystals and delactic acid fermentation broth, and purifying the calcium lactate crystals to obtain L-lactic acid; (5) using the delactic acid fermentation liquid obtained in step (4) to perform xylitol fermentation to obtain xylitol fermentation liquid, and then separating and purifying the xylitol fermentation liquid to obtain xylitol; Wherein, when the delignified lignocellulose is subjected to enzymatic hydrolysis, it is not necessary to wash and dry the delignified lignocellulose; Wherein, before the hydrolyzate is subjected to L-lactic acid fermentation, the hydrolyzate does not need to be detoxified.

2. The method according to claim 1, characterized in that In step (1), the lignocellulose includes any one or a combination of corn straw, corn cob, wheat straw, rice straw and sugarcane bagasse.

3. The method according to claim 1, characterized in that In step (1), the alkali ammonia pretreatment is to add 1 to 5 wt% NaOH and 5 to 10 wt% ammonia water to the lignocellulose, and then add water until the solid content of the lignocellulose is 200 to 300 g / L before pretreatment; the alkali ammonia pretreatment has the following pretreatment conditions: temperature 70 to 90°C, time 10 to 24 hours.

4. The method according to claim 1, characterized in that: In step (2), before the delignined lignocellulose is subjected to enzymatic hydrolysis, the moisture content of the delignined lignocellulose is adjusted to 70-80%.

5. The method according to claim 1 or 4, characterized in that: The enzymatic hydrolysis is carried out by using cellulase, and the dosage of the cellulase is: 5-20 FPU cellulase is added for every 1g of lignin-removed cellulose; the enzymatic hydrolysis conditions are: pH value 4.8-5.2, temperature 45-55°C, rotation speed 150-250rpm, and time 48-72h.

6. The method according to claim 1, 4 or 5, characterized in that: The enzymatic hydrolysis adopts a batch feeding method. In the initial stage, 100-150 g / L of delignified lignocellulose with a solid content is added for enzymatic hydrolysis. After 5-8 hours, the remaining delignified lignocellulose is supplemented to a solid content of 200-300 g / L.

7. The method according to claim 1, characterized in that In step (3), the hydrolyzate obtained in step (2) is used for L-lactic acid fermentation, specifically, a bacterial solution of Lactobacillus rhamnosus is inoculated into the hydrolyzate for L-lactic acid fermentation; Before inoculating the bacterial liquid of Lactobacillus rhamnosus into the hydrolyzate, 0.5-5 g / L yeast extract, 0.5-5 g / L trypsin, 1-3 g / L ammonium sulfate, 0.5-2.0 g / L potassium dihydrogen phosphate, and 0.2-0.8 g / L magnesium sulfate heptahydrate are added to the hydrolyzate, and after adjusting the pH to 5.5-6.5, 25-35 g / L calcium carbonate is added.

8. The method according to claim 7, characterized in that The inoculation amount is 5-15% v / v; the L-lactic acid fermentation has the following fermentation conditions: temperature 35-45° C., rotation speed 150-250 rpm, and time 96-120 h.

9. The method according to claim 1, characterized in that: In step (5), xylitol fermentation is carried out using the delactic acid fermentation liquid obtained in step (4), specifically, the bacterial liquid of tropical Candida albicans is inoculated into the delactic acid fermentation liquid to carry out xylitol fermentation; Before the bacterial liquid of tropical Candida is inoculated into the delactic acid fermentation liquid, 5-10 g / L yeast extract and 15-23 g / L tryptone are added to the delactic acid fermentation liquid to adjust the pH to 4.0-5.

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10. The method according to claim 9, characterized in that The inoculation amount is 2-8% v / v; the xylitol fermentation has the following fermentation conditions: temperature 28-32° C., rotation speed 150-200 rpm, and time 96-120 hours.

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