Lignocellulose pretreatment liquid biological detoxification and resource utilization method based on Chlorella sorokiniana

Abstract

The invention discloses a biological detoxification and resource utilization method for a lignocellulose pretreatment solution based on Chlorella sorokiniana, and belongs to the technical field of environmental protection. Microalgae are inoculated into lignocellulose pretreatment liquid, and toxic substances in the lignocellulose pretreatment liquid are subjected to biological detoxification. According to the present invention, with the application of the Chlorella sorokiniana, the furfural can be converted into the low-toxicity furfuryl alcohol, the furfuryl alcohol and the vanillin can be adopted as the organic carbon source so as to be used for biomass accumulation, the high value-added product can be produced, and the resource utilization of the lignocellulose pretreatment liquid can be achieved;

Description

Technical Field

[0001] The present invention relates to the field of ecological protection, and particularly to a method for biological detoxification and resource utilization of typical inhibitors in lignocellulose pretreatment liquor by Chlorella sorokiniana. Background Art

[0002] The resource utilization of lignocellulosic biomass conforms to the trend of realizing circular economy in modern society and has attracted more and more extensive attention. However, the complexity and rigidity of the biomass matrix make its utilization costly and environmentally risky. The rapid growth of fuel demand makes the large-scale production of biofuels necessary. Among them, lignocellulose is the most abundant biomass, with an annual total output of 15 billion tons, and is the main raw material for second-generation fuel ethanol. Lignocellulose has a lattice structure composed of cellulose, hemicellulose and lignin. The lignin layer has a hydrolysis barrier against the attack of cellulose and hemicellulose, and this barrier is closely related to the structure of the plant cell wall. Therefore, pretreatment is usually adopted to break the physical barrier and improve the accessibility of cellulose and hemicellulose. At present, many studies mainly focus on the effect of the solid residue after pretreatment on hydrolysis and saccharification, and the attention to the resource utilization of the pretreatment liquor generated during the pretreatment process is not high. However, its resource utilization is directly related to the degree of resource utilization of lignocellulosic biomass. In addition to sugars such as glucose and xylose, the pretreatment liquor also contains inhibitors such as phenols, furans and organic acids that have inhibitory effects on organisms. Therefore, detoxification treatment is crucial for the biological utilization of the pretreatment liquor.

[0003] The detoxification methods mainly include physical methods, chemical methods and biological methods. Physical detoxification includes membrane detoxification using a semi-permeable membrane to separate lignocellulose pretreatment inhibitors from monosaccharides and adsorption detoxification using adsorbents such as resins to adsorb inhibitors, but this method has high requirements for instruments and equipment, high costs, and a relatively high loss rate of monosaccharides, which is not conducive to large-scale production in factories. Chemical detoxification mainly uses chemical reagents to react with inhibitors in the lignocellulose pretreatment solution to generate low-toxic or non-toxic substances. The chemical reagents most commonly used are alkalis and reducing agents. The advantage of alkali detoxification is that alkali can react with furfural substances, phenolic substances and weak acid substances, and can also degrade lignin. The alkalis used are relatively common and the operation is relatively simple. The disadvantage is that the amount of alkali used is large and neither the alkali nor the inhibitor can be recovered. Reducing agent detoxification is a relatively commonly used detoxification method. Selecting a suitable reducing agent to reduce furfural and phenolic substances into substances with less toxicity to microorganisms such as bacteria. There are many types of reducing agents, with a large selectivity and low costs, and they can react with a variety of inhibitors, but the detoxification rate and accuracy are not high. Biological detoxification refers to using microorganisms to degrade toxins or inhibitors in lignocellulose hydrolysates. Compared with other detoxification methods, biological detoxification has the advantages of mild reaction conditions, complete conversion of inhibitors into non-toxic derivatives, low energy consumption, low wastewater production, and low biomass loss. A variety of microorganisms have been used for biological detoxification. It is reported that Enterobacter cloacae GGT036 with furfural tolerance can convert 62.8% and 64.3% of furfural into furfuryl alcohol under the conditions of furfural concentrations of 20 mM and 40 mM; the oleaginous yeast Trichosporon fermentans ferments for 12 h to convert 7 mM of furfural into furfuryl alcohol and converts furfuryl alcohol into furoic acid within 240 h. However, few microalgae are used to degrade inhibitors in lignocellulose hydrolysates, namely the generated phenolic and furan compounds. We first explored the biological detoxification and resource utilization method of lignocellulose pretreatment solution based on Chlorella sorokiniana, and at the same time explored the synergistic effect of the combination of furfural and vanillin on the growth of Chlorella sorokiniana. Summary of the Invention

[0004] The present invention provides a biological detoxification and resource utilization method of lignocellulose pretreatment solution based on Chlorella sorokiniana, which solves the problems of high costs and low detoxification rates of physical and chemical detoxification methods, and realizes the resource utilization of the pretreatment solution.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The first object of the present invention is to provide a method for biological detoxification and resource utilization of lignocellulose pretreatment liquor based on Chlorella sorokiniana, which is to inoculate microalgae into the lignocellulose pretreatment liquor to biologically detoxify the toxic substances therein.

[0007] Preferably, the microalgae is Chlorella sorokiniana.

[0008] Preferably, the toxic substances in the lignocellulose pretreatment liquor are furfural and / or vanillin.

[0009] Preferably, the toxic substances in the lignocellulose pretreatment liquor are furfural and vanillin, and their concentrations are respectively: 0.1 g / L furfural + 0.05 g / L vanillin or 0.1 g / L furfural + 0.1 g / L vanillin.

[0010] Preferably, the detoxification is to convert furfural into furfuryl alcohol through biological transformation and further into microalgae biomass.

[0011] Preferably, when inoculating the microalgae into the lignocellulose pretreatment liquor, the culture temperature is 30 °C, the light intensity is 110 - 120 μmol m -2 s -1 , and the rotation speed is 150 rpm.

[0012] The second object of the present invention is the application of microalgae in the biological detoxification of furfural and / or vanillin in lignocellulose pretreatment liquor.

[0013] Preferably, the microalgae is Chlorella sorokiniana.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0015] 1. Chlorella sorokiniana has a good effect on the biological detoxification of inhibitors in lignocellulose pretreatment liquor. When growing for 1 day, 0.1 g / L furfural can be completely biologically transformed.

[0016] 2. The biological detoxification method of Chlorella sorokiniana for inhibitors has a lower cost than chemical and physical methods, and can utilize lignocellulose pretreatment liquor as a resource to produce high-value products. Description of the Drawings

[0017] Figure 1Effects of single furfural and vanillin at different concentrations on the growth of Chlorella sorokiniana (a, effects of single furfural on the growth of Chlorella sorokiniana; b, effects of single vanillin on the growth of Chlorella sorokiniana);

[0018] Figure 2 Effects of the combination of furfural and vanillin at different concentrations on the growth of Chlorella sorokiniana;

[0019] Figure 3 Bioconversion and metabolism of inhibitors by Chlorella sorokiniana;

[0020] Figure 4 Nutrient accumulation in Chlorella sorokiniana; Detailed implementation method

[0021] The following examples are further illustrations of the present invention, rather than limitations thereof. Unless otherwise specified, the test methods in the following examples are all conventional test methods, and the experimental reagents and consumables described in the following examples are all from conventional biochemical reagent companies unless otherwise specified.

[0022] Example 1:

[0023] Application of Chlorella sorokiniana in the resource utilization of biological detoxifying agents for inhibitors in lignocellulose pretreatment liquor, the steps are as follows:

[0024] Chlorella sorokiniana (FACHB1068, Freshwater Algae Collection of the Chinese Academy of Sciences) cultured to the logarithmic growth phase in BG11 medium was washed with sterile water and inoculated into a simulated lignocellulose pretreatment liquor (0.5 g / L yeast extract, 10 g / L peptone, 4 g / L glucose, 12 g / L xylose) to make its initial inoculation amount 0.04 g / L;

[0025] B. Experimentally designed 8 experimental groups (3 biological replicates in each group) and a control group (without inhibitor addition). Furfural and vanillin were added to the experimental groups respectively. The furfural concentrations were 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.5 g / L; the vanillin concentrations were 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L. At the same time, 0.2 M phosphate buffer was used to control the pH to 7.2. The reaction system was a 250 mL conical flask, the culture temperature was 30 °C, the light intensity was 110 - 120 μmol m -2 s -1 , and the rotation speed was 150 rpm;

[0026] C. According to the results of Experiment B, 0.1 g / L furfural was compounded with 0.05 g / L and 0.1 g / L respectively, and other culture conditions were the same as those in Experiment B. Samples were taken every day to detect the OD680 value to obtain the growth of microalgae, and at the same time, the biotransformation and removal of inhibitors were analyzed.

[0027] D. Analyze the biomass accumulation of Chlorella sorokiniana, and at the same time analyze the contents of oil, protein, and polysaccharide in the algal biomass.

[0028] The results show that the addition of inhibitors will have inhibitory effects on the growth of Chlorella sorokiniana to varying degrees. As can be seen from Figure 1 a, 0.2 g / L furfural has a significant inhibitory effect on the growth of Chlorella sorokiniana; as Figure 1 shown in b, low-concentration vanillin (0.025 g / L) has a significant inhibitory effect on the growth of microalgae; Figure 2 shows the effect of the compounding of inhibitors on the growth of Chlorella sorokiniana. The compounding of inhibitors will significantly reduce its growth. When the addition amount of furfural is 0.1 g / L, with the increase of the concentration of vanillin, the inhibitory effect on Chlorella sorokiniana is more significant; Figure 3 shows the biotransformation and removal effect of microalgae on inhibitors. Chlorella sorokiniana biotransforms furfural into furfuryl alcohol during growth. When 0.1 g / L furfural is compounded with 0.05 g / L and 0.1 g / L vanillin respectively, on the 1st day of growth, only 33.64% and 21.82% of furfural are converted into furfuryl alcohol. With the growth of Chlorella sorokiniana, furfuryl alcohol is gradually metabolized. On the 6th day of growth, furfuryl alcohol in each group is completely metabolized, and the removal rate of vanillin in each culture system can reach 100% in the first 2 days. Figure 4 Shows the accumulation of nutrients in Chlorella sorokiniana. The protein content of Chlorella sorokiniana is between 23.94% and 27.83%, the carbohydrate content is between 45.84% and 52.19%, and the lipid content is between 3.10% and 8.50%. Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 It is confirmed that Chlorella sorokiniana can convert furfural into less toxic furfuryl alcohol, and at the same time can metabolize furfuryl alcohol. Vanillin can also be used as a carbon source for microalgae metabolism, and can produce high-value-added products.

Claims

1. A method for biological detoxification and resource utilization of lignocellulose pretreatment liquor, Characterized in that, Microalgae are introduced into the lignocellulose pretreatment liquor to biodegrade the toxic substances therein.

2. The method according to claim 1, Characterized in that, The microalgae is Chlorella sorokiniana.

3. The method according to claim 1, Characterized in that, The toxic substances in the lignocellulose pretreatment liquor are furfural and / or vanillin.

4. The method according to claim 1, Characterized in that, The toxic substances in the lignocellulose pretreatment liquor are furfural and vanillin, and their concentrations are respectively: 0.1 g / L furfural + 0.05 g / L vanillin or 0.1 g / L furfural + 0.1 g / L vanillin.

5. The method according to claim 1, Characterized in that, The detoxification is to convert furfural into furfuryl alcohol through biotransformation and further into microalgae biomass.

6. The method according to claim 1, Characterized in that, When the microalgae are inoculated into the lignocellulose pretreatment solution, the culture temperature is 30 °C, the light intensity is 110 - 120 μmol m -2 s -1 , and the rotation speed is 150 rpm.

7. Application of microalgae in biological detoxification of furfural and / or vanillin in lignocellulose pretreatment liquor.

8. The application according to claim 7, Characterized in that, The microalgae is Chlorella sorokiniana.

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