Lignocellulose pretreatment liquid biological detoxification and resource utilization method based on Kurthia huakuii

Abstract

The invention discloses a biological detoxification and resource utilization method for a lignocellulose pretreatment solution based on Kurthia huakuii, and belongs to the technical field of environmental protection. According to the method, bacteria are inoculated into lignocellulose pretreatment liquid, and toxic substances in the lignocellulose pretreatment liquid are subjected to biological detoxification. Kurthia huakuii can convert furfural into low-toxicity furfuryl alcohol and furoic acid, and meanwhile, furfuryl alcohol, furoic acid and vanillin can act as an organic carbon source to be used for biomass accumulation, so that resource utilization of the lignocellulose pretreatment liquid is 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 the lignocellulose pretreatment liquid by Kurthia huakuii. 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, which 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 used to break physical barriers 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 liquid 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 liquid 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 liquid.

[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 of inhibitors using adsorbents such as resins to achieve detoxification. However, 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 advantages of alkali detoxification are that alkalis 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 disadvantages are 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 substances 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 the use of 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 has been 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 oil-producing 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; the removal rate of furfural produced during the liquid hot water pretreatment of corn stover by Coniochaeta ligniaria NRRL30616 is >65%. Some studies have found that Kurthia huakuii LAM0618T can effectively convert phenolic compounds (syringaldehyde, hydroxybenzaldehyde and vanillin) in lignocellulose hydrolysates into acids with lower toxicity. However, there is still no report on the effect of Kurthia huakuii on furfural in the liquid of woody cellulose pretreatment. We first explored the biological detoxification and resource utilization method of lignocellulose pretreatment liquid based on Kurthia huakuii, and at the same time explored the synergistic effect of the combination of furfural and vanillin on the growth of Kurthia huakuii. Summary of the Invention

[0004] The present invention provides a method for biological detoxification and resource utilization of lignocellulose pretreatment liquid based on Kurthia huakuii, 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 liquid. The technical solution adopted by the present invention is as follows:

[0005] The first object of the present invention is to provide a method for biological detoxification and resource utilization of lignocellulose pretreatment liquor, which involves inoculating bacteria into the lignocellulose pretreatment liquor to biologically detoxify the toxic substances therein.

[0006] Preferably, the bacteria is Kurthia huakuii.

[0007] Preferably, the toxic substances in the lignocellulose pretreatment liquor are furfural and / or vanillin.

[0008] Preferably, the toxic substances in the lignocellulose pretreatment liquor are furfural and vanillin, and their concentrations are respectively: 1 g / L furfural + 0.5 g / L vanillin; 1 g / L furfural + 1 g / L vanillin; 3 g / L furfural + 0.5 g / L vanillin or 3 g / L furfural + 1 g / L vanillin.

[0009] Preferably, the detoxification is to convert furfural into furfuryl alcohol and furoic acid through biological transformation.

[0010] Preferably, when inoculating the bacteria into the lignocellulose pretreatment liquor, the culture temperature is 30 °C and the rotation speed is 130 rpm.

[0011] The second object of the present invention is the application of bacteria in the biological detoxification of furfural and / or vanillin in lignocellulose pretreatment liquor.

[0012] Preferably, the bacteria is Kurthia huakuii.

[0013] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0014] 1. Kurthia huakuii has a good effect on the biological detoxification of inhibitors in lignocellulose pretreatment liquor. After growing for 72 h, 3 g / L furfural can be completely biologically transformed.

[0015] 2. While achieving detoxification, Kurthia huakuii can realize the resource utilization of organic substances in lignocellulose pretreatment liquor. Description of the Drawings

[0016] Figure 1 It shows the effects of different concentrations of single furfural and vanillin, and their combinations on the growth of bacteria (a, the effect of single furfural on the growth of bacteria; b, the effect of single vanillin on the growth of bacteria; c, the effect of the combination of 1 g / L furfural and different concentrations of vanillin on the growth of bacteria; d, the effect of the combination of 3 g / L furfural and different concentrations of vanillin on the growth of bacteria).

[0017] Figure 2Bioconversion and metabolism of inhibitors by bacteria (a, bioconversion of 1 g / L furfural by bacteria; b, bioconversion of 3 g / L furfural by bacteria; c, removal of vanillin by bacteria). Detailed implementation manners

[0018] 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.

[0019] Example 1:

[0020] Application of Kurthia huakuii in biode-detoxification and resource utilization of inhibitors in lignocellulose pretreatment liquor, and the steps are as follows:

[0021] A. Kurthia huakuii (China General Microbiological Culture Collection Center, CGMCC-1.15389) cultured to the logarithmic growth phase in LB medium was washed with sterile water and inoculated into a simulated lignocellulose pretreatment liquor (0.5 g / L yeast extract, 5 g / L peptone, 4 g / L glucose, 12 g / L xylose) to make its initial inoculum concentration 0.07 g / L;

[0022] B. 6 experimental groups and 1 control group (without inhibitor addition) were designed in the experiment, with 3 replicates in each group. Furfural and vanillin were added to the experimental groups to concentrations of 1 g / L, 2 g / L, 3 g / L and 0.25 g / L, 0.5 g / L, 1 g / L respectively. The pH was controlled at 7.2 by 0.2 M phosphate buffer, and the culture was carried out at 30 °C and 130 rpm for 72 h. The OD value was measured every 12 h;

[0023] C. According to the results of experiment B, 1 g / L and 3 g / L furfural were respectively compounded with 0.5 g / L and 1 g / L, and other culture conditions were the same as those in experiment B. Samples were taken every 12 h, the OD600 value was detected to obtain the bacterial growth situation, and at the same time, the bioconversion and removal of inhibitors were analyzed;

[0024] D. Analyze the biomass accumulation situation of Kurthia huakuii, and at the same time, determine the carbon, hydrogen and nitrogen contents in the bacterial cells by elemental analysis.

[0025] The results show that the addition of inhibitors will cause different degrees of inhibitory effects on the growth of Kurthia huakuii. As can be seen from Figure 1 a, 3 g / L furfural has a significant inhibitory effect on the growth of Kurthia huakuii, while the addition of 1 g / L furfural has no obvious inhibitory effect on bacterial growth; As Figure 1As shown in Fig. b, vanillin at 0.25 g / L promoted the growth of bacteria, while vanillin at 1 g / L had a significant inhibitory effect on its growth, showing a low-promotion and high-inhibition effect. Figure 1 Figs. c and d show the effect of the combination of furfural and vanillin on the growth of bacteria. When 1 g / L furfural was combined with 0.5 g / L vanillin, the biomass reached 0.90 g / L at 72 h of growth, which was higher than that of the control group, indicating that the combination of the two had no inhibitory effect on the growth of bacteria. However, the combination of 3 g / L furfural and 1 g / L vanillin showed a significant inhibitory effect on the growth of bacteria. Figure 2 This shows the biotransformation and removal effect of bacteria on inhibitors. During the growth process, bacteria biotransformed furfural into furfuryl alcohol and furoic acid. In the group with the combination of 1 g / L furfural and 0.5 g / L vanillin, 0.5 g / L vanillin was completely metabolized at 48 h. When growing to 72 h, only furoic acid remained in the culture system as an inhibitor, with a residual amount of 0.51 g / L. As shown in Table 1, when the two inhibitors were combined, they both showed an antagonistic effect on the growth inhibition of Kurthia huakuii, and the Q values were all lower than 0.85. Figure 1 、 Figure 2 It was confirmed that Kurthia huakuii could convert furfural into less toxic furfuryl alcohol or furoic acid, and could also metabolize furfuryl alcohol and furoic acid. Vanillin could also be used as a carbon source for bacterial metabolism; as shown in Table 1, when furfural and vanillin were combined, they both showed an antagonistic effect on the growth inhibition of Kurthia huakuii.

[0026] Table 1

[0027]

[0028]

[0029] Note: Q < 0.85, antagonistic effect; 0.85 ≤ Q < 1.15, additive effect; Q ≥ 1.15, synergistic effect.

Claims

1. A method for biological detoxification and resource utilization of lignocellulose pretreatment liquor, characterized in that, bacteria are introduced into the lignocellulose pretreatment liquor to biologically detoxify the toxic substances therein.

2. The method according to claim 1, characterized in that, the bacteria are Kurthia huakuii.

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: 1 g / L furfural + 0.5 g / L vanillin; 1 g / L furfural + 1 g / L vanillin; 3 g / L furfural + 0.5 g / L vanillin or 3 g / L furfural + 1 g / L vanillin.

5. The method according to claim 1, characterized in that, the detoxification is to convert furfural into furfuryl alcohol and furoic acid through biological transformation.

6. The method according to claim 1, characterized in that, when the bacteria are introduced into the lignocellulose pretreatment liquor, the culture temperature is 30 °C and the rotation speed is 130 rpm.

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

8. The application according to claim 7, characterized in that, the bacteria are Kurthia huakuii.

Images