CN114196588A - Thermophilic anaerobic succinic acid clostridium strain and method for producing succinic acid by using lignocellulose - Google Patents
Thermophilic anaerobic succinic acid clostridium strain and method for producing succinic acid by using lignocellulose Download PDFInfo
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Abstract
The present invention provides a thermophilic anaerobic succinic acid clostridium strain (for producing succinic acid)Pseudoclostridium thermosuccinogenes) PT-GD-2. The strain PT-GD-2 can produce succinic acid with high yield under high temperature conditions, consumes 100 g of glucose and can produce nearly 70g of succinic acid, and compared with the existing clostridium succinicum strain, the strain PT-GD-2 realizes the remarkable improvement of the yield of succinic acid under the high temperature conditions. In addition, the strain PT-GD-2 has a wider optimal pH condition, and can obtain higher biomass under the condition of pH 6.2-8.0. Based on the mismatch of this optimal pH condition with that of clostridium thermocellum (neutral), the present application provides a method for producing succinic acid by coupling the strain PT-GD-2 with clostridium thermocellum in a "one-pot" process. The method takes lignocellulose as a substrate, and has simple operation,The production cost is reduced, the yield of the succinic acid is improved to the maximum extent, the generation of byproducts is reduced, and the method has important practical application value.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a thermophilic anaerobic succinic acid clostridium strain (Pseudomonas thermosacclimates) for producing succinic acid and a method for producing succinic acid by using lignocellulose as a substrate through clostridium thermocellum coupling of the strain.
Background
Succinic acid (also called Succinic acid) is an important C4 platform compound, can derive a plurality of daily necessities and special chemicals, and is widely applied to the fields of food, medicine and agriculture. Wherein, the product is mainly used for flavor improvement of liquid seasonings and refined products in the field of food, is mainly used for medicaments such as erythromycin ethylsuccinate and the like in the field of medicine, and is mainly used for producing plant growth regulators, pesticides and the like in the field of agriculture. In addition, succinic acid is an important basic chemical raw material, and can be used as an intermediate product and a professional chemical product for synthesizing 1, 4-butanediol, tetrahydrofuran, a degradable biopolymer material polybutylene succinate (PBS) and the like. In recent years. With the continuous expansion of the application field, the demand of the international market for succinic acid is strongly increased. At present, succinic acid has been recognized as the most promising bio-based chemical produced biologically or chemically from carbohydrates.
Compared with the high-temperature high-pressure reaction conditions of a chemical method, the biological fermentation method for preparing the succinic acid has the advantages of mild conditions, low energy consumption and environmental friendliness. The existing succinic acid production strains are mainly selected strains or metabolic engineering strains of actinomycetes, Mannheim, Corynebacterium glutamicum, Escherichia coli and the like. However, the processes for preparing succinic acid by biological fermentation all adopt glucose from starch as a carbon source, so that the cost is high, and the problems of competing with people for grains and competing with grains for land exist.
To address this problem, researchers developed methods for the conversion of lignocellulosic feedstock to sugars and cultured strains to produce succinic acid using lignocellulose-based fermentable sugars (typically mixed sugars of five and six carbon sugars) as a carbon source. However, there are many problems in the fermentation culture of succinic acid-producing strains using a hydrolysate obtained by hydrolyzing lignocelluloses such as straw with fungal-derived commercial cellulase and hemicellulase as a sugar source. Most importantly, the enzyme preparation from fungi is high in cost, so that the fermentable sugar from lignocellulose does not have obvious market competitiveness compared with starch sugar, and the preparation technology of succinic acid from lignocellulose is difficult to really realize practical application. In addition, the yield is also not ideal. Bukhari, N.A. et al (Industrial Crops and Products 2021,171) reported that pretreated oil palm biomass was used as a raw material, cellulase was added to hydrolyze the raw material to obtain a sugar-containing hydrolysate, and then an actinomycete producing succinic acid was cultured using the hydrolysate as a carbon source to produce succinic acid. However, the succinic acid yield was only 17.5 g per 100 g of oil palm substrate, wherein the main problem was that the yield of fermentable sugars from the substrate was only 45% (mass ratio).
The invention patents 201810939329.2 and 201810939479.3 propose lignocellulose saccharification methods independent of free enzymes, and mainly utilize high-temperature cellulose degradation bacteria such as clostridium thermocellum and the like as whole bacteria catalysts. The invention patents 201810939294.2, 201810939182.7, 201810939517.5, 201810939181.2, 201810939518.X and the like further show that the lignocellulose saccharification method can be matched with a downstream fermentation technology for use. However, the known succinic acid producing strains are all bacteria growing under a medium temperature condition, and the known succinic acid producing strains and a system based on high-temperature cellulose degrading bacteria have the problem of non-uniform culture conditions, such as forced matching, and the known succinic acid producing strains need to undergo the processes of temperature rise, temperature reduction and the like in actual production, so that the energy consumption cost is high. Therefore, strains for producing succinic acid from lignocellulose and related application techniques are lacking.
Disclosure of Invention
Aiming at the problems in the prior art that lignocellulose is used as a raw material to produce succinic acid, the invention provides a thermophilic anaerobic succinic acid clostridium strain (Pseudomonas thermosulficogenes) PT-GD-2 for producing succinic acid. Meanwhile, the application provides a method for producing succinic acid by the strain PT-GD-2 through combined fermentation of lignocellulose serving as a substrate and a cellulose degradation high-temperature bacterium clostridium thermocellum, and the method has important practical application value.
The technical scheme of the invention is as follows:
the inventor selects and obtains a thermophilic anaerobic succinic acid shuttle strain (Pseudochlorococcum thermosuccinogenes) capable of producing succinic acid with high yield from farmland in Maoming city, Guangdong province, the thermophilic anaerobic succinic acid shuttle strain is named as thermophilic anaerobic succinic acid shuttle strain PT-GD-2, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of West Lu No. 1 institute in sunny region, Beijing city, the preservation number is CGMCC No.23684, and the preservation date is 28 days 10 months in 2021 year. The thermophilic anaerobic succinic acid clostridium strain PT-GD-2 has a wider optimal pH condition, and can obtain higher biomass under the condition of pH 6.2-8.0; and the optimum pH condition is staggered with the optimum pH condition (neutral) of the clostridium thermocellum, thereby providing possibility for the coupling of the two. This is because the growth of Clostridium thermocellum is inhibited at pH6.2, whereas the cellulolytic enzyme system, cellulosome, produced by Clostridium thermocellum has a higher activity under these conditions, enabling the degradation of lignocellulosic substrates, thus providing a carbon source for the growth of PT-GD-2 strain.
A microbial inoculum comprising said thermophilic anaerobic succinic acid clostridium strain PT-GD-2.
Use of a strain of Clostridium thermophilum PT-GD-2 as described hereinbefore, for the fermentative production of succinic acid. The method specifically comprises the following steps: inoculating the thermophilic anaerobic succinic acid clostridium strain into a fermentation culture medium for fermentation growth, and separating from fermentation liquor to obtain succinic acid after fermentation is finished; the carbon source in the fermentation medium is glucose, fructose, cellobiose or xylose. Wherein the concentration of the carbon source is 10-20 g/L; the temperature of the fermentation growth is 55-65 ℃; the pH of the fermentation growth is 6.2-8. The fermentation medium comprises the following components: 10-20g/L of carbon source, 2.9g/L of dipotassium phosphate, 1.5g/L of monopotassium phosphate, 2.1g/L of urea, 150mg/L of calcium chloride, 1.0g/L of magnesium chloride, 1.25mg/L of ferrous sulfate, 1.0g/L of cysteine, 1.0mg/L of resazurin and 6.0g/L of yeast extract.
Preferably, the carbon source is one or both of glucose and xylose.
The application of the thermophilic anaerobic succinic acid clostridium strain PT-GD-2 in the method for producing succinic acid by coupling the thermophilic anaerobic succinic acid clostridium strain PT-GD-2 with clostridium thermocellum in a one-pot method specifically comprises the following steps:
(1) saccharification of lignocellulose: adding a GS-2 culture medium and the pretreated lignocellulose substrate into a fermentation tank, and uniformly mixing; inoculating clostridium thermocellum strain, saccharifying at pH of 6.2-8.0 to obtain lignocellulose fermentation liquor. Wherein, the lignocellulose substrates are straws and corncobs; the inoculation proportion of the clostridium thermocellum is 5-10%. Since the optimal growth conditions for Clostridium thermocellum are neutral in pH, cell growth and metabolic arrest occur at pH 6.2; meanwhile, the produced cellulose degradation enzyme system-cellulosome has higher activity under the condition, and is beneficial to the process of saccharification of lignocellulose.
(2) And (3) producing succinic acid by fermentation: when the concentration accumulation of glucose in the lignocellulose fermentation liquor reaches 10-20g/L, controlling the pH value to be 6.2-6.5, and inoculating a thermophilic anaerobic succinic acid clostridium strain PT-GD-2 according to the volume ratio of 1-10%; and when the concentration of the glucose in the lignocellulose fermentation liquid is zero, ending the fermentation to obtain the succinate. Wherein the temperature for producing succinic acid by fermentation is 55-65 ℃.
The invention has the beneficial effects that:
(1) the invention provides a thermophilic anaerobic succinic acid clostridium strain PT-GD-2, which can produce succinic acid with high yield under high temperature, has few byproducts and high succinic acid yield, consumes 100 g of glucose and can produce approximately 70g of succinic acid, and compared with the existing clostridium succinicum strain, the yield under the high temperature is obviously improved.
(2) The thermophilic anaerobic succinic acid clostridium strain PT-GD-2 has a wider optimal pH condition, and can obtain higher biomass under the condition of pH 6.2-8.0; and the optimum pH condition is staggered with the optimum pH condition of the clostridium thermocellum, thereby providing possibility for the coupling of the two.
(3) The invention provides a method for producing succinic acid by coupling thermophilic anaerobic succinic acid clostridium strain PT-GD-2 and thermocellum through a one-pot method, which utilizes the characteristic that the optimal pH conditions of the thermocellum and PT-GD-2 are staggered, realizes the production of succinic acid by using lignocellulose as a substrate through the control of pH, has simple operation, reduces the production cost, improves the yield of succinic acid to the maximum extent, reduces the generation of byproducts, and has important practical application value.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1 isolation and screening of Strain PT-GD-2
1) Primary screening:
in a clean bench, 2g of soil is added into 48mL of sterile water and fully shaken and uniformly mixed, then 5mL of suspension is added into 20mL of screening medium (2.9 g of dipotassium hydrogen phosphate, 1.5g of monopotassium phosphate, 2.1g of urea, 150mg of calcium chloride, 1.0g of magnesium chloride, 1.25mg of ferrous sulfate, 1g of cysteine, 1.0mg of resazurin, 2g of pyridoxamine hydrochloride, 0.2g of biotin, 0.4g of p-aminobenzoic acid, 120.2 g of vitamin B, 5g of glucose and pH 6.2) and fully shaken and uniformly mixed, and soil suspension is obtained. Gradient dilution of soil suspension with screening Medium 102-105And spreading the mixture evenly on a screening culture medium plate of agar with the mass volume ratio of 0.8%. The plate was placed in an anaerobic box and incubated in a 60 ℃ incubator for 10 days under anaerobic conditions.
2) Re-screening:
the larger colonies were picked and expanded in 5mL of selection medium, followed by shaking at a speed of 170rpm for 24 hours at 60 ℃ and then 0.05mL of the culture was re-inoculated into 5mL of selection medium for subculture, followed by shaking at a speed of 170rpm for 24 hours at 60 ℃ and then the different cultures were analyzed for OD600 and the supernatant for the type and amount of organic acids by HPLC.
Example 2 identification of strains
A strain having a large biomass (OD600) and producing succinic acid in the supernatant was selected, genomic DNA was extracted, and 16S rRNA gene amplification was performed using 27F/1492R as a primer set. The homology of the 16S rRNA gene sequence of one strain and a Clostridium succinogenes Pseudomonas thermosulficcoles DSM 5809 strain (Genbank serial number: NR _119284.1) is more than 99 percent through Blast sequence alignment of NCBI, so that the strain is judged to belong to the Clostridium succinogenes and is named as PT-GD-2. The strain is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) No. 3 of Xilu No. 1 on North Cheng, the area facing Yang, Beijing, and the preservation number is CGMCC No.23684, and the preservation date is 2021 year, 10 months and 28 days.
Example 3 physiological and Biochemical Properties of Strain PT-GD-2
The optimal growth conditions of the strain PT-GD-2 are analyzed, and the strain is determined to grow in glucose, fructose, cellobiose and xylose, wherein the optimal carbon source type is xylose and glucose, the two sugars can be commonly utilized, the carbon repression phenomenon does not exist, the optimal growth temperature is 55-65 ℃, and the optimal growth pH is 6.2-8. At a pH below 5.5, the strain was unable to grow. The xylose or the glucose is used as a carbon source for batch fermentation, and the optimal initial carbon source concentration is 10-20 g/L.
The strain PT-GD-2 was cultured for 30 hours at an initial carbon source of 10g/L glucose, an inoculum size of 1% (volume ratio), and a temperature of 60 ℃ in a fermentation medium (2.9 g of dipotassium hydrogen phosphate, 1.5g of monopotassium phosphate, 2.1g of urea, 150mg of calcium chloride, 1.0g of magnesium chloride, 1.25mg of ferrous sulfate, 1g of cysteine, 1.0mg of resazurin, 6.0g of yeast extract, and a pH of 7.4) at a fermentation temperature of 60 ℃, and the pH OD600 was reduced to 5.5, but the biomass was stable up to 60 hours and exceeded 2.5, indicating that the strain was robust to fermentation.
Batch fermentation was carried out with glucose as initial carbon source at an initial pH of 6.5, an inoculum size of 10% (vol/vol) and a temperature of 60 ℃, by adding 20g/l magnesium carbonate as pH buffer. When the initial glucose concentration was 20g/L, the succinic acid yield was 7.6 g/L, wherein 5g/L of glucose remained due to the strain growth arrest caused by the pH drop to 5.5 during the fermentation, and the succinic acid yield was 50.6 g per 100 g of glucose, about 0.84 mol of succinic acid per mol of glucose.
Example 4 succinic acid fermentation Performance optimization of Strain PT-GD-2 (control of pH, batch fermentation)
Carrying out batch fermentation by using 20g/L glucose as an initial carbon source under the conditions of initial pH of 7.0, inoculum size of 1% (volume ratio) and temperature of 60 ℃; the pH was controlled at 7.0 by feeding sodium carbonate to a 1L fermenter on the basis of 7.5 g/L magnesium carbonate addition. After 36 hours of fermentation, 11.8g/L succinate was obtained after complete consumption of glucose, with a yield of 59.2 g per 100 g of glucose. The strain can realize complete consumption of glucose under the condition of controlling pH, and meanwhile, the yield of succinate is obviously improved. Succinate production was increased by 8.6g per 100 g glucose by about 17% compared to no pH control.
Example 5 succinic acid fermentation Performance optimization of Strain PT-GD-2 (feed, continuous fermentation)
In contrast to example 4, a continuous feed experiment was carried out in a 1L fermenter at an initial pH of 7.4, an inoculum size of 5% (by volume) and a temperature of 55 ℃ so that the glucose concentration was controlled at 10 g/L. After 36 hours of fermentation, the temperature was increased to 65 ℃. After 72 hours of fermentation, 55.4g/L succinate is produced,the yield was 69.6 grams per 100 grams of glucose.The concentration of succinate produced by the strain is obviously improved under the condition of controlling the concentration of the carbon source. Succinate production was increased by 19.0g per 100 g glucose by about 38% compared to uncontrolled carbon source concentration.
Example 6 Whole lignocellulose catalyzed saccharification of Clostridium thermocellum
Carrying out whole-bacterium saccharification of alkali pretreated lignocellulose substrates by using a clostridium thermocellum strain expressing glucosidase, and specifically operating as follows: under the condition of initial pH7.4, a clostridium thermocellum recombinant strain for expressing glucosidase is cultured in a GS-2 culture medium taking 5 grams per liter of microcrystalline cellulose as a carbon source to a logarithmic phase in advance, then the strain is inoculated into 80 grams per liter of dry weight of GS-2 culture medium taking pretreated straw or xylose residue (the xylose residue is corncob waste after xylose is extracted by acid hydrolysis) as a carbon source according to the inoculation amount of 10 percent (volume ratio), and the strain is cultured in a shaking table at 60 ℃ and 170r/min until the concentration of reducing sugar in hydrolysate is not changed any more. To be provided withStraw is taken as substrate, and the total sugar content is 69%The concentration of reducing sugar in the hydrolysate is 50.1 g/L, the sugar yield is 90.6 percent,wherein glucose is 38.1 grams per liter, and xylose is 12 grams per liter.The total sugar content of the xylose residue is 78 percent by taking the xylose residue as a substrateThe concentration of reducing sugar in the hydrolysate is 55.5 g/L, and the sugar yield is 89%, wherein the glucose is 52 g/L, and the xylose is 3.5 g/L. (see the reference for details.)
Example 7 production of succinic acid by coupled saccharification and coupling of Clostridium thermocellum straw with PT-GD-2 batch fermentation
The hydrolysate obtained in example 6 was diluted according to the glucose concentration until the glucose concentration was 20g/L, and the batch fermentation experiment as described in example 4 was carried out using the diluted hydrolysate as the culture medium, and both glucose and xylose were completely consumed and 10.9g/L succinate was obtained, yielding 54.5 g/100 g glucose. It can be seen that the strain PT-GD-2 described in the present application, using glucose derived from lignocellulose as a carbon source, produced succinic acid in a slightly lower yield under the same conditions than that obtained in example 4. This indicates that the strain is feasible to produce succinic acid by fermentation under the condition of controlling pH and using glucose of lignocellulose as a carbon source.
Example 8 production of succinic acid by Clostridium thermocellum straw saccharification coupling PT-GD-2 continuous fermentation
The hydrolysate obtained in example 6 was concentrated by rotary evaporation to increase the sugar concentration to 200 g per liter. According to the continuous fed-batch fermentation experiment described in example 5, concentrated hydrolysate was added to 1L of fermentation system to control the glucose concentration at 10g/L and a total of 85 g of glucose was consumed after 72 hours of fermentation to obtain 46.3 g of succinate in 53.8 g per 100 g of glucose, with pH being controlled at 6.5. It can be seen that the strain PT-GD-2 described in the present application, using glucose and xylose of lignocellulosic origin as carbon sources, produced succinic acid in a lower yield under the same conditions than that obtained in example 5, but still not lower than that obtained in example 3. This indicates that the strain is feasible to produce succinic acid by fermentation using glucose and xylose of lignocellulose source as carbon source under the condition of controlling the glucose concentration.
Example 9 Clostridium thermocellum coupled PT-GD-2 one-pot Process for the production of succinic acid
Adding into 2L fermentation tankGS-2 medium (ingredients: 2.9g of dipotassium hydrogen phosphate per liter, 1.5g of monopotassium phosphate, 2.1g of urea, 150mg of calcium chloride, 1.0g of magnesium chloride, 1.25mg of ferrous sulfate, 1g of cysteine, 3g of sodium citrate, 6.0g of yeast extract and 1.0mg of resazurin) is added with 10% by mass and volume of alkali pretreated xylose residues as a substrate (the xylose residues are corn cob waste after xylose is extracted by acid hydrolysis), the mixture is subjected to air culture for 12 hours at 60 ℃ under anaerobic conditions, the rotation speed of a stirring paddle is 150 revolutions per minute, the substrates are fully mixed, and the initial pH is 7.4. Then inoculating the clostridium thermocellum strain described in the embodiment 5 according to the volume ratio of 5% for culture, inoculating PT-GD-2 according to the volume ratio of 10% when the glucose accumulation concentration reaches 20g per liter, and controlling the pH value to be 6.2-6.5, wherein the PT-GD-2 can normally grow and the growth of the clostridium thermocellum can be inhibited under the condition, and meanwhile, a cellulose degradation enzyme system, namely a cellulose body produced by the clostridium thermocellum can still normally play a role, so that the synchronous realization of cellulose substrate degradation and succinic acid fermentation production is realized. The fermentation was terminated when the glucose concentration in the fermentation broth was zero and the succinic acid concentration did not rise any more for 2 consecutive hours. The final succinate yield was 42.1 grams per liter and the xylose residue substrate contained about 78% total sugars, with a succinic acid yield of 42 grams per 100 grams of xylose residue dry weight, calculated on the lignocellulosic feedstock. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,succinic acid to obtain The rate was 59.9 grams per 100 grams of glucose。
Example 10 Clostridium thermocellum coupled PT-GD-2 one-pot Process for the production of succinic acid
In contrast to example 9, GS-2 medium and 4% by mass/volume alkali-pretreated straw as substrate were added to a 2L fermenter and the substrates were mixed thoroughly at 60 ℃ with an initial pH of 7.4. Then, the strain Clostridium thermocellum described in example 5 was inoculated at 10% by volume, and when the concentration of accumulated glucose reached 10g/l, PT-GD-2 was inoculated at 5% by volume, the temperature was adjusted to 65 ℃ and the pH was controlled to 6.2 to 6.5, and when the glucose concentration in the fermentation broth became zero and the succinic acid concentration did not rise any more for 2 hours, the fermentation was terminated. The final succinate yield was 13.2 grams per liter and the succinic acid yield was 33.1 grams per 100 grams dry weight of pretreated straw, calculated on the lignocellulosic feedstock. According to the condition that the sugar yield is 90 percentCalculating the equivalent weight of the glucose,the yield of succinic acid was 53.3 g per 100 g of glucose。
Example 11 Clostridium thermocellum coupled PT-GD-2 one-pot Process for the production of succinic acid
In contrast to example 10, GS-2 medium and 8% by mass/volume alkali-pretreated straw as substrate were added to a 1L fermenter, and the substrates were mixed thoroughly at 60 ℃ with an initial pH of 7.4. Then, the strain Clostridium thermocellum described in example 5 was inoculated at 10% by volume, PT-GD-2 was inoculated at 1% by volume when the glucose accumulation concentration reached 15 g/L, the pH was controlled at 6.2 to 6.5, and the fermentation was terminated when the glucose concentration in the fermentation broth was zero and the succinic acid concentration did not rise any more for 2 hours. The final succinate yield was 30.1 grams per liter and the succinic acid yield was 37.6 grams per 100 grams dry weight of pretreated straw, calculated on the lignocellulosic feedstock. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,yield of succinic acid 60.5 grams per 100 grams of glucose。
In conclusion, the optimum growth temperature of the clostridium succinicum strain PT-GD-2 is 55-65 ℃, and the yield of succinic acid produced by fermentation can reach approximately 70g per 100 g of glucose under the conditions of proper pH and glucose concentration. Compared with the existing Clostridium mesophilic succinate strain, the Clostridium succinicum strain PT-GD-2 realizes the obvious improvement of yield under the high-temperature condition, and has important practical application value. In addition, based on the high-temperature characteristic of the strain PT-GD-2 and the characteristic that the optimum pH value of the strain PT-GD-2 is staggered with the optimum pH value of the clostridium thermocellum, the method for producing succinic acid by coupling the strain PT-GD-2 with the clostridium thermocellum is provided, the succinic acid is produced by a one-pot method by taking lignocellulose as a substrate only by controlling the pH value, the temperature is not required to be increased or reduced in the whole process, and a production device is not required to be replaced, so that the operation is simple, the production cost is reduced, and the generation of byproducts is also reduced. Meanwhile, the method adopts lignocellulose as a substrate, so that the production cost is further reduced, and the method has important practical application value.
Claims (10)
1. A thermophilic anaerobic clostridium strain for producing succinic acid, which is characterized in that: said thermophilic anaerobeThe Clostridium strain is named as thermophilic anaerobic Clostridium strain PT-GD-2: (Pseudoclostridium thermosuccinogenes) The culture medium is preserved in China general microbiological culture Collection center of China general microbiological culture Collection center, No. 3 of Xilu No. 1 of Beijing, Chaoyang, with the preservation number of CGMCC number 23684 and the preservation date of 2021 year, 10 months and 28 days.
2. A microbial inoculant comprising the thermophilic anaerobic clostridium strain of claim 1.
3. Use of the thermophilic anaerobic shuttle strain according to claim 1, characterized in that: the strain of thermophilic anaerobic clostridium is used for producing succinic acid by fermentation.
4. Use of a thermophilic anaerobic shuttle strain according to claim 3, characterized in that: inoculating the strain of Clostridium thermoanaerobicum according to claim 1 into a fermentation medium for fermentation growth, and separating succinic acid from the fermentation broth after fermentation; the carbon source in the fermentation medium is glucose, fructose, cellobiose or xylose.
5. Use of the thermophilic anaerobic shuttle strain according to claim 4, characterized in that: the carbon source is one or two of glucose and xylose.
6. Use of a thermophilic anaerobic shuttle strain according to claim 5, characterized in that: the concentration of the carbon source is 10-20 g/L; the temperature of the fermentation growth is 55-65 ℃; the pH of the fermentation growth is 6.2-8.
7. Use of a thermophilic anaerobic shuttle strain according to any one of claims 4 to 6, characterized in that: the fermentation medium comprises the following components: 10-20g/L of carbon source, 2.9g/L of dipotassium phosphate, 1.5g/L of monopotassium phosphate, 2.1g/L of urea, 150mg/L of calcium chloride, 1.0g/L of magnesium chloride, 1.25mg/L of ferrous sulfate, 1.0g/L of cysteine, 1.0mg/L of resazurin and 6.0g/L of yeast extract.
8. Use of a thermophilic anaerobic shuttle strain according to claim 3, characterized in that: coupling the thermophilic anaerobic clostridium strain PT-GD-2 with clostridium thermocellum to produce succinic acid by a one-pot method, and specifically comprising the following steps:
(1) saccharification of lignocellulose: adding a GS-2 culture medium and the pretreated lignocellulose substrate into a fermentation tank, and uniformly mixing; inoculating a clostridium thermocellum strain for saccharification to obtain a lignocellulose fermentation liquor;
(2) and (3) producing succinic acid by fermentation: inoculating thermophilic anaerobic clostridium strain PT-GD-2 according to the volume ratio of 1-10% under the condition that the pH =6.2-6.5 when the concentration accumulation of glucose in the lignocellulose fermentation liquor reaches 10-20 g/L; and when the concentration of the glucose in the lignocellulose fermentation liquid is zero, ending the fermentation to obtain the succinate.
9. Use of a thermophilic anaerobic shuttle strain according to claim 8, characterized in that: the temperature for producing the succinic acid by the fermentation in the step (2) is 55-65 ℃.
10. Use of a thermophilic anaerobic shuttle strain according to claim 8, characterized in that: the lignocellulose substrates in the step (1) are straws and corncobs; the inoculation proportion of the clostridium thermocellum is 5-10%.
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| CN115029264A (en) * | 2022-06-14 | 2022-09-09 | 中国科学院青岛生物能源与过程研究所 | Acid-resistant clostridium thermocellum and method for degrading lignocellulose by using same |
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