Thermoclostridium succinogenes and application of thermoclostridium succinogenes in succinic acid production by utilizing lignocellulose

Abstract

The invention relates to the field of straw conversion and utilization and microbial fermentation engineering, in particular to thermoclostridium succinogenes and application of the thermoclostridium succinogenes in succinic acid production by utilizing lignocellulose. The thermoclostridium succinogenes is classified and named as Clostridium succinogenes, the preservation date of the thermoclostridium succinogenes is November 4, 2024, and the preservation number of the thermoclostridium succinogenes is CGMCC (China General Microbiological Culture Collection Center) No.32487. The thermoclostridium succinogenes obtained by the invention is relatively high in growth speed and strong in stress resistance, and can be normally fermented under the conditions of low pH and high aldehyde (furfural, 5-hydroxymethylfurfural and vanillin) of a steam explosion substrate, and the highest succinic acid produced by consuming 100g of a pretreated straw substrate under a high-temperature anaerobic condition can reach 25.32 g.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, specifically to a succinic acid-producing bacterium and its application in the production of succinic acid using lignocellulose. Background Technology

[0002] Succinic acid (also known as succinic acid) is an important platform compound widely used in the food, pharmaceutical, daily chemical, and chemical industries. Succinic acid can be used to produce numerous high-value products. For example, in the food industry, it can be used to improve the flavor of liquid seasonings and refined products; in the pharmaceutical industry, it can be used to produce erythromycin ethylsuccinate, plant growth regulators, and pesticides; and in the chemical industry, it can be used to synthesize 1,4-butanediol, tetrahydrofuran, and the biodegradable biopolymer polybutylene succinate (PBS). Currently, the main methods for producing succinic acid are chemical and biological methods. Compared to the high-temperature and high-pressure reaction conditions of chemical methods, the biological fermentation method for producing succinic acid has the advantages of milder conditions, lower energy consumption, and environmental friendliness. However, the biological fermentation method for producing succinic acid mainly uses glucose derived from starch as a carbon source, which is costly and poses a challenge of competition for food with humans and livestock.

[0003] As zero-carbon renewable resources, non-grain biomass resources such as straw are an inevitable trend in future resource structure adjustment, and their efficient conversion and utilization to replace petrochemical resources is crucial. The enzymatic hydrolysis of biomass into a sugar solution to replace glucose from grains, followed by microbial fermentation to produce succinic acid, is a promising new production method that has attracted much attention in recent years. Currently, the main method uses free cellulase preparations derived from fungi to hydrolyze straw, using the straw saccharification liquid as a carbon source for the fermentation and cultivation of succinic acid-producing strains. However, this method still has many problems: the high cost of fungal enzyme preparations makes fermentable sugars from lignocellulose less competitive in the market compared to starch sugars, thus hindering the practical application of lignocellulose-based succinic acid preparation technology. In addition, invention patents CN108977421A, CN109097417A, and CN114196588A propose a lignocellulose saccharification method that does not rely on fungal free enzymes, primarily utilizing thermophilic cellulose-degrading bacteria such as Clostridium thermophilum as whole-cell catalysts. However, Clostridium thermophilum has poor stress resistance and is greatly affected by inhibitors such as lignin and furfural, which is incompatible with the mainstream pretreatment methods in industry, such as steam explosion, making it difficult to achieve industrial application.

[0004] Biomass has a complex structure and composition, requiring physical, chemical, or biological pretreatment to remove degradation barriers before efficient enzymatic hydrolysis and saccharification can be achieved. Steam explosion is one of the important straw pretreatment methods and is currently the mainstream pretreatment process used in industry. However, during high-temperature and high-pressure pretreatment, biomass inevitably produces numerous inhibitory small molecules, mainly of three types: small organic acids such as formic acid and acetic acid; sugar derivatives such as furfural and 5-hydroxymethylfurfural; and lignin derivatives such as vanillin and p-hydroxybenzaldehyde. These oxygenated compounds not only lead to high product viscosity and strong corrosiveness, but more importantly, they strongly inhibit the lignocellulose degrading enzyme system and fermentation strains. Existing methods to overcome inhibition mainly employ physical, chemical, or biological detoxification methods, such as washing, activated carbon or resin adsorption, nanofiltration membranes, or reverse osmosis membranes to remove inhibitors. However, these methods incur significant additional processing costs and ecological burdens. Therefore, effectively overcoming the impact of small molecule inhibitors is a crucial issue for the sustainable production of straw bioconversion.

[0005] In summary, the complex biomass system leads to harsh conditions and complex side reactions during pretreatment, resulting in numerous inhibitors with complex compositions that are difficult to remove efficiently. These inhibitors strongly suppress lignocellulose-degrading enzymes and fermentation strains. If physical, chemical, or biological methods are used for detoxification, it will increase treatment costs and ecological burden, which is not conducive to the sustainable production of succinic acid. Summary of the Invention

[0006] To address the problems existing in the production of succinic acid from lignocellulose in the prior art, this invention provides a succinic acid-producing *Clostridium thermophilum* strain that can tolerate inhibitors in steam explosion pretreatment substrates and can be directly used for the steam explosion pretreatment of straw fermentation to produce succinic acid. Clostridium thermosuccinogenes ) sdu068, no detoxification process required, high yield of succinic acid.

[0007] The technical solution of this invention: A type of succinic acid-producing Clostridium thermophilum, classified as Clostridium thermosuccinogenes The date of deposit is November 4, 2024, and the accession number is CGMCC No. 32487.

[0008] The application of the succinic acid-producing Clostridium thermophilum in the production of succinic acid from lignocellulose.

[0009] Using the succinic acid-producing *Clostridium thermophilum* strain as the starting strain, succinic acid was obtained by fermentation in a fermentation medium containing straw substrate pretreated by steam explosion.

[0010] The straw substrate is one of the following: corn stalks, corn cobs, wheat straw, cotton stalks, or sugarcane bagasse.

[0011] When the straw substrate is corn stalk, corn cob, wheat straw, or cotton stalk, the method for preparing the steam explosion pretreated straw substrate is to chop the straw substrate into fragments smaller than 2 cm, rehydrate it to a moisture content of 48-52%, and then perform steam explosion pretreatment at 168-172℃ for 18-22 minutes. When the straw substrate is sugarcane bagasse, the method for preparing the steam explosion pretreated straw substrate is to directly rehydrate the straw substrate to a moisture content of 48-52%, and then perform steam explosion pretreatment at 168-172℃ for 18-22 minutes.

[0012] The fermentation medium consists of: 10-80 g / L of steam-exploded pretreated straw substrate, 5-10 g / L of yeast extract, 3.8-4.2 g / L of potassium dihydrogen phosphate, 2-2.2 g / L of urea, 0.9-1.1 g / L of cysteine, 0.9-1.1 mg / L of resazurin, 0.9-1.1 g / L of magnesium chloride, 148-152 mg / L of calcium chloride, and 1.24-1.26 mg / L of ferrous sulfate.

[0013] The fermentation temperature is 58-62℃, the fermentation pH is 5.4-5.6, and the fermentation time is 4-10 days.

[0014] The succinic acid-producing *Clostridium thermophilum*, named *Clostridium thermophilum* SDU068 in the laboratory, was isolated from rice paddies in Dianbai District, Maoming City, Guangdong Province. It was obtained by mutagenesis induced by dielectric barrier discharge (DBD) plasma device and high-temperature anaerobic enrichment and screening on low-pH high-aldehyde microcrystalline cellulose medium.

[0015] The preservation information is as follows: Clostridium thermophilum producing succinic acid sdu068, classified and named Clostridium thermosuccinogenes It was deposited on November 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32487.

[0016] Compared with the prior art, the present invention achieves the following beneficial effects: (1) In this invention, a succinic acid-producing thermoclosing strain SDU068 was obtained by mutagenesis induced by dielectric barrier discharge (DBD) device and then by high-temperature anaerobic enrichment and screening in low pH high aldehyde medium. The single colony of this strain was cultured in 50 mL of low pH high aldehyde medium at 60 °C for 6 days, and the succinic acid content in the fermentation broth reached 3.05%.

[0017] (2) This invention obtains a succinic acid-producing thermoclosing strain SDU068 by mutagenesis induced by a dielectric barrier discharge (DBD) device and then by high-temperature anaerobic enrichment and screening in a low-pH high-aldehyde medium. The strain can directly degrade steam-explosion pretreated straw to produce succinic acid under high-temperature anaerobic conditions, realizing the integrated bioprocessing of straw to produce succinic acid in one pot. The equipment and process steps are few, the high-temperature anaerobic conditions are not easy to be contaminated by bacteria, and there is no need for ventilation and strong stirring. It is especially suitable for semi-solid fermentation with high solid content. The cost is low and the succinic acid yield is high. Consuming 100 grams of steam-explosion pretreated straw substrate can produce 25.32 grams of succinic acid, which has a significant improvement in cost reduction and efficiency.

[0018] (3) The succinic acid-producing thermoclostridium strain SDU068 described in this invention has good stress resistance and can tolerate the inhibitors produced by the straw substrate under steam explosion pretreatment. It can grow normally under the conditions of conventional concentrations of common steam explosion inhibitors such as formic acid, acetic acid, 5-hydroxymethylfurfural, furfural, vanillin, and p-hydroxybenzaldehyde. It does not require a detoxification process, which greatly simplifies the process flow, makes the operation simple, and further reduces equipment and production costs. It has important practical application value. Attached Figure Description

[0019] Figure 1 Photographs of the culture plate of the succinic acid-producing bacterium SDU068 and cell morphology under an optical microscope. Figure 2 The results are based on BLAST sequence alignment. Figure 3 This is a schematic diagram of a dielectric barrier discharge (DBD) plasma mutation device. Detailed Implementation

[0020] The present invention will be further described below with reference to the embodiments.

[0021] Example 1: Isolation and screening of strain SDU068 1) Initial screening: 10g of soil sample taken from paddy fields in Dianbai District, Maoming City, Guangdong Province was suspended in 100mL of sterile water and allowed to stand at 180r / min. After standing, 1mL of the supernatant was taken and spread on a primary screening medium plate (each liter contains 4g potassium dihydrogen phosphate, 2.1g urea, 1g L-cysteine ​​hydrochloride, 6g yeast powder, 2mg resazurin, 1g MgCl2·6H2O, 0.15g CaCl2·2H2O, 1.25mg FeSO4·7H2O, 5g microcrystalline cellulose, and 10g high-temperature agar).

[0022] The culture plate was placed upright in the anaerobic chamber and anaerobically cultured at 60℃ for 5 days. A culture plate with a suitable colony density was selected, and single colonies of different morphologies were picked up with an autoclaved toothpick and streaked again on a new primary screening medium plate. The culture was anaerobically cultured at 60℃ for 5 days. The streaking purification was performed a total of 3 times on the primary screening medium plate to finally obtain a pure culture strain that efficiently degrades cellulose.

[0023] 2) Mutagenesis and secondary screening: The pure culture colonies obtained in the previous step were inoculated and expanded, then mutagenized using a dielectric barrier discharge (DBD) plasma device, and enriched in a low-pH, high-aldehyde medium for targeted screening of target strains.

[0024] The specific grouping and operations are as follows: Group A: Step 1. Inoculate a single colony into an anaerobic bottle containing 50 mL of culture medium. This medium contains 4 g potassium dihydrogen phosphate, 2.1 g urea, 1 g L-cysteine ​​hydrochloride, 6 g yeast extract, 2 mg resazurin, 1 g MgCl2·6H2O, 0.15 g CaCl2·2H2O, 1.25 mg FeSO4·7H2O, and 5 g microcrystalline cellulose per liter. Incubate anaerobically at 60 °C. When the OD reaches 0.1, proceed to the next step.

[0025] Step 2. Take 50 mL of bacterial solution and perform dielectric barrier discharge (DBD) plasma device mutagenesis. The voltage is 3 KV, the frequency is 2.5 KHz, the distance between the outer edge of the inner electrode and the inner edge of the glass tube is 0.5 mm, the working gas is argon, the gas flow rate is 0.05 L / min, and the processing time is 60 seconds.

[0026] Step 3. Pour 50 mL of the treated bacterial culture into 150 mL of enrichment medium for enrichment culture at 60 °C. Each liter of medium contains: 4 g potassium dihydrogen phosphate, 2.1 g urea, 1 g formic acid, 1.5 g 5-hydroxymethylfurfural, 2.5 g furfural, 3 g vanillin, 3 g p-hydroxybenzaldehyde, 1 g L-cysteine ​​hydrochloride, 6 g yeast extract, 3 g acetic acid, 2 mg resazurin, 1 g MgCl2·6H2O, 0.15 g CaCl2·2H2O, 1.25 mg FeSO4·7H2O, and 5 g microcrystalline cellulose. The pH is 5.5, and the culture time is 6 days.

[0027] The culture medium was deoxygenated with high-purity nitrogen for 5 minutes before inoculation. By deoxygenating the culture medium with nitrogen in advance, the purpose of anaerobic culture was achieved.

[0028] Group B: The steps 1, 2, and 3 of the procedure are the same as those of Group A, except that the mutagenesis conditions in step 2 are changed to a voltage of 10KV, a frequency of 5KHz, a distance of 4mm between the outer edge of the inner electrode and the inner edge of the glass tube, a working gas of nitrogen, a gas flow rate of 0.05L / min, and a processing time of 30 seconds.

[0029] Group C: The steps 1, 2, and 3 of the procedure are the same as those of Group A, except that the mutagenesis conditions in step 2 are changed to a voltage of 25KV, a frequency of 10KHz, a distance of 8mm between the outer edge of the inner electrode and the inner edge of the glass tube, an argon working gas, a gas flow rate of 0.05L / min, and a processing time of 10 seconds.

[0030] Group D: The steps 1, 2, and 3 of the procedure are the same as those of Group A, except that the mutagenesis conditions in step 2 are changed to a voltage of 10KV, a frequency of 10KHz, a distance of 4mm between the outer edge of the inner electrode and the inner edge of the glass tube, an argon working gas, a gas flow rate of 0.05L / min, and a processing time of 30 seconds.

[0031] Group E: The procedure is the same as for group D, except that step 3 is changed to: culture temperature 50℃, culture time 12 days.

[0032] Group F: The procedure is the same as for group D, except that step 3 is changed to: culture temperature 65℃, culture time 8 days.

[0033] After the above-mentioned groups have been cultured, 100 μL of bacterial suspension was diluted and plated onto screening plates (the culture medium for the plates was the screening medium below with 10 g / L high-temperature agar added). The culture plates were placed upright in an anaerobic chamber and anaerobically cultured at 60℃ for 5 days. Single colonies were picked and inoculated into 50 mL of screening medium for further culture at 60℃. Each liter of medium contained: 4 g potassium dihydrogen phosphate, 2.1 g urea, 1 g formic acid, 1.5 g 5-hydroxymethylfurfural, 2.5 g furfural, 3 g vanillin, 3 g p-hydroxybenzaldehyde, 1 g L-cysteine ​​hydrochloride, 6 g yeast extract, 3 g acetic acid, 2 mg resazurin, 1 g MgCl2·6H2O, and 0.15 g... CaCl2·2H2O, 1.25 mg FeSO4·7H2O, 5 g microcrystalline cellulose, pH 5.5, anaerobic culture for 6 days, organic acids were extracted from the fermentation broth for analysis. The two strains with the best results from each group were selected for preservation and statistical analysis. The results are shown in Table 1. A1 and A2 were derived from group A, and so on.

[0034] Table 1. Succinic acid content in fermentation broth of mutant strains

[0035] The strain D2 with the highest succinic acid production was selected from Table 1 and named sdu068. Morphological and structural observations under an optical microscope revealed (…). Figure 1 ): sdu068 cells are short rod-shaped, with a diameter of 0.5-1 micrometer. Sequencing was performed, and BLAST sequence alignment was conducted (alignment results are shown in...). Figure 2 The strain was identified as *Clostridium thermophilum*, a succinic acid-producing bacterium. Its 16S rDNA sequence is shown in SEQ ID NO.1. It was deposited with the China General Microbiological Culture Collection Center, and the deposit information is as follows: Clostridium thermophilum producing succinic acid sdu068, classified and named Clostridium thermosuccinogenes It was deposited on November 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32487.

[0036] Example 2: Effects of different culture conditions on succinic acid production by strain sdu068 Cultivation Condition 1: 100 μL of the sdu068 strain preserved in glycerol tubes was inoculated into a 250 mL anaerobic bottle containing 100 mL of seed culture medium. The culture was carried out in a shaker at 60 °C and 100 rpm for 48 h to obtain the seed culture. Add 1.2L of fermentation medium to a 3L bioreactor and inoculate with 100mL of the seed liquid obtained above. During fermentation, the micro-positive pressure (30kPa) and anaerobic state in the tank are controlled by high-purity nitrogen gas, the temperature is controlled at 60℃, the pH value is 5.5, and the fermentation time is 4 days.

[0037] The seed culture medium consisted of 5 g / L microcrystalline cellulose, 6 g / L yeast extract, 4 g / L potassium dihydrogen phosphate, 2.1 g / L urea, 1 g / L cysteine, 2 mg / L resazurin, 1 g / L magnesium chloride, 150 mg / L calcium chloride, 1.25 mg / L ferrous sulfate, and pH 5.5.

[0038] The fermentation medium consisted of 10 g / L of steam-exploded pretreated corn stalk substrate, 5 g / L of yeast extract, 4 g / L of potassium dihydrogen phosphate, 2.1 g / L of urea, 1 g / L of cysteine, 1 mg / L of resazurin, 1 g / L of magnesium chloride, 150 mg / L of calcium chloride, 1.25 mg / L of ferrous sulfate, and pH 5.5.

[0039] Cultivation condition 2: The culture conditions were the same as in culture condition 1, except that the concentration of the steam-exploded corn straw substrate in the fermentation medium was adjusted to 20 g / L, the concentration of the yeast extract was adjusted to 10 g / L, and the fermentation time was adjusted to 6 days.

[0040] Cultivation condition 3: The culture conditions were the same as in culture condition 1, except that the concentration of the steam-exploded corn straw substrate in the fermentation medium was adjusted to 60 g / L, the concentration of the yeast extract was adjusted to 10 g / L, and the fermentation time was adjusted to 8 days.

[0041] Cultivation condition 4: The culture conditions were the same as in culture condition 1, except that the concentration of the steam-exploded corn straw substrate in the fermentation medium was adjusted to 80 g / L, the concentration of the yeast extract was adjusted to 10 g / L, and the fermentation time was adjusted to 10 days.

[0042] Cultivation condition 5: The culture conditions are the same as in culture condition 2, except that the steam-explosion pretreated corn stalk substrate is replaced with steam-explosion pretreated corn cob substrate in the fermentation medium.

[0043] Cultivation condition 6: The conditions are the same as in culture condition 2, except that the steam-explosion pretreated corn straw substrate is replaced with steam-explosion pretreated wheat straw substrate in the fermentation medium.

[0044] Cultivation condition 7: The culture conditions are the same as in culture condition 2, except that the steam-explosion pretreated corn stalk substrate is replaced with steam-explosion pretreated cotton stalk substrate in the fermentation medium.

[0045] Cultivation condition 8: The conditions are the same as in culture condition 2, except that the steam-explosion pretreated corn stalk substrate is replaced with steam-explosion pretreated sugarcane bagasse substrate in the fermentation medium.

[0046] The preparation method of the steam explosion pretreated corn stalk substrate, steam explosion pretreated wheat stalk substrate, steam explosion pretreated cotton stalk substrate, and steam explosion pretreated corn cob substrate is as follows: the corn stalk, wheat stalk, cotton stalk or corn cob is chopped into fragments less than 2 cm, rehydrated to 50% moisture content, and steam explosion pretreated at 170℃ for 20 minutes.

[0047] The method for preparing the sugarcane bagasse substrate for steam explosion pretreatment is to rehydrate the sugarcane bagasse to a moisture content of 50% and then perform steam explosion pretreatment at 170°C for 20 minutes.

[0048] After fermentation, the fermentation product at the end of the fermentation was separated by centrifugation. The supernatant was taken and sent for testing of succinate content. The results were converted into grams of succinic acid per 100 grams of oven-dry substrate. The results are shown in Table 2.

[0049] Table 2. Effects of different culture conditions on succinic acid production by strain sdu068

[0050] The experimental results above show that when steam explosion pretreatment of wheat straw is used as the substrate, the succinic acid conversion rate reaches the highest level, reaching 25.32 g succinic acid / 100 g substrate.

Claims

1. A succinic acid-producing Clostridium thermophilum, characterized in that: The succinic acid-producing Clostridium thermophilum is classified as follows: Clostridium thermosuccinogenes The date of deposit is November 4, 2024, and the accession number is CGMCC No.32487.

2. The application of the succinic acid-producing Clostridium thermophilum according to claim 1 in the production of succinic acid from lignocellulose.

3. The application according to claim 2, characterized in that: Using the succinic acid-producing *Clostridium thermophilum* strain as the starting strain, succinic acid was obtained by fermentation in a fermentation medium containing straw substrate pretreated by steam explosion.

4. The application according to claim 3, characterized in that: The straw substrate is one of the following: corn stalks, corn cobs, wheat straw, cotton stalks, or sugarcane bagasse.

5. The application according to claim 4, characterized in that: When the straw substrate is corn stalk, corn cob, wheat straw, or cotton stalk, the method for preparing the steam explosion pretreated straw substrate is to chop the straw substrate into fragments smaller than 2 cm, rehydrate it to a moisture content of 48-52%, and then perform steam explosion pretreatment at 168-172℃ for 18-22 minutes. When the straw substrate is sugarcane bagasse, the method for preparing the steam explosion pretreated straw substrate is to directly rehydrate the straw substrate to a moisture content of 48-52%, and then perform steam explosion pretreatment at 168-172℃ for 18-22 minutes.

6. The application according to claim 3, characterized in that: The fermentation medium consists of: 10-80 g / L of steam-exploded pretreated straw substrate, 5-10 g / L of yeast extract, 3.8-4.2 g / L of potassium dihydrogen phosphate, 2-2.2 g / L of urea, 0.9-1.1 g / L of cysteine, 0.9-1.1 mg / L of resazurin, 0.9-1.1 g / L of magnesium chloride, 148-152 mg / L of calcium chloride, and 1.24-1.26 mg / L of ferrous sulfate.

7. The application according to claim 3, characterized in that: The fermentation temperature is 58-62℃, the fermentation pH is 5.4-5.6, and the fermentation time is 4-10 days.

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