Bacillus coagulans and application thereof in biosynthesis of L-lactic acid by using non-grain sugar

Abstract

The invention relates to the technical field of biology, and particularly discloses bacillus coagulans and application thereof in biosynthesis of L-lactic acid by using non-grain sugar. According to the invention, a high-temperature-resistant strain bacillus coagulans BC-A is obtained through breeding. The strain has broad-spectrum substrate adaptability, can efficiently utilize different sources of agricultural waste saccharification hydrolysate, can convert different mixed sugars composed of glucose / xylose into L-lactic acid, the sugar-acid conversion rate is over 96%, the yield of L-lactic acid reaches 90-200g / L, and the strain is suitable for industrial production. Therefore, the strain has remarkable advantages and wide industrial application prospects in the aspect of producing high-value L-lactic acid by using complex and cheap non-grain biomass raw materials (such as lignocellulose hydrolysate). Meanwhile, the strain is perfectly matched with the optimal temperature of trichoderma reesei cellulase, cellulose enzymolysis saccharification and lactic acid fermentation are synchronously completed in the same reaction system, and the efficiency limitation of a traditional step-by-step process is broken through.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a Bacillus coagulans and its application in fermenting agricultural waste hydrolysate to produce L-lactic acid. Background Art

[0002] With the increasing global emphasis on sustainable development and environmental protection, the biobased and biodegradable material polylactic acid (PLA) is regarded as an ideal alternative to traditional petroleum-based plastics due to its excellent properties and green environmental protection characteristics, and the market demand shows an explosive growth. However, the core bottleneck in the booming development of the polylactic acid industry lies in the production cost of its monomer - highly optically pure L-lactic acid. Traditional L-lactic acid fermentation mainly relies on starch sugars provided by food crops such as corn and potatoes as carbon sources, which not only results in the raw material cost accounting for more than 50% of the production cost, but also triggers the resource sustainability problem of "competing with humans for food and with livestock for feed", severely restricting the large-scale development and market competitiveness of the polylactic acid industry. Therefore, developing non-grain raw materials, especially using lignocellulosic biomass (such as crop straws, bagasse, forestry waste, etc.) which is widely sourced, low-cost and does not conflict with food production to produce L-lactic acid, has become a key strategic direction to break through the industrial bottleneck and achieve the economic feasibility and environmental friendliness of polylactic acid.

[0003] However, fermenting L-lactic acid using lignocellulose hydrolysate (i.e., "straw sugar") faces a series of severe technical challenges: the toxic inhibition of complex inhibitors (such as furfural, 5-hydroxymethylfurfural, etc.) in the hydrolysate on the growth and metabolism of microorganisms; the need for strains to have efficient co-utilization ability for the simultaneously present pentose sugars (xylose, arabinose) and hexose sugars (glucose) in the hydrolyzed sugars; the complex structure of the lignocellulose raw material itself, with high pretreatment and enzymatic hydrolysis costs, requiring the fermentation process to have high conversion efficiency to spread the costs; at the same time, in order to meet the requirements of polylactic acid polymerization grade, the optical purity of L-lactic acid must be extremely high (usually >99%), and the fermentation process needs to be simplified as much as possible to reduce energy consumption and operating costs. Therefore, screening or constructing microbial strains with strong environmental robustness, co-utilization of pentose / hexose sugars, and low-cost production of highly optically pure L-lactic acid, and developing a matching efficient and low-cost fermentation process, are the core research objectives and technical difficulties for realizing the economical production of L-lactic acid using non-grain lignocellulose resources. Summary of the Invention

[0004] The present invention obtains a high-temperature resistant strain through breeding, which has a broad-spectrum substrate adaptability and can efficiently utilize agricultural waste saccharification hydrolysates from different sources, and can convert mixed sugars with different compositions of glucose / xylose into L-lactic acid, thus the present invention is proposed.

[0005] The present invention first provides a Bacillus coagulans ( Bacillus coagulans), BC-A, which was deposited on August 14, 2023 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number: CGMCC No. 28166, and the taxonomic name is Bacillus coagulans ( Bacillus coagulans ).

[0006] The present invention further provides the application of the above-mentioned Bacillus coagulans in the preparation of L-lactic acid.

[0007] The present invention particularly provides a method for preparing L-lactic acid using straw, which comprises the following steps: Using a fermentation medium with straw hydrolysate as the sole carbon source, inoculating the above-mentioned Bacillus coagulans for fermentation to obtain L-lactic acid.

[0008] The straw is selected from corn straw, corn cob, rice straw or bagasse.

[0009] Specifically, the fermentation temperature is 45°C - 60°C, anaerobic fermentation for 24 - 48 h, the stirring speed is 40 - 80 r / min. When the glucose residue in the fermentation broth is 10 - 20 g / L, add corn straw hydrolysate and continue fermentation until the fermentation ends.

[0010] The present invention also provides a method for synchronous saccharification and fermentation of straw to produce L-lactic acid, which comprises the following steps: S1 Using one or more of steam-exploded corn straw, corn cob, rice straw, bagasse as substrates, adding Trichoderma reesei A2H (Trichodermareesei) complex cellulase in the reaction system; Among them, Trichoderma reesei strain A2H, which was deposited on March 17, 2021 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number: CGMCC No. 21470, and the taxonomic name is Trichoderma reesei Trichoderma reesei .

[0011] S2 Then inoculate the above-mentioned Bacillus coagulans seed culture solution for fermentation to synchronously saccharify and obtain L-lactic acid.

[0012] Specifically, in step S2, the fermentation temperature is 45°C - 60°C, anaerobic fermentation for 24 - 48 h, and the stirring speed is 40 - 80 r / min.

[0013] Optionally, it further includes step S3 of further separating the produced L-lactic acid.

[0014] Specifically, the Trichoderma reesei A2H complex cellulase is obtained by solid-liquid separation of the fermentation broth of Trichoderma reesei A2H strain and collecting the supernatant.

[0015] More specifically, the liquid fermentation culture conditions of Trichoderma reesei strain A2H are as follows: the temperature is 24°C to 28°C, the pH is 4.8 to 5.2, the rotation speed is 250 to 300 rpm, the dissolved oxygen content in the fermentation broth is 25% to 35% (v / v), and the culture time is 24 to 120 hours; the culture medium used in the fermentation culture contains the following components: glucose 20 to 30 g / L, corn steep liquor dry powder 2 to 6 g / L, KOH 1.60 to 1.72 g / L, (NH4)2SO4 2.6 to 3.0 g / L, and MgSO4 0.4 to 0.8 g / L. For example, the culture conditions of Trichoderma reesei strain A2H are: the temperature is 26°C, the pH is 5.0, and the dissolved oxygen content in the fermentation broth is 30% (v / v); the culture medium contains the following components: glucose 25 g / L, corn steep liquor dry powder 4 g / L, KOH 1.66 g / L, (NH4)2SO4 2.8 g / L, and MgSO4 0.6 g / L.

[0016] The present invention selects a specific strain of Bacillus coagulans BC-A that is heat-resistant (such as 55°C). Its special features are strong robustness, such as tolerance to inhibitors in straw sugar, high temperature tolerance, and high lactic acid production. This strain can perfectly match the optimal temperature for the enzymatic hydrolysis of the composite cellulase of Trichoderma reesei (such as 50°C), and simultaneously complete cellulose enzymatic hydrolysis saccharification and lactic acid fermentation in the same reaction system, breaking through the efficiency limitation of the traditional step-by-step process; secondly, this strain has a broad substrate adaptability and can efficiently utilize saccharified hydrolysis liquids from different sources of agricultural waste, and can convert mixed sugars with different compositions of glucose / xylose into L-lactic acid. The sugar-acid conversion rate exceeds 96%, and the L-lactic acid production reaches 90 - 200 g / L, indicating that this strain has significant advantages and broad industrial application prospects in the production of high-value L-lactic acid using complex and inexpensive non-grain biomass raw materials (such as lignocellulose hydrolysis liquid). More crucially, this process realizes biological fermentation under high-temperature anaerobic and low-rotation speed conditions, without the need for strict sterilization equipment, significantly reducing energy consumption. No D-lactic acid is detected in the product, and the optical purity reaches over 99.9%, directly meeting the polymerization-grade requirements of downstream production, and can be directly used for the polymerization preparation of polylactic acid without product separation and purification. The industrial significance of this technological breakthrough is profound: it not only opens up the conversion path from non-grain biomass to high-end bio-based materials, significantly reducing the production cost of polylactic acid, but also provides a universal solution for regional biorefining by establishing a standardized process to adapt to diverse agricultural waste, promoting the transformation of the bio-based materials industry towards a green, low-carbon, and circular economy model. Description of the Drawings

[0017] Figure 1 : Growth of Bacillus coagulans BC-A on YPD plates containing furfural or hydroxymethylfurfural.

[0018] Figure 2:Liquid chromatogram of lactic acid produced by Bacillus coagulans BC-A.

[0019] Figure 3 :Detection of lactic acid production process by Bacillus coagulans.

[0020] Biological material preservation information: Bacillus coagulans BC-A, which was deposited on August 14, 2023, at the General Microbiology Center of the China National Center for Biotechnology Development (abbreviated as CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number: CGMCC No. 28166, and the taxonomic name is Bacillus coagulans ( Bacillus coagulans ).

[0021] Trichoderma reesei strain A2H, which was deposited on March 17, 2021, at the General Microbiology Center of the China National Center for Biotechnology Development (abbreviated as CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number: CGMCC No. 21470, and the taxonomic name is Trichoderma reesei Trichoderma reesei ). Detailed implementation manners

[0022] The present invention will be described below through specific implementation manners in order to better understand the present invention, but it does not constitute a limitation to the present invention.

[0023] Example 1: Obtaining of strain BC-A I. Primary screening: In order to screen the wild strain with the strongest lactic acid production ability from natural habitats, compost samples were collected from many places such as Hebei, Shandong, and Guangxi, and placed into a triangular flask containing 95 mL of sterile water and 10 glass beads, and shaken at 30 °C and 180 rpm for 30 min. Take 1 mL of the bacterial suspension for 10 -1 -10 -7 serial concentration gradient dilutions, and then take 10 -5 、10 -6 、10 -7 three dilution degrees and coat them onto the culture medium plate with straw sugar as the sole carbon source, and incubate them in an inverted position at 55 °C for 2 d.

[0024] Purification: After the colonies formed on the culture medium plate with straw sugar as the sole carbon source, select the colonies with high temperature resistance and fast growth rate for purification culture. Inoculate the purified strain onto the YPD plate and incubate it statically at 55 °C for 48 h. After repeated screening for many times, 10 wild strains that can grow at high temperature are selected, preserved and subjected to subsequent experiments.

[0025] II. Re-screening: 1. Select strains that are resistant to high temperatures of 55 °C and can utilize straw sugar as the sole carbon source. First, inoculate them into a test tube containing 5 mL of YPD medium for activation and amplification culture. 2. Inoculate the strains into 50 mL of fermentation medium and ferment at 55 °C and 200 rpm for 1 day to obtain a bacterial solution. 3. Take 10000 rpm of the fermentation broth and centrifuge for 10 minutes. Take the supernatant and dilute it by an appropriate multiple, and measure the lactic acid content with a Hillman M-1000.

[0026] 4. Comprehensive evaluation based on lactic acid production and strain growth characteristics. The results are shown in Table 1. Only strain A produces lactic acid most significantly, and the optimal strain A is determined.

[0027] Table 1: Lactic acid production by wild bacteria screened initially using straw sugar

[0028] III. Strain identification In order to more accurately identify the isolated strain A, the 16S rDNA sequence of this strain A was amplified and compared. First, extract the genomic DNA of this strain A, which is used as a template for 16S rDNA sequence amplification.

[0029] Collect the bacterial cells of strain A cultured statically overnight by centrifuging at 12000 rpm at room temperature for 1 min, and extract DNA using a bacterial DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The method is carried out according to the instructions.

[0030] Reaction conditions: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, a total of 35 cycles; then extension at 72 °C for 10 min.

[0031] After gel recovery of the target fragment, it was sent for sequencing. The sequenced 16S sequence determination results were subjected to BLAST comparison in NCBI. The results showed that the obtained sequence had a homology of up to 99.9% with Bacillus coagulans. Combining with the morphological, physiological and biochemical determination results, referring to Bergey, D.H. and Holt, J.G. (1994) Bergey’s Manual of Determinative Bacteriology. 9th Edition, Williams&Wilkins, Baltimore, Maryland. It was determined that strain A was Bacillus coagulans Bacillus coagulans , and it was named Bacillus coagulansBC-A was sent to the preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the preservation date: August 14, 2023, the preservation number: CGMCC No. 28166.

[0032] Example 2: Strain robustness analysis To evaluate the adaptability of Bacillus coagulans BC-A in the complex environment of actual lignocellulosic hydrolysate, a tolerance experiment of Bacillus coagulans BC-A against typical inhibitors in agricultural waste hydrolysates such as straw was carried out. In the basic growth medium, furfural (5 mM final concentration) and 5-hydroxymethylfurfural (HMF) were added respectively to simulate the common toxic components in the pretreated straw hydrolysate. The activated Bacillus coagulans BC-A strain was inoculated onto the culture medium plate containing the above inhibitors and statically cultured under anaerobic conditions at 55 °C for 24 hours, and the number and size of colonies formed on the plate were observed.

[0033] The experimental results are shown in Figure 1 It shows that in the medium containing 10 mM furfural or 10 mM 5-hydroxymethylfurfural, there were no significant differences in the colony morphology, colony size and number of Bacillus coagulans BC-A compared with the control group without inhibitors. Colonies of Bacillus coagulans BC-A on different culture media were picked, suspended in sterile water, and then observed under a microscope. It was found that the cell morphology was intact, and there were no obvious cell lysis or malformation phenomena. This indicates that even under the stress conditions equivalent to the inhibitor concentration in typical straw hydrolysates, the strain BC-A can still maintain strong metabolic activity and stable growth and reproduction ability, showing excellent tolerance and environmental robustness. This characteristic is crucial for its efficient fermentation directly in the crude straw sugar hydrolysate without deep detoxification, significantly reducing the pretreatment cost and improving the economy of the process.

[0034] Example 3: Simultaneous saccharification and fermentation of straw to produce L-lactic acid Using steam-exploded corn straw, corn cob, rice straw, and sugarcane bagasse as substrates, the high-solid content of the substrate in the reaction system was 30%, the dosage of Trichoderma reesei A2H complex cellulase was 20 FPU / g substrate, and the pH was 5.0. After saccharification for 24 hours, the dosage of Trichoderma reesei A2H complex cellulase was supplemented once at 10 FPU / g.

[0035] After the steam explosion pretreatment of corn stover, corn cob, rice straw, and sugarcane bagasse, compound cellulase was used for enzymatic hydrolysis and saccharification for 24 hours. Then, the culture medium of Bacillus coagulans seeds was inoculated at an inoculation amount of 10% (v / v), and synchronous saccharification was carried out at 50 °C for 96 h. At the same time, three parallel experiments were conducted. Samples were taken at 24, 48, 72, and 96 h respectively. After centrifugation, the supernatant was taken, passed through a 0.22 μm membrane, and the L-lactic acid production was measured by HPLC.

[0036] The components of the Bacillus coagulans seed culture medium are as follows: glucose 80 g / L, yeast powder 10 g / L, anhydrous calcium carbonate 30 g / L.

[0037] The components of the Bacillus coagulans fermentation culture medium are as follows: different crop straw sugars, yeast powder: 10 g / L, peptone 5 g / L, anhydrous calcium carbonate: 100 g / L After anaerobic fermentation at low speed (50 rpm) for 96 hours, the L-lactic acid production from steam-exploded corn stover, corn cob, rice straw, and sugarcane bagasse was measured to reach 110.5 g / L, 93.1 g / L, 90.2 g / L, and 108.9 g / L respectively. By calculating the released glucose and xylose contents based on the remaining substrate cellulose and hemicellulose contents, it can be seen from the L-lactic acid production results that this strain converted both pentose and hexose in straw sugar into L-lactic acid, and the sugar-acid conversion rate reached 0.96 g lactic acid / g straw sugar.

[0038] Furthermore, through the liquid phase analysis results Figure 2 and as shown in Table 2, it was found that the lactic acid produced by the fermentation of Bacillus coagulans BC-A was all L-lactic acid, and no D-lactic acid was detected, and its optical purity was over 99.9%. Most importantly, Bacillus coagulans BC-A could convert non-grain sugars with different glucose / xylose compositions into L-lactic acid, indicating that this strain has significant advantages and broad industrial application prospects in the production of high-value L-lactic acid using complex and inexpensive non-grain biomass raw materials (such as lignocellulose hydrolysate).

[0039] Table 2: Simultaneous saccharification and fermentation of different crop straws to produce L-lactic acid

[0040] Example 4: Stepwise production of L-lactic acid by first saccharifying straw and then fermenting lactic acid The Bacillus coagulans BC-A stored in glycerol at -80°C was streaked and inoculated into the growth medium, and the strain was activated by culturing at 50°C for 18 - 24 h; under sterile conditions, 1 loop of the strain was inoculated into the seed medium, and after culturing at 50°C at a low rotation speed for 20 h, it was used as the seed; the seed was inoculated into the fermenter containing the fermentation medium at an inoculation amount of 5 - 20%, and the corn straw hydrolysate was used as the sole carbon source in the fermentation medium. Anaerobic fermentation was carried out at 55°C with low-speed stirring, and the stirring speed was 80 r / min. When the residual glucose in the fermentation broth was 20 g / L, the corn straw hydrolysate was supplemented, and fermentation was continued until the fermentation ended after 96 h.

[0041] The results are as Figure 3 shown. When the residual amount of glucose or reducing sugar is lower than 20 g / L, it indicates that the residual fermentable sugar is insufficient and straw sugar needs to be continuously supplemented, and the yield of L-lactic acid accumulates continuously. When the fermentation lasts for 96 hours, the yield of L-lactic acid can approach 200 g / L. By liquid-phase determination of the fermentation supernatant, it was found that only L-lactic acid was present and no D-lactic acid was detected, which was consistent with the production of lactic acid by simultaneous saccharification and fermentation of straw, further proving the ability of Bacillus coagulans BC-A to produce L-lactic acid using straw sugar, and the optical purity of L-lactic acid reached over 99.5%.

Claims

1. A Bacillus coagulans ( Bacillus coagulans ), characterized in that Its preservation number is CGMCC No. 28166.

2. The application of the Bacillus coagulans according to claim 1 in the preparation of L-lactic acid.

3. A method for preparing L-lactic acid using straw, characterized in that, It includes the following steps: Using a fermentation medium with straw hydrolysate as the sole carbon source, inoculating the Bacillus coagulans according to claim 1 for fermentation to obtain L-lactic acid.

4. The method according to claim 3, wherein The straw is selected from corn straw, corn cob, rice straw or bagasse.

5. The method according to claim 3, characterized in that, The fermentation temperature is 45°C - 60°C, anaerobic fermentation for 24 - 48 h, the stirring speed is 40 - 80 r / min. When the glucose residue in the fermentation broth is 10 - 20 g / L, add corn straw hydrolysate and continue fermentation until the fermentation ends.

6. A method for the synchronous saccharification and fermentation of straw to produce L-lactic acid, characterized in that, It includes the following steps: S1 uses one or more of corn straw, corncob, rice straw, and bagasse pretreated by steam explosion as substrates, and Trichoderma reesei ( Trichoderma reesei ) A2H composite cellulase is added to the reaction system; Then inoculate the seed culture solution of the Bacillus coagulans according to claim 1 in S2 for fermentation to obtain L-lactic acid by synchronous saccharification.

7. The method according to claim 6, characterized in that In step S2, the fermentation temperature is 45°C - 60°C, anaerobic fermentation for 24 - 48 h, and the stirring speed is 40 - 80 r / min.

8. The method according to claim 6 or 7, characterized in that, It also includes step S3 of further separating the produced L-lactic acid.

9. The method according to claim 6 or 7, characterized in that The Trichoderma reesei A2H composite cellulase is obtained by solid-liquid separation of the fermentation broth of the Trichoderma reesei A2H strain through liquid fermentation and collecting the supernatant.

10. The method according to claim 9, characterized in that The liquid fermentation culture conditions of the Trichoderma reesei A2H strain are: the temperature is 24°C - 28°C, pH 4.8 - 5.2, the rotation speed is 250 - 300 rpm, the dissolved oxygen content in the fermentation broth is 25 - 35% (v / v), and the culture time is 24 - 120 hours; the medium used in the fermentation culture contains the following components: glucose 20 - 30 g / L, corn steep liquor dry powder 2 - 6 g / L, KOH 1.60 - 1.72 g / L, (NH4)2SO4 2.6 - 3.0 g / L, and MgSO4 0.4 - 0.8 g / L.

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