CN114181859A - Geobacillus stearothermophilus and method for producing lactic acid by utilizing lignocellulose - Google Patents
Geobacillus stearothermophilus and method for producing lactic acid by utilizing lignocellulose Download PDFInfo
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Abstract
The invention provides a high-temperature anaerobic bacterium Geobacillus stearothermophilus GS-2H-3 capable of highly producing lactic acid, which is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No.23683 and the preservation date of 2021, 10 months and 28 days. The Geobacillus stearothermophilus GS-2H-3 has a wide optimal pH condition, can produce lactic acid with high yield under a high-temperature condition, and has few byproducts and high lactic acid yield; compared with the existing lactic acid producing strains, the yield under the high temperature condition is remarkably improved. Meanwhile, the invention provides a method for producing lactic acid by coupling the geobacillus stearothermophilus GS-2H-3 with clostridium thermocellum through a one-pot method. The method realizes the production of lactic acid by using lignocellulose as a substrate by utilizing the characteristic that the optimal growth conditions of clostridium thermocellum and GS-2H-3 are staggered and controlling the pH or the temperature, not only has simple operation and reduces the production cost, but also improves the yield of the lactic acid to the maximum extent and reduces the generation of byproducts, thereby having important practical application value.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a bacillus stearothermophilus strain for producing lactic acid and a method for producing lactic acid by using the strain coupled with clostridium thermocellum and using lignocellulose as a substrate.
Background
Lactic acid is an important fine chemical intermediate, and has important application in the fields of food, medicine, daily chemicals, degradable materials and the like. Polylactic acid (PLA), which is a high molecular polymer obtained by polycondensation of lactic acid monomers, is an important degradable plastic and is consistently recognized by the industry as a new "bio-based material" with the most promising future in the new century. As PLA has gained increasing attention, lactic acid production technology has also received much attention. At present, most of the industrial production of lactic acid all over the world is carried out by a microbial fermentation method; the method has the advantages of low production cost, high optical purity and safety of the product, mild production conditions, small pollution and the like.
However, the existing biological fermentation preparation of lactic acid takes glucose as a carbon source to produce, and has the problems that the cost is influenced by the fluctuation of the price of grains and the problems of competing with people for grains and competing with grains for land. The raw material cost is reported to exceed 34% of the lactic acid production cost. To address this problem, researchers have developed methods of converting lignocellulosic feedstocks to sugars to achieve the substitution of non-grain feedstocks for starch sugars. At present, the method mainly utilizes commercial cellulase and hemicellulase from fungi to hydrolyze pretreated lignocellulose raw materials such as agricultural straws and the like to obtain hydrolysis liquid containing sugar, and then takes the hydrolysis liquid as a sugar source to perform downstream fermentation culture. For example, patent CN201510025738.8 uses mechanical treatment of lignocellulose, followed by addition of free enzymes to obtain fermentable sugars, which are then used in lactic acid fermentation. However, the cost of enzyme preparations derived from fungi is high, so that the fermentable sugar derived from lignocellulose has no market competitiveness compared with starch sugar, and further, the fermentation technology of lactic acid derived from lignocellulose is difficult to really realize practical application. In addition, the fermentation technology of lignocellulose-derived lactic acid in the prior art has the problems of complex process, unsatisfactory yield and the like. In the invention patent CN202110660995.4, lignocellulose raw material is treated by acid to obtain hydrolysate, detoxification is realized by pseudomonas putida biological treatment, and then lactic acid is generated by bacillus coagulans, so that the process is complex, the period is long, and acid-containing sewage is easy to generate. Sun, Y., et al (Bioprocess biosystem Eng. (2021)10.1007/s00449-021-02616-5) disclose a technique for cellulose degradation and lactic acid synthesis using a complex flora of corn cobs pretreated with sulfuric acid as a raw material. In this technique, the reaction temperature is around 45 ℃, but the yield of lactic acid is only 0.5 g per gram of substrate.
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, strains for producing succinic acid from lignocellulose and related application techniques are still quite lacking. Therefore, obtaining the novel efficient lactic acid fermentation production strain which has the characteristics of high temperature resistance, acid resistance, low culture cost and the like is very important for industrial production.
Disclosure of Invention
Aiming at the problems in the prior art that lignocellulose is used as a raw material to produce lactic acid, the invention provides a Geobacillus stearothermophilus strain GS-2H-3 for producing lactic acid, which has the characteristics of high stress resistance and capability of producing lactic acid by fermentation by utilizing various carbon sources under a high-temperature condition. Meanwhile, the application provides a method for producing lactic acid by jointly fermenting the strain GS-2H-3 and cellulose degradation high-temperature bacterium clostridium thermocellum with lignocellulose as a substrate, and the method has important practical application value.
The technical scheme of the invention is as follows:
the inventor screens a high-temperature anaerobic bacterium Geobacillus stearothermophilus GS-2H-3 capable of producing lactic acid with high yield from a gap of an anaerobic fermentation tank stirring paddle in Qingdao city in Shandong province, and the high-temperature anaerobic bacterium Geobacillus stearothermophilus GS-2H-3 is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center No. 3 of Xilu No. 1 North Chen located in the south China, Beijing city, the preservation number of the high-temperature anaerobic bacterium is CGMCC No.23683, and the preservation date is 2021 year, 10 months and 28 days.
The Geobacillus stearothermophilus GS-2H-3 has a wide optimal pH condition, can obtain high biomass under the condition of pH5.2-7.5, and can still normally grow under the condition of pH 5.2. The presence of a range (pH 5.2-6.2) that is out of phase with the optimum pH condition (neutral) of clostridium thermocellum provides a possibility for coupling the two. This is because the growth of Clostridium thermocellum is inhibited at pH 6.2, and the cellulose-degrading enzyme system produced by Clostridium thermocellum, cellulosome, still has high activity under this condition, and can achieve degradation of lignocellulosic substrates, thereby providing a carbon source for the growth of the GS-2H-3 strain. Meanwhile, the Geobacillus stearothermophilus GS-2H-3 also has a wider optimal growth temperature, and can obtain higher biomass under the condition of 55-85 ℃. Similarly, the optimum temperature condition is in a range (70-85 ℃) which is out of the optimum temperature condition (55-65 ℃) of Clostridium thermocellum, and provides possibility for coupling the two.
A microbial inoculum containing the Geobacillus stearothermophilus GS-2H-3.
The geobacillus stearothermophilus GS-2H-3 is applied to the fermentation production of lactic acid by using the strain of the geobacillus stearothermophilus GS-2H-3. The method specifically comprises the following steps: inoculating the Geobacillus stearothermophilus strain into a fermentation culture medium for fermentation growth, and separating lactic acid from fermentation liquor after fermentation is finished; the carbon source in the fermentation medium is glucose, cellobiose and fructose. Wherein the concentration of the carbon source is 10-20 g/L; the temperature of the fermentation growth is 55-85 ℃; the pH of the fermentation growth is 5.2-7.5. The fermentation medium comprises the following components: 10-20g/L of carbon source, 2.9g/L of dipotassium phosphate, 1.5g/L of potassium dihydrogen 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 2-6g/L of nitrogen source.
Preferably, the carbon source in the fermentation medium is glucose; the nitrogen source in the fermentation medium is yeast extract or corn steep liquor.
The application of the geobacillus stearothermophilus GS-2H-3, namely the geobacillus stearothermophilus GS-2H-3 and clostridium thermocellum are coupled to produce the lactic acid by a one-pot method, and the 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 7.0-8.0 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 at 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) fermentation production of lactic acid: when the concentration of glucose in the lignocellulose fermentation liquor is accumulated to 10-20g/L, (a): controlling pH to 5.5-6.2, controlling temperature to 55-85 deg.C, inoculating GS-2H-3 according to volume ratio of 1-10%, and simultaneously adding 10-60g/L calcium carbonate; when the glucose concentration in the lignocellulose fermentation liquid is zero, the fermentation is finished, and lactate is obtained. Or (b): controlling the temperature at 70-85 ℃ and the pH at 5.2-7.5, inoculating 1-10% of GS-2H-3 by volume ratio, and adding 10-60g/L calcium carbonate; when the glucose concentration in the lignocellulose fermentation liquid is zero, the fermentation is finished, and lactate is obtained. By adding a suitable amount of calcium carbonate, carbonate and stable pH are provided, contributing to cell growth and acid production.
The invention has the beneficial effects that:
(1) the invention provides geobacillus stearothermophilus GS-2H-3 which can produce lactic acid at a high yield under a high temperature condition, not only is few in by-product, but also is high in lactic acid yield, 115g of lactic acid can be produced by consuming 100 g of glucose under the conditions of adding carbonate and low pH, the conversion rate exceeds 100%, and compared with the existing lactic acid-producing strain, the yield under the high temperature condition is remarkably improved.
(2) The Geobacillus stearothermophilus GS-2H-3 has wider optimal pH condition and temperature condition, can obtain higher biomass under the conditions of pH5.5-7.5 and temperature of 55-85 ℃, and has higher fermentation capacity; according to this feature, fermentation conditions can be established which are in phase with the optimal growth conditions (temperature or pH) of Clostridium thermocellum, providing the possibility of coupling the two.
(3) The invention provides a method for producing lactic acid by utilizing Geobacillus stearothermophilus GS-2H-3 through fermentation by utilizing various carbon sources such as glucose, cellobiose, fructose and the like, and coupling the method with Clostridium thermocellum to produce the lactic acid by a one-pot method, which realizes the production of the lactic acid by taking lignocellulose as a substrate through the control of pH or temperature by utilizing the characteristic that the optimal growth conditions of the Clostridium thermocellum and GS-2H-3 are staggered, not only has simple operation and reduced production cost, but also improves the yield of the lactic acid to the greatest 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 the Strain GS-2H-3
1) Primary screening:
in the process of carrying out research on high-temperature fermentation of anaerobic microorganisms, a fermentation system is found to have high content of lactic acid, and because the fermentation target microorganism does not have the capacity of high yield of lactic acid, unknown strains are considered to exist in a tank body. The obtained stain is added into 1mL of sterile water by using an inoculating loop in a gap of a stirring paddle of a fermentation tank, sufficiently and uniformly shaken, and then 100 microliters of the obtained stain is uniformly coated on a screening culture medium (2.9 g of dipotassium hydrogen phosphate, 1.5g of potassium dihydrogen 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, 120.2g of vitamin B, 5g of glucose and 5.5 of pH) of agar with the mass-to-volume ratio of 0.8 percent. The plate was placed in an anaerobic box and incubated in a 60 ℃ incubator for 3 days under anaerobic conditions.
2) Re-screening:
10 colonies were picked and expanded in 5mL of selection medium, and cultured with shaking horizontally at 170rpm at 60 ℃ for 24 hours, after which 0.05mL of the culture was re-inoculated into 5mL of selection medium for subculture, and also cultured with shaking horizontally at 170rpm at 60 ℃ for 24 hours, and then the OD600 of the different cultures was analyzed while analyzing the organic acid species and lactic acid content in the supernatant by HPLC.
Example 2 identification of strains
The strain with a large biomass (OD600) and the highest lactic acid production 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 Geobacillus stearothermophilus strain (Genbank serial number: CP034952.1) is more than 99 percent through Blast sequence comparison of NCBI, so that the strain is judged to belong to the Geobacillus stearothermophilus and is named as GS-2H-3. 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, the preservation number is CGMCC No.23683, and the preservation date is 2021 year, 10 months and 28 days.
Example 3 physiological and Biochemical Properties of Strain GS-2H-3
The optimal growth conditions of the strain GS-2H-3 are analyzed, and the strain can be grown in glucose, fructose and cellobiose; wherein the most suitable carbon source type is glucose, the most suitable growth temperature is 55-85 deg.C, and the most suitable growth pH is 5.5-7.5. At a pH below 5.0, the strain was unable to grow. The glucose is used as a carbon source for batch fermentation, and the optimal initial carbon source concentration is 5-10 g/L.
10g/L glucose is used as an initial carbon source, the inoculation amount is 1 percent (volume ratio), the temperature is 60 ℃, and the strain GS-2H-3 is cultured for 20 hours under the conditions of 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 7.4 of pH), the pH value is reduced to 5.0, but the OD600 is more than 2.5, and the biomass is stable up to 60 hours, thereby indicating that the strain has fermentation robustness.
Under the conditions of initial pH of 6.0, the inoculation amount of 10 percent (volume ratio) and the temperature of 60 ℃, the batch fermentation is carried out by taking glucose as an initial carbon source, and 20 grams of calcium carbonate per liter is added. When the initial glucose concentration was 20g/L, the lactic acid production reached 8.2 g per liter at 12 hours of fermentation, the pH dropped below 5.0, thus causing the growth of the strain to be arrested, at which time 9.6g/L of glucose remained, thus giving a lactic acid yield of 78.8 g per 100 g of glucose.
Example 4 lactic acid fermentation Performance optimization of Strain GS-2H-3 (pH control, batch fermentation)
Carrying out batch fermentation by using 20g/L glucose as an initial carbon source under the conditions of initial pH (7.0), inoculum size of 1% (volume ratio) and temperature of 70 ℃; the pH was controlled at 7.0 by feeding sodium hydroxide to a 1L fermentor based on the addition of 20 grams per liter of calcium carbonate. After 24 hours of fermentation, glucose is completely consumed, the yield of lactic acid reaches 15.8 grams per liter, and the yield of lactic acid is 79.2 grams per 100 grams of glucose. The strain can realize the complete consumption of glucose under the condition of controlling pH; meanwhile, the yield of lactate is obviously improved. Compared with the method without controlling the pH, the yield of the lactic acid is improved by 7.6g per liter and is improved by about 92.7 percent.
Example 5 lactic acid fermentation Performance optimization of Strain GS-2H-3 (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 60 ℃ so that the glucose concentration was controlled at 5 g/L. After 24 hours of fermentation, the yield of lactic acid reached 17.0 g/L, and 40g/L calcium carbonate was added. After 72 hours fermentation, 50.2 grams of lactic acid per liter was produced, giving a yield of 89.6 grams per 100 grams of glucose. The production of lactic acid by the strain is obviously improved under the condition of controlling the concentration of the carbon source. The lactate yield was increased by 10.8% compared to example 3 without control of the 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. Wherein, in orderStraw is taken as substrate (cellulose content is 59%)The glucose in the hydrolysate is 42.5 grams per liter, and the sugar yield is 90 percent;taking the xylose residue as a substrate (the cellulose content is 76%)The glucose content in the hydrolysate was 54.1 g/l, and the sugar yield was 89%. (see the reference for details.)
Example 7 production of lactic acid by fermentation of Clostridium thermocellum straw saccharification coupled strain GS-2H-3 batch
Diluting the hydrolysate obtained in example 6 according to the concentration of glucose until the concentration of glucose is 20 grams per liter, and performing the batch fermentation experiment as described in example 4 by using the diluted hydrolysate as a culture medium; glucose was completely consumed and 14.9g/L lactic acid was obtained, yielding 74.5 g per 100 g glucose. It is thus clear that the strain GS-2H-3 according to the present invention, which uses glucose derived from lignocellulose as a carbon source, produces lactic acid in a yield slightly lower than that obtained in example 4 under the same conditions. This indicates that the strain is feasible to produce lactic acid by fermentation under the condition of controlling pH and using glucose of lignocellulose as a carbon source.
Example 8 production of lactic acid by continuous fermentation of Clostridium thermocellum straw saccharification coupled strain GS-2H-3
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 feeding fermentation experiment described in example 5, concentrated hydrolysate was added to 1L fermentation system to control glucose concentration at 10g/L, and after 24 hours of fermentation, the temperature was raised to 80 ℃ and 40g/L calcium carbonate was added. After 72 hours of fermentation a total of 75.4 g of glucose were consumed, yielding 66.3 g of lactic acid in 88 g per 100 g of glucose. Therefore, the strain GS-2H-3 provided by the application adopts glucose from lignocellulose as a carbon source, and the yield of lactic acid obtained under the same conditions is higher than that obtained in example 5. This indicates that the strain is feasible to produce lactic acid by fermentation using glucose derived from lignocellulose as a carbon source under the condition of controlling the glucose concentration.
Example 9 production of lactic acid by Clostridium thermocellum-coupled Strain GS-2H-3 one-pot Process
Adding a culture medium (components: 2.9g of dipotassium hydrogen phosphate, 1.5g of monopotassium phosphate, 1.0g of urea, 150mg of calcium chloride, 1.0g of magnesium chloride, 1.25mg of ferrous sulfate, 1g of cysteine, 3g of sodium citrate, 60g of calcium carbonate and 6.0g of corn steep liquor powder) into a 2L fermentation tank, simultaneously adding 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), carrying out air culture for 12 hours at 60 ℃ under an anaerobic condition, rotating the speed of a stirring paddle at 150 revolutions per minute to fully mix the substrate, and setting the initial pH to be 7.4. Then inoculating the clostridium thermocellum strain described in example 5 according to the volume ratio of 5% for culture, and inoculating GS-2H-3 according to the volume ratio of 10% when the glucose accumulation concentration reaches 20g/L, controlling the pH at 5.5-6.2, and raising the temperature to 55 ℃; at the same time, 60g/L calcium carbonate is added. Under the condition, GS-2H-3 can normally grow and clostridium thermocellum can inhibit the growth, and a cellulose degradation enzyme system, namely a cellulosome, produced by the clostridium thermocellum can still normally play a role, so that the degradation of a cellulose substrate and the fermentation production of lactic acid are synchronously realized. When the glucose concentration in the fermentation liquid is zero and the lactic acid concentration does not rise any more for 2 hours continuously, the fermentation is finished. The final lactic acid yield was 87.3 g/L in xylose residue substrateThe cellulose content is 76%The yield of lactic acid is calculated according to the raw material of glucose75.5 g per 100 g of dry weight of xylose residue. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,the yield of lactic acid was 115g per 100 g glucose。
Example 10 Clostridium thermocellum coupling GS-2H-3 one-pot method for producing lactic 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 inoculating the clostridium thermocellum strain of example 5 according to the volume ratio of 10% for culture, and inoculating GS-2H-3 according to the volume ratio of 5% when the glucose accumulation concentration reaches 10g per liter, keeping the temperature at 65 ℃, controlling the pH at 5.5-6.2, and simultaneously adding 50g/L calcium carbonate; when the glucose concentration in the fermentation liquid is zero and the lactic acid concentration does not rise any more for 2 hours continuously, the fermentation is finished. The final yield of lactic acid was 22.8 grams, calculated as straw feedstock, with a lactic acid yield of 56.7 grams per 100 grams of dry weight of pretreated straw. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,the yield of lactic acid was 107 g per 100 g glucose。
Example 11 Clostridium thermocellum coupling GS-2H-3 one-pot method for producing lactic acid
Different from the example 10, when the glucose accumulation concentration reaches 15 g/L, GS-2H-3 is inoculated according to the volume ratio of 1 percent, the temperature is kept at 60 ℃, the pH is controlled at 5.5-6.2, and 50g/L calcium carbonate is added; when the glucose concentration in the fermentation liquid is zero and the lactic acid concentration does not rise any more for 2 hours continuously, the fermentation is finished. The final yield of lactic acid was 21.5 g, calculated on straw material, lactic acid yield was 53.5 g per 100 g dry weight of pretreated straw. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,the yield of lactic acid is 100.9 g per 100 g glucose。
Example 12 Clostridium thermocellum coupling GS-2H-3 one-pot method for producing lactic acid
In contrast to example 9, a 1L fermenter was charged with GS-2 medium and 8% by mass/volume alkali pretreated straw as substrate, 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 glucose accumulation concentration reached 15 g/L, GS-2H-3 was inoculated at 1% by volume, pH was controlled to 6.5-7.5, and the temperature was raised to 85 ℃ while adding 30g/L of calcium carbonate. When the glucose concentration in the fermentation broth is zero and the lactic acid concentration does not rise any more for 2 hours,and (5) finishing the fermentation. The final lactate yield was 40.6 grams per liter, and the lactic acid yield was 50.7 grams per 100 grams dry weight of pretreated straw, calculated on the basis of lignocellulosic feedstock. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,the yield of lactic acid was 95.6 g per 100 g glucose。
Example 13 Clostridium thermocellum coupling GS-2H-3 one-pot method for producing lactic 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 5% by volume, and when the glucose accumulation concentration reached 15 g/L, GS-2H-3 was inoculated at 10% by volume, pH was controlled to 6.5-7.5, and the temperature was raised to 70 ℃ while adding 20g/L of calcium carbonate. When the glucose concentration in the fermentation liquid is zero and the lactic acid concentration does not rise any more for 2 hours continuously, the fermentation is finished. The final lactate yield was 41.5 grams per liter, and the lactic acid yield was 51.7 grams per 100 grams dry weight of pretreated straw, calculated on the basis of lignocellulosic feedstock. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,the yield of lactic acid was 97.5 g per 100 g glucose。
Example 14 Clostridium thermocellum coupling GS-2H-3 one-pot method for producing lactic acid
Unlike example 13, when the glucose accumulation concentration reached 15 g/L, GS-2H-3 was inoculated at 5% by volume, pH was controlled at 5.2 to 6.0, and the temperature was raised to 75 ℃ while adding 40g/L calcium carbonate. When the glucose concentration in the fermentation liquid is zero and the lactic acid concentration does not rise any more for 2 hours continuously, the fermentation is finished. The final lactate yield was 42.8 grams per liter, and the lactic acid yield was 53.5 grams per 100 grams dry weight of pretreated straw, calculated on the basis of lignocellulosic feedstock. Calculated according to the glucose equivalent in the case of a sugar yield of 90%,the yield of lactic acid is 100.8 g per 100 g glucose。
In conclusion, the growth temperature of the lactic acid producing strain GS-2H-3 is 55-85 ℃, and the yield of lactic acid produced by fermentation can reach 115g per 100 g of glucose under the conditions of proper pH and glucose concentration. Compared with the existing lactic acid producing strain at medium temperature, the lactic acid producing strain GS-2H-3 realizes the obvious improvement of yield under the high temperature condition, and has important practical application value. In addition, based on the characteristics that the high-temperature characteristic and the growth condition of the strain GS-2H-3 are staggered with the optimal pH and temperature of clostridium thermocellum, the application provides the method for producing the lactic acid by coupling the strain GS-2H-3 with the clostridium thermocellum, the lactic acid is produced by using lignocellulose as a substrate in a one-pot method only by controlling the pH or the temperature, and a production device does not need to be replaced in the whole process, so 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 lactic acid-producing Geobacillus stearothermophilus strain is characterized in that: the Geobacillus stearothermophilus strain is named as a thermophilic strain GS-2H-3 (Geobacillus stearothermophilus) The microbial inoculum is preserved in China general microbiological culture Collection center of China Committee for culture Collection, No. 3 of Xilu No. 1 of Beijing, Chaoyang, with the preservation number of CGMCC number 23683 and the preservation date of 2021 year, 10 months and 28 days.
2. A microbial agent comprising the Geobacillus stearothermophilus strain of claim 1.
3. Use of the geobacillus stearothermophilus strain according to claim 1, characterized in that: the geobacillus stearothermophilus strain is used for producing lactic acid by fermentation.
4. Use of the Geobacillus stearothermophilus strain according to claim 3, characterized in that: inoculating the Geobacillus stearothermophilus strain of claim 1 into a fermentation medium for fermentation growth, and separating lactic acid from fermentation liquor after fermentation is completed; the carbon source in the fermentation medium is glucose, cellobiose or fructose.
5. Use of the Geobacillus stearothermophilus strain according to claim 4, characterized in that: the carbon source in the fermentation medium is glucose; the nitrogen source in the fermentation medium is yeast extract or corn steep liquor.
6. Use of the Geobacillus stearothermophilus 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 55-85 ℃; the pH of the fermentation growth is 5.2-7.5.
7. Use of the Geobacillus stearothermophilus strain according to any one of claims 4-6, 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 potassium dihydrogen 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 2-6g/L of nitrogen source.
8. Use of the Geobacillus stearothermophilus strain according to claim 3, characterized in that: the Geobacillus stearothermophilus strain GS-2H-3 and clostridium thermocellum are coupled to produce lactic acid by a one-pot method, and the 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 a clostridium thermocellum strain, and saccharifying the clostridium thermocellum strain at the pH =7.0-8.0 to obtain a lignocellulose fermentation liquid;
(2) and (3) fermentation production of lactic acid: when the concentration accumulation of glucose in the lignocellulose fermentation liquor reaches 10-20g/L, inoculating 1-10% of Geobacillus stearothermophilus strain GS-2H-3 according to the volume ratio under the condition that the pH =5.5-6.2 or the fermentation temperature is 70-85 ℃, and simultaneously adding 10-60g/L calcium carbonate; when the glucose concentration in the lignocellulose fermentation liquid is zero, the fermentation is finished, and lactate is obtained.
9. Use of the geobacillus stearothermophilus strain according to claim 8, characterized in that: when the pH =5.5-6.2 in the step (2), the temperature for producing the lactic acid by the fermentation is 55-85 ℃; when the fermentation temperature is controlled to be 70-85 ℃ in the step (2), the pH = 5.2-7.5.
10. Use of the geobacillus stearothermophilus 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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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667110A (en) * | 2013-10-23 | 2014-03-26 | 中国科学院过程工程研究所 | Bacillus coagulans strain and integrated process for producing lactic acid by using same through synchronous saccharification and fermentation of lignocellulose |
| CN104911128A (en) * | 2015-06-10 | 2015-09-16 | 华南理工大学 | Thermoanaerobacterium aotearoense and application thereof in lactic acid production |
| KR101811263B1 (en) * | 2016-09-08 | 2017-12-26 | 고려대학교 산학협력단 | A sterilization composition for Geobacillus stearothermophilus and sterilization method using the composition |
| CN107603920A (en) * | 2017-11-02 | 2018-01-19 | 中山国晟生物工程有限公司 | A kind of composite bacteria agent and its application in fermented organic waste increases numerous probiotics production biological organic fertilizer |
| CN108690819A (en) * | 2018-05-30 | 2018-10-23 | 叶荫人 | A kind of Geobacillus stearothermophilus bacterial strain and its apply and methods for using them |
| CN108865938A (en) * | 2018-07-11 | 2018-11-23 | 青岛高迈生物科技有限公司 | A kind of bacillus stearothermophilus of high yield gemma |
| CN109536409A (en) * | 2018-12-17 | 2019-03-29 | 吉林中粮生化有限公司 | A kind of resistance is high and using the Pedicoccus acidilacticii strain of several kinds of carbon source and the method for producing lactic acid using the bacterial strain |
-
2021
- 2021-12-10 CN CN202111503374.1A patent/CN114181859B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667110A (en) * | 2013-10-23 | 2014-03-26 | 中国科学院过程工程研究所 | Bacillus coagulans strain and integrated process for producing lactic acid by using same through synchronous saccharification and fermentation of lignocellulose |
| CN104911128A (en) * | 2015-06-10 | 2015-09-16 | 华南理工大学 | Thermoanaerobacterium aotearoense and application thereof in lactic acid production |
| KR101811263B1 (en) * | 2016-09-08 | 2017-12-26 | 고려대학교 산학협력단 | A sterilization composition for Geobacillus stearothermophilus and sterilization method using the composition |
| CN107603920A (en) * | 2017-11-02 | 2018-01-19 | 中山国晟生物工程有限公司 | A kind of composite bacteria agent and its application in fermented organic waste increases numerous probiotics production biological organic fertilizer |
| CN108690819A (en) * | 2018-05-30 | 2018-10-23 | 叶荫人 | A kind of Geobacillus stearothermophilus bacterial strain and its apply and methods for using them |
| CN108865938A (en) * | 2018-07-11 | 2018-11-23 | 青岛高迈生物科技有限公司 | A kind of bacillus stearothermophilus of high yield gemma |
| CN109536409A (en) * | 2018-12-17 | 2019-03-29 | 吉林中粮生化有限公司 | A kind of resistance is high and using the Pedicoccus acidilacticii strain of several kinds of carbon source and the method for producing lactic acid using the bacterial strain |
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