阅读说明：本技术 一种梨囊鞭菌发酵秸秆生产琥珀酸的新方法和应用 (Novel method for producing succinic acid by fermenting straws with Verbena pyricularis and application ) 是由 魏亚琴 王治业 牛永艳 秦鹏 张静荣 于 2019-07-16 设计创作，主要内容包括：本发明涉及生物技术可再生能源领域,具体涉及一株梨囊鞭菌发酵秸秆生产琥珀酸的新方法和应用。本发明公开了一种梨囊鞭菌Piromyces CY1厌氧发酵秸秆生产琥珀酸的方法,所述的梨囊鞭菌Piromyces CY1保藏于中国普通微生物菌种保藏管理中心,保藏编号为：CGMCC NO.18141。本发明所公开的梨囊鞭菌Piromyces CY1可以通过保藏在体外传代存活,发酵秸秆可产生高浓度的琥珀酸,且发酵工艺简单,对设备要求低,便于推广,在工业领域具有重要工业应用价值和开发前景。(The invention relates to the field of biotechnology renewable energy, and particularly relates to a novel method for producing succinic acid by fermenting straws with a strain of Verbena pyricularis and application thereof. The invention discloses a method for producing succinic acid by anaerobic fermentation of straws by using Verbena pyricularis (Piromyces CY 1), wherein the Verbena pyricularis (Piromyces CY 1) is preserved in China general microbiological culture preservation management center, and the preservation numbers are as follows: CGMCC NO. 18141. The invention discloses a penis et testis Pyri Piromyces CY1 which can survive through in vitro passage, the fermented straw can produce high-concentration succinic acid, the fermentation process is simple, the requirement on equipment is low, the popularization is convenient, and the invention has important industrial application value and development prospect in the industrial field.)

1. A method for producing succinic acid by anaerobic fermentation of straws by using Pityrosporum ovale Piromyces CY1 is characterized by comprising the following steps:

(1) preparation of pure culture microbial inoculum of Pitaya virgata Piromyces CY1

Inoculating 10% v/v inoculum size of a pure culture bacterial liquid of Piromyces CY1 into a liquid minimal medium, adding 1% w/v dry and crushed straw as a substrate, simultaneously adding a compound antibiotic for subculture, and performing anaerobic culture to obtain a high-activity microbial inoculum;

(2) production of succinic acid by anaerobic fermentation of straw

And (2) absorbing the microbial inoculum prepared in the step (1), inoculating the microbial inoculum into a liquid minimal medium taking 1% w/v straws as a substrate according to the inoculation amount of 10% v/v, and adding a compound antibiotic for anaerobic culture.

2. The method of claim 1, wherein the Verbena pyricularis Piromyces CY1 is preserved in China general microbiological culture Collection center with the preservation numbers: CGMCC NO. 18141.

3. The method of claim 2, wherein the liquid minimal medium formulation is: yeast extract 1.0g, peptone 1.0g, NaHCO₃7.0g, 1.0g/L resazurin 1mL, L-cysteine hydrochloride 1.7g, 8000 Xg of rumen fluid collected before morning feeding, 170mL of supernatant after centrifugation at 4 ℃ for 20min, saline solution I165mL, 165mL of saline solution II, and distilled water to reach a constant volume of 1000 mL.

4. The method of claim 3, wherein said salt solution I comprises 6g NaCl, (NH4)₂SO₄ 3g，KH₂PO₄ 3g，CaCl₂·2H₂O 0.4g，MgSO₄·2H₂0.6g of O and distilled water with constant volume of 1000 mL; the salt solution II comprises 4g K₂HPO₄And distilled water is added to the volume of 1000 mL.

5. The method of claim 1, wherein the antibiotic cocktail is penicillin, streptomycin sulfate, and chloramphenicol; the final concentration of the penicillin and the streptomycin sulfate in an anaerobic culture medium is 1600IU/mL and 2000IU/mL respectively, and the final concentration of the chloramphenicol in the culture medium is 50 mu g/mL.

6. The method of claim 1, wherein the straw added in step (1) is wheat straw.

7. The method of claim 1, wherein the straw added in the step (2) is any one of oat straw, wheat straw, corn straw and rice straw.

8. The method of claim 7, wherein the substrate added in step (2) is oats.

9. The method of claim 1, wherein in step (2) the straw substrate is added, deoxygenated, and autoclaved.

10. The use of the fermented straw of Verbena pyricularis (Piromyces CY 1) of claim 1 in the preparation of succinic acid.

Technical Field

The invention relates to the field of biotechnology renewable energy sources, in particular to a novel method for producing succinic acid by anaerobic fermentation of straws and application thereof.

Background

Oat is one of the main food crops in China, the seeding area is wide, and the quantity of straws which are produced along with the oat every year is very large. At present, a large amount of rice straw resources in rural areas in China are completely in the conditions of high consumption, high pollution, low utilization rate and low yield, and oat straws as energy substances are not reasonably developed and utilized. Oat straw can be subjected to resource regeneration through anaerobic digestion treatment, but the existing anaerobic digestion technology has the problems of low technical efficiency and great popularization difficulty.

Lignocellulose is the main component of the straw, and the hydrolysis of the lignocellulose is the rate-limiting step in the whole anaerobic digestion and is also the difficulty of the whole technology. The lignocellulose biomass mainly comprises cellulose, hemicellulose and lignin, cellulose molecules are embedded in the lignin by covalent bonds combined by the lignin and the hemicellulose, ether bonds and carbon-carbon bonds in the lignin form macromolecular aromatic compounds with a three-dimensional structure, and the strong chemical bonds inhibit the action of hydrolase. Thus, pretreatment of lignocellulose is required. Common methods for pretreating lignocellulose include mechanical methods, heat treatment methods, and chemical treatments, all of which are effective in promoting anaerobic digestion, but these pretreatment methods are costly and not environmentally friendly. The common microbial treatment has more defects, the single microbial treatment effect is not good, the effect of the composite flora of an artificial component is not ideal, and the strains have antagonistic expressions, so that the pretreatment time is long, the conversion efficiency is low, and no complete scheme is provided for carrying out anaerobic fermentation on the oat straws to produce the succinic acid at present.

The method is a relatively new and effective means for treating oat straws by adopting anaerobic fungi, the inventor researches anaerobic fermentation of a co-culture of yak rumen anaerobic fungi and methane bacteria and an anaerobic fungi pure culture by taking corn straws, rice straws and wheat straws as substrates during the period of doctor (Weiyaqin, the diversity of the co-culture of the yak rumen anaerobic fungi and the methane bacteria and the fiber degradation characteristics thereof are researched [ D ].2016 ]), the straw degradation effect of the co-culture of the anaerobic fungi and the methane bacteria and the anaerobic fungi pure culture is evaluated by detecting gas production, polysaccharide hydrolase activity, esterase activity, dry matter degradation rate, phenolic acid release amount, methane and acetic acid yield, and the research results show that: the P genus anaerobic fungus pure culture Piromyces Yak18 capable of efficiently degrading three straws is subjected to anaerobic fermentation by taking wheat straws as a substrate, the highest yield of succinic acid in a 7-day culture period is 0.09mM, the anaerobic fermentation is performed by taking corn straws as the substrate, the highest yield of succinic acid in the 7-day culture period is 0.11mM, rice straws are taken as the substrate, and the highest yield of succinic acid in the 7-day culture period is 0.04 mM; the N-genus anaerobic fungus pure culture (N.frontalis) Yak16 capable of efficiently degrading the three straws also respectively takes the three straws as substrates, and the yield of succinic acid produced by fermentation with the corn straws as the substrates reaches the highest yield of only 0.21mM within 7 days of culture, and is higher than that of succinic acid produced with the wheat straws and the rice straws as the substrates. According to the invention, the anaerobic fungus Piromyces CY1 separated from the rumen of dzo is fermented to produce succinic acid by taking oat straws as a substrate, the highest yield reaches 4.9mM, and an unexpected effect is achieved.

Disclosure of Invention

The strain used in the anaerobic fermentation is a pure culture Piromyces CY1 of the pizobia pyricularis separated from the rumen content of the cattle in the whole grazing dzo in the Changqing village of Tianzhu county, Gansu province, Qinghai and Gaoyuan, the preservation number of the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC NO.18141, the preservation date is 7 months and 9 days in 2019, and the preservation unit address is as follows: the classification name of the Xilu No.1 Hospital No. 3, Beijing, Chaoyang, is: piromyces CY 1.

The invention specifically discloses a method for producing succinic acid by fermenting straws with Pityrosmyces piricola CY1, which specifically comprises the following steps:

(1) preparation of pure culture microbial inoculum of Piromyces CY1

Inoculating 10% v/v inoculum size of a pure culture bacterial liquid of Piromyces CY1 into a liquid minimal medium, adding 1% w/v dry and crushed straw as a substrate, simultaneously adding a compound antibiotic for subculture, and placing at 39 ℃ for anaerobic culture for 72h to obtain the high-activity microbial inoculum.

(2) Production of succinic acid by fermentation

And (2) absorbing the microbial inoculum prepared in the step (1), inoculating the microbial inoculum into the liquid minimal medium which takes 1% w/v straws as a substrate and is the same as the liquid minimal medium prepared in the step (1) according to the inoculation amount of 10% v/v, simultaneously adding the compound antibiotic, and carrying out anaerobic culture at 39 ℃ for 7 days.

Preferably, the liquid minimal medium formula comprises: yeast extract 1.0g, peptone 1.0g, NaHCO₃ 7.0g，1.0g/L resazurin 1mL, L-cysteine hydrochloride 1.7g, collected rumen fluid 8000 Xg before morning feeding, supernatant 170mL after centrifugation for 20min at 4 ℃, salt solution I165mL, salt solution II 165mL, distilled water constant volume to 1000 mL.

Preferably, the salt solution I comprises 6g of NaCl, (NH4)₂SO₄ 3g，KH₂PO₄ 3g，CaCl₂·2H₂O 0.4g，MgSO₄·2H₂O0.6 g and distilled water to 1000 mL.

Preferably, the salt solution II comprises 4g K₂HPO₄And distilled water is added to the volume of 1000 mL.

Preferably, the straws added in the step (1) are wheat straws.

Preferably, the straw added in the step (2) is any one of oat straw, wheat straw, corn straw and rice straw.

Preferably, the substrate is added in step (2) followed by oxygen removal and then autoclaving.

Preferably, the compound antibiotics are penicillin, streptomycin sulfate and chloramphenicol, and are added in the fermentation process, so that the co-culture system can be prevented from being polluted by bacteria and methane bacteria, and the anaerobic fermentation efficiency is improved.

Preferably, the final concentration of penicillin and streptomycin sulfate in the composite antibiotic in the anaerobic culture medium is 1600IU/mL and 2000IU/mL respectively, and the final concentration of chloramphenicol in the culture medium is 50 mug/mL.

The invention has the beneficial effects that: the yield of succinic acid produced by fermenting straws by using the Pityrosporum ovale Piromyces CY1 disclosed by the invention is remarkably improved, particularly, the straws of oats are used as a substrate for anaerobic fermentation, and the concentration of the produced succinic acid is 4.9 mM; the pear sac whip adopted in the invention can survive through preservation outside the body for passage, the oat straw can be fermented to generate high-concentration succinic acid, the fermentation process is simple, the requirement on equipment is low, the popularization is convenient, and the pear sac whip has important industrial application value and development prospect in the industrial field; thirdly, through the anaerobic fermentation of oat straws by the sorrel of the rumen pear of dzo grazing, high-concentration succinic acid can be generated, the utilization rate of the oat straws can be further improved, and the economic benefit is obviously improved.

Detailed Description

The technical solutions claimed in the present invention will be described below with reference to specific examples, but the scope of the claimed invention is not limited to the following examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials and reagents used in the following examples are commercially available, unless otherwise specified.

The anaerobic medium used in the following examples is as follows:

the formula of the liquid minimal medium is as follows: yeast extract 1.0g, peptone 1.0g, NaHCO₃7.0g, 1.0g/L resazurin 1mL, L-cysteine hydrochloride 1.7g, 8000 Xg of rumen fluid collected before morning feeding, 170mL of supernatant after centrifugation at 4 ℃ for 20min, saline solution I165mL, 165mL of saline solution II, and distilled water to reach a constant volume of 1000 mL.

Salt solution I contains 6g of NaCl, (NH)₄)₂SO₄ 3g，KH₂PO₄ 3g，CaCl₂·2H₂O 0.4g， MgSO₄·2H₂O0.6 g and distilled water to 1000 mL.

Salt solution II comprises 4g K₂HPO₄And distilled water is added to the volume of 1000 mL.

Separating and purifying the culture medium: adding 1.0g/L glucose into a liquid minimal medium without adding straws, and then sterilizing after removing oxygen.

Agar roller tube medium: 1.0g/L glucose and 20g/L agar were added to the liquid minimal medium, which was then sterilized after deoxygenation.

Straw culture medium: adding 1% w/v of crushed and air-dried oat straw, wheat straw, corn straw and rice straw into a liquid minimal medium respectively without adding glucose, and then sterilizing after removing oxygen.

Subculture medium: adding 1% w/v of crushed air-dried wheat straw into a liquid minimal medium, and then sterilizing after removing oxygen.

The oxygen removing method comprises the following steps: subpackaging the liquid minimal mediumIntroducing into Henschel anaerobic tube or bottle, introducing the anaerobic tube or bottle into a vacuum pump and high-purity CO via a needle₂The air extractor(s) removes oxygen from the culture medium. Firstly, the color of the culture medium is changed when the gas in the vacuum pump extraction pipe reaches the negative pressure, and then high-purity CO is filled in₂. And 3 times of air pumping and inflating for each tube, wherein the 1 st time is about 15min, the rest 2 times are 5min each time, the anaerobic tube is inflated for the last 1 time, then the air is deflated again by using a sterile strain needle to balance the internal and external pressures of the anaerobic tube, and then the anaerobic tube is sterilized by moist heat at the high temperature of 121 ℃ for 20min for later use.