阅读说明：本技术 一种梨囊鞭菌发酵秸秆生产乳酸的新方法和应用 (Novel method for producing lactic acid by fermenting straw with sorangium japonicum and application ) 是由 魏亚琴 王治业 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,具体涉及一种梨囊鞭菌发酵秸秆生产乳酸的方法和应用。本发明公开了梨囊鞭菌Piromyces CY1厌氧发酵秸秆生产乳酸的方法及其在制备乳酸中的应用。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,还公开了本发明所公开的梨囊鞭菌可以通过保藏在体外传代存活。发酵秸秆可产生大量高浓度乳酸,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, in particular to a method for producing lactic acid by fermenting straws with pear bursa. The invention discloses a method for producing lactic acid by anaerobic fermentation of straws of Pityrosporum ovale Piromyces CY1 and application of the method in preparation of the lactic acid. The Verbena pyricularis Piromyces CY1 is preserved in China general microbiological culture collection center with the preservation numbers as follows: CGMCC NO.18141, also discloses that the disclosed Campylobacter pear can survive through in vitro passage by preservation. The fermented straw can produce a large amount of high-concentration lactic acid, and the fermentation process is simple, has low requirements on equipment, is convenient to popularize, and has important industrial application value and development prospect in the industrial field.)

1. A method for producing lactic 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 lactic acid by anaerobic fermentation of straw

And (2) absorbing the microbial inoculum prepared in the step (1), respectively 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, adding a compound antibiotic, and carrying out anaerobic culture at 39 ℃.

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 said antibiotic cocktail is penicillin sodium, streptomycin sulfate, and chloramphenicol; the final concentrations of the penicillin sodium and the streptomycin sulfate in an anaerobic culture medium are 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 stalks added in the step (2) are any one of sorghum stalks, wheat stalks, corn stalks and rice stalks, respectively.

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

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) according to claim 1 in the preparation of lactic acid.

Technical Field

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

Background

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 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 the artificial component is not ideal, and the strains have antagonistic performance, so that the pretreatment time is long, the conversion efficiency is low, and no complete scheme is provided for producing lactic acid by performing anaerobic fermentation on straws at present.

Sorghum is a main food crop in China, the seeding area is wide, and the quantity of straws which are produced along with each year is very large. At present, a large amount of sorghum straw resources in rural areas in China are completely in the conditions of high consumption, high pollution, low utilization rate and low yield, and sorghum straws as energy substances are not reasonably developed and utilized. The sorghum straws 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.

Dzo is the first generation of the cross between yak and cattle. Dzo (male) and milk cow (female) have obvious hybridization advantages, and the meat and milk production capacity and working capacity are close to those of yak. Wild blood yak frozen semen is used for hybridizing western siemens cattle in rural areas, the filial generation of the wild blood yak frozen semen is dzo, and the dzo contains 50% of wild yak blood, so that the wild yak has high environment adaptability to Qinghai-Tibet plateau. The rumen of dzos inhabits uniquely, complexly and various, a large number of microbial communities synergistically metabolize wild pasture to efficiently degrade so as to provide survival energy and nutrient substances for yaks, and the rumen of dzos becomes an efficient lignocellulose degradation enzyme system through long-term natural selection and evolution, so that the rumen of dzos has unique advantages and efficient lignocellulose degradation capability.

The method is a new and effective means for treating straws by adopting anaerobic fungi, the inventor researches the anaerobic fermentation of a co-culture of the rumen anaerobic fungi of yaks 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 (Wei Yao musical instrument, the diversity of the co-culture of the rumen anaerobic fungi and the methane bacteria and the fiber degradation characteristics thereof research [ D ].2016 ]), and evaluates the straw degradation effects of the co-culture of the anaerobic fungi and the methane bacteria and the anaerobic fungi pure culture by detecting the gas production, the activity of polysaccharide hydrolase, the activity of esterase, the degradation rate of dry substances, the release amount of phenolic acid and the yield of methane and acetic acid, 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 within a 7-day culture period, the highest yield of lactic acid is 5.3mM, the anaerobic fermentation is performed by taking corn straws as the substrate, the highest yield of lactic acid is 5.2mM, and the highest yield of lactic acid is 3.2mM by taking rice straws as the substrate. And in the 7-day culture period, the N-genus anaerobic fungus pure culture (N.frontalis) Yak16 for efficiently degrading the three straws is fermented by taking the wheat straws as a substrate, the highest yield of lactic acid is 15.9mM, the corn straws are fermented by taking the corn straws as the substrate, the highest yield of lactic acid is 12.6mM, the rice straws are fermented by taking the rice straws as the substrate, and the highest yield of lactic acid is 8.4 mM.

According to the invention, the sorangium pyricularis Piromyces CY1 separated from the rumen of dzos is fermented to produce lactic acid by taking sorghum straws as a substrate, the highest yield reaches 27.5mM, and an unexpected effect is achieved.

Disclosure of Invention

The strain used in the anaerobic fermentation is a pure culture Piromyces CY1 of Lepidotis pyriformis separated from rumen content of cattle in the whole grazing dzo of the Changjingmo county of the Nanmuda village of the Anyuan county of Gansu, Tibet plateau, is preserved in the China general microbiological culture collection center, and the preservation number is as follows: 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 provides a method for producing lactic acid by carrying out anaerobic fermentation on pear bursa bacteria by using straws, which specifically comprises the following steps:

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

Inoculating the pure culture bacterial liquid of Piromyces CY1 into a liquid minimal medium at an inoculation amount of 10% v/v (explaining that 1% w/v of dry and crushed straws are added into the liquid minimal medium in advance as a substrate, and composite antibiotics are added for subculture, and the high-activity microbial inoculum is obtained after anaerobic culture at 39 ℃ for 72 hours.

(2) Production of lactic acid by straw 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 formulation: 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, 165mL of salt solution I, 165mL of salt solution II, and distilled water to reach the constant volume of 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 straws added in the step (2) are sorghum straws, wheat straws, rice straws and corn straws respectively.

Preferably, the straw added in step (2) is sorghum straw.

Preferably, the straw substrate is added in the step (2), then oxygen is removed, and high-temperature and high-pressure sterilization is carried out.

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 concentrations of penicillin and streptomycin sulfate in the anaerobic medium are 1600IU/mL and 2000IU/mL, respectively, and the final concentration of chloramphenicol in the medium is 50. mu.g/mL.

The invention has the beneficial effects that: firstly, the sorangium pyricularis (Piromyces CY 1) disclosed by the invention performs anaerobic fermentation by taking sorghum straws as a substrate, the lactic acid amount generated by degrading the sorghum straws in a 7-day culture period reaches the highest value of 27.5mM, and the lactic acid amount is remarkably improved compared with the prior art; the sorrel adopted in the invention can survive passage by being preserved in vitro, the fermented sorghum straw can produce a large amount of high-concentration lactic acid, the fermentation process is simple, the requirement on equipment is low, the popularization is convenient, and the sorrel has important industrial application value and development prospect in the industrial field; thirdly, through the anaerobic fermentation of sorghum straws by the sorrel of the rumen pear of dzo grazing, a large amount of lactic acid can be produced, the utilization rate of the sorghum 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.

Materials, reagents and the like 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: 1.0g of yeast extract, 1.0g of peptone, 37.0 g of NaHCO, 1mL of Resazurin (1.0g/L), 1.7g of L-cysteine hydrochloride, 8000 Xg of rumen fluid collected before morning feeding, 170mL of supernatant after centrifugation for 20min at 4 ℃, 165mL of salt solution I, 165mL of salt solution II and constant volume of distilled water to 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 the liquid minimal medium without adding straws, and sterilizing under high pressure after removing oxygen.

Agar roller tube medium: adding 1.0g/L glucose and 20g/L agar powder into the liquid minimal medium, and sterilizing under high pressure after oxygen removal.

Straw culture medium: adding 1% (w/v) of crushed and air-dried sorghum straws, wheat straws, corn straws and rice straws into a liquid minimal medium respectively, adding no glucose, and carrying out high-pressure sterilization after removing oxygen.

Subculture medium: adding 1% (w/v) of crushed air-dried wheat straw into a liquid minimal medium, deoxidizing, and then sterilizing under high pressure.

The oxygen removing method comprises the following steps: the anaerobic tube or the anaerobic bottle is connected with the high-purity CO with the vacuum pump through the 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 other two times are 5min, 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 the anaerobic tube is sterilized by moist heat at the high temperature of 121 ℃ for 20min for later use.