CN110591922B - Pear penis and straw fermentation method for producing hydrogen and application thereof - Google Patents
Pear penis and straw fermentation method for producing hydrogen and application thereof Download PDFInfo
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- CN110591922B CN110591922B CN201910638854.5A CN201910638854A CN110591922B CN 110591922 B CN110591922 B CN 110591922B CN 201910638854 A CN201910638854 A CN 201910638854A CN 110591922 B CN110591922 B CN 110591922B
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- 239000010902 straw Substances 0.000 title claims abstract description 92
- 239000001257 hydrogen Substances 0.000 title claims abstract description 42
- 229910052739 hydrogen Inorganic materials 0.000 title claims abstract description 42
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 235000014443 Pyrus communis Nutrition 0.000 title claims abstract description 30
- 238000000855 fermentation Methods 0.000 title claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 26
- 230000004151 fermentation Effects 0.000 title abstract description 11
- 210000003899 penis Anatomy 0.000 title abstract description 9
- 235000007212 Verbena X moechina Moldenke Nutrition 0.000 claims abstract description 15
- 235000001594 Verbena polystachya Kunth Nutrition 0.000 claims abstract description 15
- 235000007200 Verbena x perriana Moldenke Nutrition 0.000 claims abstract description 15
- 235000002270 Verbena x stuprosa Moldenke Nutrition 0.000 claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 36
- 241000193632 Piromyces sp. Species 0.000 claims description 23
- 235000007164 Oryza sativa Nutrition 0.000 claims description 22
- 235000009566 rice Nutrition 0.000 claims description 22
- 241000233866 Fungi Species 0.000 claims description 19
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- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229960005091 chloramphenicol Drugs 0.000 claims description 6
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 6
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- 229960002385 streptomycin sulfate Drugs 0.000 claims description 6
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- 239000001888 Peptone Substances 0.000 claims description 3
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- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 claims description 3
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 241001081203 Verbena Species 0.000 claims 7
- 240000007594 Oryza sativa Species 0.000 claims 2
- 241000596422 Pithecellobium Species 0.000 claims 1
- 241001530126 Scrophularia Species 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 9
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- 150000002431 hydrogen Chemical class 0.000 abstract description 4
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 14
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- 241001260012 Bursa Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 235000010837 Echinocereus enneacanthus subsp brevispinus Nutrition 0.000 description 2
- 235000006850 Echinocereus enneacanthus var dubius Nutrition 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
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- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/59—Biological synthesis; Biological purification
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- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention relates to the field of biotechnology renewable energy sources, and particularly relates to a novel method and application for producing hydrogen by fermenting straws of sorangium pyricularis. The invention discloses a penis et testis Pyri Piromyces CY1 preserved in China general microbiological culture preservation management center with the preservation numbers as follows: CGMCC NO.18141, and also discloses a method for producing hydrogen by anaerobic fermentation of straws of Verbena pyricularis Piromyces CY1 and application thereof in biological hydrogen production. The pear flagellate disclosed by the invention can survive through in vitro passage by being preserved, the fermented straw can generate a large amount of high-concentration hydrogen, the fermentation process is simple, the requirement on equipment is low, the popularization is convenient, and the pear flagellate has important industrial application value and development prospect in the industrial field.
Description
Technical Field
The invention relates to the field of biotechnology renewable energy sources, and particularly relates to a method for producing hydrogen by using pear bursa and straw fermented by the pear bursa.
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 cellulose by covalent bonds combined by the lignin and the hemicellulose, ether bonds and carbon-carbon bonds in the lignin form macromolecular aromatic compounds with three-dimensional structures, and the strong bonds inhibit the action of hydrolase. Therefore, pretreatment of lignocellulose is required in anaerobic fermentation of straw. 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. Common microbial treatment also has many defects, for example, the single microbial treatment effect is not good, the effect of the composite flora of an artificial component is not ideal, and mutual antagonism performance exists among strains, so that the pretreatment time is long, the conversion efficiency is low, and no complete scheme is provided for producing hydrogen by anaerobic fermentation of straws at present.
Rice is the main grain crop in China, the seeding area is wide, and the quantity of straws which are produced along with the rice every year is very large. At present, a large amount of jade rice straw resources in rural areas of China are completely in the conditions of high consumption, high pollution, low utilization rate and low yield, and the rice straw as an energy substance is not reasonably developed and utilized. The corn 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.
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, researches are carried out on a co-culture of the yak rumen anaerobic fungi and the methane bacteria and an anaerobic fungi pure culture by taking corn straws, rice straws and wheat straws as substrates during the period of doctor of the inventor (Wei Yao Qin. the diversity of the co-culture of the yak rumen anaerobic fungi and the methane bacteria and the fiber degradation characteristics of the co-culture of the yak rumen anaerobic fungi and the methane bacteria are researched [ D ].2016 ]), the effects of the anaerobic fungi and the methane bacteria co-culture and the straw degradation of the anaerobic fungi pure culture are evaluated by detecting the gas production, the activity of polysaccharide hydrolase, the esterase activity, the dry matter degradation rate, the release amount of phenolic acid and the yield of: n-genus anaerobic fungus pure culture (N.frontalis) Yak16 for efficiently degrading three straws is fermented by taking the three straws as substrates respectively, wherein the amount of hydrogen produced in a 7-day culture period by taking the corn straws as the substrates is up to 2.3mmol/g DM, which is higher than the amount of hydrogen produced by taking the wheat straws and the rice straws as the substrates; the P-genus anaerobic fungus pure culture Piromyces Yak18 capable of efficiently degrading three straws is fermented by taking wheat straws as a substrate, the hydrogen production rate in a 7-day culture period is 0.7mmol/g DM, the corn straws are fermented by taking the corn straws as the substrate, the hydrogen production rate in the 7-day culture period is 1.5mmol/g DM, the rice straws are fermented by taking the rice straws as the substrate, and the hydrogen production rate in the 7-day culture period is only 0.7mmol/g DM; in the existing document, straw is used as a substrate for anaerobic fermentation, the amount of produced hydrogen is low, and the wide application of biological hydrogen production cannot be met.
Aiming at the technical problems, the invention develops a new way, separates the pure culture of the pear sac whip from the rumen of dzo, applies the pure culture to the straw anaerobic fermentation, and unexpectedly obtains the pear sac whip (Piromyces sp.) CY1 which can efficiently decompose the rice straw and generate a large amount of hydrogen.
Disclosure of Invention
The invention discloses a pear sac fungus (Piromyces sp.) CY1 which is separated from the rumen content of cattle on the grazing dzo in the Changqing village of the southern mud gulf village in Tianzhu county, Gansu province, Qinghai and Gansu province, wherein the pear sac fungus (Piromyces sp.) CY1 is preserved in the China general microbiological culture preservation management center with the preservation number of CGMCC NO.18141 and the preservation date of 2019, 7 and 9 days, and the preservation unit address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The invention also discloses a method for producing hydrogen by using straw as a substrate through anaerobic fermentation of the stigmaria pyricularis (Piromyces sp.) CY1, which specifically comprises the following steps:
(1) preparation of pure culture microbial inoculum of Campylobacter pyrenoidosa (Piromyces sp.) CY1
Inoculating 10% v/v inoculum of pure culture bacterial liquid of Campylobacter pyrenoidosa (Piromyces sp.) CY1 into a liquid minimal medium, adding 1% w/v dry and crushed straw as a substrate, simultaneously adding compound antibiotic for subculture, and placing at 39 ℃ for anaerobic culture for 72h to obtain the high-activity microbial inoculum.
(2) Production of hydrogen by anaerobic fermentation of straw
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 formula of the liquid minimal medium is as follows: yeast extract 1.0g, peptone 1.0g, NaHCO37.0g of resazurin 1m L-cysteine hydrochloride of 1.7g and 1.0 g/L g, 8000 × g of rumen fluid collected before morning feeding,after centrifugation at 4 ℃ for 20min, the supernatant was 170m L, salt solution I165 m L, salt solution II 165m L and distilled water to 1000m L.
Preferably, the salt solution I comprises 6g of NaCl, (NH4)2SO43g，KH2PO43g，CaCl2·2H2O0.4g，MgSO4·2H2O0.6g and distilled water to 1000m L.
Preferably, the salt solution II comprises K2HPO44g of distilled water, and the volume is up to 1000m L.
Preferably, the straws added in the step (1) are wheat straws.
Preferably, the straws added in the step (2) are wheat straws, corn straws and rice straws respectively.
Preferably, the straws added in the step (2) are rice straws.
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; the composite antibiotic is 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 liquid minimal medium is 1600IU/m L and 2000IU/m L respectively, and the final concentration of chloramphenicol in the medium is 50 μ g/m L.
The application of the Campylobacter pyrus CY1 in biological hydrogen production.
① the invention discloses a new bacterial strain of pear bag penis (Piromyces sp.) CY1 extracted from the gastric juice of dzo, the invention is preserved in China general microbiological culture collection management center, the preservation number is CGMCC NO.18141, ② the pear bag penis (Piromyces sp.) CY1 produces hydrogen with extremely high amount, the highest amount reaches 4.3mmol/g DM, compared with the prior art, ③ the pear bag penis employed in the invention can survive passage outside the body through preservation, the fermented rice straw can produce a large amount of high concentration hydrogen, and the fermentation process is simple, the requirement on equipment is low, the invention is convenient to popularize, has important industrial application value and development prospect in the industrial field, ④ can produce a large amount of hydrogen through the anaerobic fermentation of Tianzhu rumen pear bag penis bacteria, the utilization rate of rice straw 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:
composition of liquid minimal medium: yeast extract 1.0g, peptone 1.0g, NaHCO37.0g of resazurin (1.0 g/L) 1m L-cysteine hydrochloride 1.7g, 8000 × g of rumen fluid collected before morning feeding, supernatant 170m L obtained after centrifugation at 4 ℃ for 20min, salt solution I165 m L, salt solution II 165m L and distilled water to reach the constant volume of 1000m L.
Salt solution I contains 6g of NaCl, (NH)4)2SO43g，KH2PO43g，CaCl2·2H2O 0.4g，MgSO4·2H2O0.6g, distilled water to 1000m L.
Salt solution II comprises 4g K2HPO4And distilled water is added to reach the constant volume of 1000m L.
Separating and purifying culture medium, adding 1.0 g/L glucose into liquid minimal medium without straw, then removing oxygen and sterilizing.
Agar tube rolling culture medium 1.0 g/L glucose and 20 g/L agar powder were added to the liquid minimal medium, then deoxygenated and sterilized.
Straw culture medium: adding 1% w/v of crushed air-dried wheat straw, corn straw and rice straw into a liquid minimal medium respectively without adding glucose, and then deoxidizing and sterilizing.
Subculture medium: adding 1% w/v of crushed air-dried wheat straw to a liquid minimal medium, then deoxygenating and sterilizing.
The oxygen removing method comprises the following steps: subpackaging the liquid culture medium into Henschel anaerobic tube or anaerobic bottle, which is connected with vacuum pump and high purity CO via 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. EXAMPLE I extraction and isolation of Campylobacter Pyricularis (Piromyces sp.) CY1
1. Collection of dzo rumen fluid
After the cattle are grazed freely in a pasture, rumen fluid is collected, one end of a stainless steel stomach tube is inserted into the rumen of the cattle through a metal mouth gag, the other end of the stainless steel stomach tube is connected with an air pumping device to pump air out of the stomach tube to form negative pressure, rumen chyme fluid flows out of the stainless steel stomach tube, 1m L rumen chyme fluid is quickly sucked by an aseptic injector and is quickly inoculated into an anaerobic tube which takes 0.1g of wheat straw as a substrate and comprises 9m L basic culture medium of pear sac whip fluid, about 10 anaerobic tubes are inoculated and are placed at 39 ℃ for constant temperature culture, and the pear sac whip fluid is used for separating and purifying the pear sac whip in the next step.
2. Separation and purification of pear sac whip fungus
Sucking 1m L yak rumen chyme sample with sterile injector, inoculating into 9m L liquid minimal medium preheated at 39 deg.C and using wheat straw as substrate, and making into 10-1Diluting the diluted solution, and sequentially diluting the diluted solution to 10 degrees in a gradient manner-2、10-31m L dilution was inoculated into the agar roller tube medium of Henkett anaerobic tubes containing 9m L liquid minimal medium, glucose (1 g/L) and melted agar (20 g/L). In the inoculation, compound antibiotic solution was added to each tube of liquid medium by a seven gauge syringe needle sterile syringeAnd 2-3 days later, selecting single bacteria by using an inoculating loop under anaerobic conditions, inoculating the single bacteria into a straw-free separation and purification liquid culture medium added with glucose (1 g/L), and adding a compound antibiotic solution into each tube of the liquid culture medium by using a No. 7 injection needle sterile injector while inoculating, repeating the process for 4-5 times between a Hencatel agar rolling tube culture medium and the separation and purification liquid culture medium until the shapes of fungus colonies on the Hencatel agar rolling tube are consistent after microscopic examination, so that fungus single bacteria are obtained, inoculating the obtained single bacteria into a passage culture medium, culturing at 39 ℃, carrying out passage for 1 time every 4 days, adding the compound antibiotic solution to eliminate bacteria and methane bacteria during each passage, and observing whether fermentation liquor of each 1 separated pear penis single strain has blue or blue-green fluorescence under 420nm by using a fluorescence microscope (Eclipse 80i, Nikon, Japan) to determine whether each 1 separated pear penis single strain produces methane bacteria, so as to ensure that no pure pear strains exist in the separated pear strains, thereby obtaining the pure pear strains.
3. Extraction of pear sac whip fungus total DNA
The liquid nitrogen is adopted to grind the thalli, and then a Tiangen plant genome kit (Tiangen biochemical technology, Beijing) is used for extracting the total DNA of the pear sac whip fungus.
(1) Inoculating 10m L pure culture bacterial liquid of Leptosphaeria pyriformis into 90m L without straws, adding 0.1% w/v glucose into a liquid culture medium, culturing for 4-5 days, transferring the bacterial liquid into a centrifugal tube, centrifuging for 5min at 12000 × g, removing supernatant, collecting bacterial precipitates, transferring the bacterial precipitates into a mortar, adding liquid nitrogen, and quickly and fully grinding the bacterial into powder.
(2) The milled powder was quickly transferred to a centrifuge tube pre-filled with 700 μ L65 ℃ pre-heated buffer GP1 (mercaptoethanol was added to pre-heated GP1 to a final concentration of 0.1% w/v before the experiment, and after mixing by rapid inversion, the tube was placed in a 65 ℃ water bath for 20min, during which the tube was inverted to mix the samples.
(3) Adding 700 mu L chloroform, mixing well, centrifuging at 12000 × g for 5 min.
(4) The aqueous phase obtained in the previous step was transferred to a new centrifuge tube, and 700. mu. L buffer GP2 was added and mixed well.
(5) Transferring the mixed liquid into an adsorption column CB3, centrifuging for 30s at 12000 × g, and discarding waste liquid (the volume of the adsorption column is about 700 mu L, and centrifuging can be added in times.
(6) Add 500. mu. L buffer GD (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12000 × g for 30s, pour off waste, place adsorption column CB3 in the collection tube.
(7) Adding 600 μ L of rinsing liquid PW (whether absolute ethyl alcohol is added or not is checked before use) into the adsorption column CB3, centrifuging for 30s at 10000 × g, pouring off waste liquid, and putting the adsorption column CB3 into a collection tube.
(8) And (5) repeating the operation step (7).
(9) And (3) putting the adsorption column CB3 back into the collecting pipe, centrifuging for 2min at 12000 × g, pouring waste liquid, placing the adsorption column CB3 at room temperature for a plurality of minutes, and completely airing the residual rinsing liquid in the adsorption material.
(10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 5min, centrifuging at 12000 × g for 2min, and collecting the solution into the centrifuge tube.
(11) The solution obtained by centrifugation was added to an adsorption column CB3, and left at room temperature for 2min, and centrifuged at 12000 × g for 2 min.
(12) The DNA product should be stored at-20 ℃ to prevent DNA degradation.
4. PCR amplification of the sequence of Verbena pyricularis ITS1
Amplification of the complete sequence of the ITS1 of the Verbena pyricularis by using primers Neo18S For (5'-AAT CCT TCG GAT TGG CT-3') and Neo5.8S Rev (5'-CGA GAA CCA AGA GAT CCA-3') specific to the ITS1 of Verbena pyricularis, amplification was carried out using a MyCycler PCR apparatus (Bio-rad, USA) in which a reaction buffer (total volume 10. mu. L) containing 40m of MTricine-KOH (pH 8.0),16mM of KCI, 3.5mM of MgCl2100 μ g/M L BSA, 800 μ M dNTP, 500nM upstream and downstream primers, 0.2 μ L50 × TITANIUM Taq enzyme and template DNA approximately 50ng PCR was performed at 95 ℃ for 5min, then 10 cycles of 95 ℃ for 30s, 68 ℃ for 30s and 72 ℃ for 30s, then 25 cycles of 95 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30s, and finally 72 ℃ for 6min, and the PCR reaction system without template was extended as a blank.
5. Identification result of Verbena pyricularis
The morphological characteristics of the Verbena pyricularis (Piromyces sp.) CY1 are that the Verbena pyricularis (Piromyces sp.) is of a single-center growth type, an endophytic ellipsoidal sporangium (50 × 100 mu m), hypha is bent, hypha is highly branched and not separated, and a single-penis hair zoospore is morphologically and initially identified as a strain of Piromyces sp.
EXAMPLE two method for producing hydrogen by fermenting straw with Pityrosporum ovale (Piromyces sp.) CY1
1. Preparation of a Pitaya pear bacterium (Piromyces sp.) CY1 microbial inoculum:
sucking 1m L Verbena pyricularis (Piromyces sp.) CY1 culture into a Henry anaerobic tube with the volume of 20m L, inoculating into 9m L liquid basic culture medium with air-dried and crushed 0.1g of wheat straw as a substrate, and simultaneously adding compound antibiotics to ensure that the final concentrations of penicillin and streptomycin sulfate in the liquid basic culture medium are 1600IU/m L and 2000IU/m L respectively, and the final concentration of chloramphenicol in the culture medium is 50 mu g/m L, carrying out anaerobic culture at 39 ℃ for 72h, namely reaching the growth peak, wherein the fermentation liquid is a high-activity microbial inoculum.
2. Method for producing hydrogen by fermenting straw
The method comprises the steps of filling 90m L liquid basic culture medium in an anaerobic fermentation bottle with the volume of 100m L, taking 1.0g of crushed and air-dried corn straw, wheat straw and rice straw as substrates, deoxidizing, sterilizing, sucking 10m L of sorangium pyricularis (Piromycesp.) CY1 subjected to subculture for 72h by using a sterile injector, respectively inoculating the sorangium pyricularis (Piromyces sp.) CY1 into the liquid basic culture medium added with the corn straw, the wheat straw and the rice straw, simultaneously adding compound antibiotic, enabling the final concentration of the solution of the liquid basic culture medium to be penicillin 1600IU/m L, streptomycin sulfate 2000IU/m L and the final concentration of chloramphenicol in the culture medium to be 50 mu g/m L, carrying out anaerobic culture at 39 ℃ for 7 days, setting 3 parallel experiments altogether, and measuring the hydrogen yield in the fermentation liquid at 24h intervals.
3. Method for measuring hydrogen produced by fermentation
The gas chromatograph model is GC522, a Wufeng instrument, China, a GPS101 column (2m × 3mm) and a GC112A-TCD thermal conductivity cell detector of Shanghaineuojing, wherein the detection conditions comprise that a vaporization chamber is 250 ℃, the column temperature is 100 ℃, the detector temperature is 150 ℃, carrier gas is nitrogen, and the sample injection amount is 10 mu L.
Experimental results show that the Campylobacter pyrus (Piromyces sp.) CY1 can efficiently degrade rice straws and simultaneously generate a large amount of hydrogen, the hydrogen production amount is up to 4.3mmol/g DM, the yield is obviously higher than that of hydrogen produced by degrading various straws by the Campylobacter pyrus reported in the prior art, and the yield is also higher than that of the hydrogen produced by degrading corn straws and rice straws by the Campylobacter pyrus (Piromyces sp.) CY 1. The specific results are as follows:
TABLE 17 Hydrogen yield of Pitaya pear (Piromyces sp.) CY1 fermented wheat straw, rice straw, and corn straw within the culture period
Through the above embodiments, it can be seen that the Piromyces sp CY1 of dzo rumen degrades rice straws and simultaneously generates a large amount of hydrogen, which is obviously superior to the yield of hydrogen generated by microbial fermentation disclosed in the prior art, and has important industrial application value and development prospect in the industrial field.
Sequence listing
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<120> new method for producing hydrogen by anaerobic fungus and straw fermentation thereof and application
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Claims (8)
1. A kind of pear sac fungus: (A)Piromyces sp.) CY1, wherein the bacterium is Pithecellobium pyricularis (C)Piromyces sp.) CY1 is preserved in China general microbiological culture collection center with the preservation number: CGMCC No. 18141.
2. The Verbena pyricularis (Fr) Sing of claim 1Piromyces sp.) CY1 method for producing hydrogen by anaerobic fermentation of straws, which is characterized by comprising the following steps:
(1) pear scrophularia (B) ((B))Piromyces sp.) Preparation of CY1 pure culture microbial inoculum
Mixing the pear sac fungus (B)Piromyces sp.) Inoculating the CY1 pure culture bacterial liquid into a liquid minimal medium at an inoculation amount of 10% v/v, 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 hydrogen by anaerobic fermentation of straw
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 compound antibiotics for anaerobic culture;
the formula of the liquid minimal medium is as follows: yeast extract 1.0g, peptone 1.0g, NaHCO37.0g of 1.0 g/L g of resazurin 1m L-cysteine hydrochloride 1.7g, 8000 × g of rumen fluid collected before morning feeding, supernatant 170m L obtained after centrifugation at 4 ℃ for 20min, salt solution I165 m L, salt solution II 165m L, and distilled water with constant volume of 1000m L;
the salt solution I comprises 6g of NaCl, (NH)4)2SO43 g，KH2PO43 g，CaCl2•2H2O 0.4 g，MgSO4•2H20.6g of O, distilled water with constant volume of 1000m L, wherein the salt solution II comprises K2HPO44g of distilled water, and the volume is up to 1000m L.
3. The Verbena pyricularis (Fr) Sing of claim 2Piromyces sp.) The method for producing hydrogen by CY1 anaerobic fermentation of straws is characterized in that the straws added in the step (1) are wheat straws.
4. The Verbena pyricularis (Fr) Sing of claim 2Piromyces sp.) The method for producing hydrogen by CY1 anaerobic fermentation of straws is characterized in that the straws added in the step (2) are any one of wheat straws, corn straws and rice straws.
5. The Verbena pyricularis (Fr) Sing of claim 4Piromyces sp.) The method for producing hydrogen by CY1 anaerobic fermentation of straws is characterized in that the straws added in the step (2) are rice straws.
6. The Verbena pyricularis (Fr) Sing of claim 2Piromyces sp.) The method for producing hydrogen by CY1 anaerobic fermentation of straws is characterized in that oxygen is removed after straw substrates are added in the step (2), and high-temperature and high-pressure sterilization is carried out.
7. The Verbena pyricularis (Fr) Sing of claim 2Piromyces sp.) The method for producing hydrogen by anaerobic fermentation of CY1 straws is characterized in that the compound antibiotics are penicillin, streptomycin sulfate and chloramphenicol, the final concentration of the penicillin and the streptomycin sulfate in a liquid minimal medium is 1600IU/m L and 2000IU/m L respectively, and the final concentration of the chloramphenicol in the medium is 50 mu g/m L.
8. The Verbena pyricularis (Fr) Sing of claim 1Piromyces sp.) Use of CY1 in the biological production of hydrogen.
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