CN113789284B - Acid-producing bacteria for treating livestock and poultry manure, acid-producing bacterial agent and application thereof - Google Patents

Acid-producing bacteria for treating livestock and poultry manure, acid-producing bacterial agent and application thereof Download PDF

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Publication number
CN113789284B
CN113789284B CN202111222495.9A CN202111222495A CN113789284B CN 113789284 B CN113789284 B CN 113789284B CN 202111222495 A CN202111222495 A CN 202111222495A CN 113789284 B CN113789284 B CN 113789284B
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acid
leuconostoc
livestock
producing
poultry manure
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CN113789284A (en
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孙英
马艳雯
袁博
张月
杜贤
陈俊豪
王婷婷
张轶欣
冯绍轩
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China Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • C02F11/04—Anaerobic treatment; Production of methane by such processes
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

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Abstract

The invention relates to the technical field of microorganisms, in particular to acid-producing bacteria and acid-producing bacteria agent for treating livestock and poultry manure and application thereof. The preservation number of the acidogenic bacteria in China general microbiological culture collection center is CGMCC NO:21664 is a fraudulent Leuconostoc mesenteroides (Leuconostoc fallax) NX13, can improve the low-temperature anaerobic fermentation efficiency, is salt-resistant and low-temperature-resistant, can efficiently produce acid in a low-temperature anaerobic reactor, shortens the anaerobic fermentation starting time, improves the gas production efficiency, is favorable for the popularization of biogas anaerobic fermentation technology, promotes the recycling of livestock and poultry manure, and has wide market prospect.

Description

Acid-producing bacteria for treating livestock and poultry manure, acid-producing bacterial agent and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to acid-producing bacteria and acid-producing bacteria agent for treating livestock and poultry manure and application thereof.
Background
Leuconostoc is a gram-positive lactic acid bacteria for abnormal fermentation, and is a microorganism which plays a main role in the pickle fermentation process. The leuconostoc is wide in living environment, mainly exists on the surface and the root of plants, can metabolize and produce organic acid, alcohols, diacetyl and other aromatic compounds by fermentation, can regulate intestinal balance, promote nutrient absorption, improve the flavor of products, and has antioxidant and antiviral capabilities, so that the leuconostoc is widely applied to the food industry, especially the production of probiotics.
In recent years, large-scale and intensive livestock and poultry raising industry has become the most active growing point and main pillar industry in rural economy in China, and livestock and poultry manure has become one of main factors of non-point source pollution in China. The biogas technology is widely applied to the field of livestock and poultry manure treatment because the biogas technology has low cost and high efficiency and can generate methane with energy value. However, when the temperature is lower than 20 ℃ and even lower than 10 ℃, the growth and metabolism capacities of acid-producing bacteria such as Anaerolineaceae, erysipelothrix and methane-producing bacteria such as Methanosaeta, methanosarcina of key microorganisms in the anaerobic digestion process are reduced, so that the anaerobic digestion reactor is long in starting time and insufficient in methane production, and the biogas technology is difficult to popularize.
Biogas fermentation is a process of the co-coordination of a plurality of microorganisms, wherein the degradation of macromolecular organic matters is the speed limiting step in the whole anaerobic fermentation process, and acidogenic bacteria play an important role in the process. The acidogenic bacteria decompose the organic matter to produce volatile fatty acids, which are supplied to the methanogenic bacteria for methane production. Therefore, the separation and identification of the low-temperature acidogenic bacteria in the anaerobic reactor has very important significance for improving the gas production efficiency and solving the problem of difficult low-temperature fermentation of the anaerobic reactor.
Disclosure of Invention
In view of the above, the invention provides an acidogenic bacterium for treating livestock and poultry manure, an acidogenic bacterium agent and application thereof. The acidogenic bacteria are leuconostoc fram (Leuconostoc cfalax) NX13, can improve the low-temperature anaerobic fermentation efficiency,
in order to achieve the above object, the present invention provides the following technical solutions:
leuconostoc fram (leuconostoc cfalax) NX13 with a preservation number of CGMCC NO:21664.
the invention screens out a microorganism strain from cattle farm wastewater domesticated at low temperature, and the separation method comprises the following steps: under aseptic condition, 1ml of cattle farm wastewater is sucked, diluted by a 10-fold dilution method with normal saline, single bacterial colony is picked up and inoculated in MRS culture medium, and the cattle farm wastewater is inversely cultured for 48 hours at 15 ℃. Two drops of methyl red indicator are dripped, red colonies are picked up, streaked and cultured on a solid medium containing 30ug/ml vancomycin, and the culture is separated and purified three times, so that a single pure culture is confirmed to be obtained, and the morphological characteristic colony of the strain has smooth surface, round shape, milky white color, opacity and neat edge, as shown in figure 1. The nucleotide sequence of the 16S rDNA of the strain is shown as SEQ ID NO.1, blast comparison is carried out on the sequence, and the sequence has the highest sequence similarity with the 16S rDNA gene of Leuconostoc fram (Leuconostoc cfalax) AB023239.1, so the strain is identified as Leuconostoc fram (Leuconostoc cfalax) and is named as NX13.
The invention also provides application of the leuconostoc fram NX13 in treating livestock and poultry manure or preparing an acid-producing microbial inoculum for improving low-temperature anaerobic digestion of the livestock and poultry manure. The livestock and poultry manure comprises, but is not limited to, wastewater and sewage generated by livestock and poultry farms.
In some embodiments, the livestock and poultry manure has a solids content of 4wt%.
In some embodiments, the salt concentration of the wastewater is preferably 1-4% (w/w) of the high salt wastewater, and may specifically be 1% -or 4%.
The invention also provides an acid-producing microbial agent for treating livestock and poultry manure, comprising leuconostoc fraudum NX13.
The invention also provides a preparation method of the acid-producing microbial inoculum, and the preservation number is CGMCC NO:21664 is inoculated with the leuconostoc fram NX13 in MRS culture medium for culture, and is centrifuged and resuspended to obtain OD 600 5-6 acid-producing bacteria.
Wherein, the cultivation is: shake culturing at constant temperature of 10-20deg.C and 150-200r/min for 48-72 hr.
The invention also provides a method for treating livestock and poultry manure, which comprises the steps of putting the leuconostoc fram fraud NX13 and the acid-producing microbial inoculum prepared by the leuconostoc fram fraud NX13 into the high-salt livestock and poultry manure, and performing anaerobic digestion treatment.
In some embodiments, the input amount of the acid-producing microbial inoculum accounts for preferably 10-20% of the volume of the livestock and poultry manure, and can be specifically 10% or 20%.
In some embodiments, the anaerobic digestion treatment is carried out at a temperature of 10 to 20℃for a period of 20 to 40 days.
The preservation number of the acidogenic bacteria in China general microbiological culture collection center is CGMCC NO:21664 is Leuconostoc fram (Leuconostoc cfalax) NX13. Experiments show that the strain can improve the low-temperature anaerobic fermentation efficiency, is salt-resistant and low-temperature-resistant, can efficiently produce acid in a low-temperature anaerobic reactor, shortens the anaerobic fermentation starting time, improves the gas production efficiency, is favorable for the popularization of a biogas anaerobic fermentation technology, promotes the recycling of livestock and poultry manure, and has wide market prospect.
Description of biological preservation
Biological material: NX13, class naming: leuconostoc fram Leuconostoc cfalax, deposited in China general microbiological culture Collection center, at 18, 1, 2021, was addressed: the collection number of the microbiological institute of China academy of sciences is CGMCCNo.21664, and the collection number of the microbiological institute of China is No. 3 of North West Lu 1 in the Chaoyang area of Beijing city.
Drawings
FIG. 1 is a colony morphology of strain NX13 of the present invention isolated on MRS medium;
FIG. 2 is a phylogenetic tree of strain NX13.
Detailed Description
The invention provides acid-producing bacteria and acid-producing bacteria agent for treating livestock and poultry manure and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The reagents and materials used in the present invention are commercially available.
The processes not mentioned in the preparation process of the present invention are all conventional processes.
The invention is further illustrated by the following examples:
example 1 application of Low temperature acid-producing microbial agent in anaerobic digestion
Adjusting the solid content of the culture manure to 4wt%, adding bottom mud into the reactor, inoculating 10% of the volume of the biogas slurry, adding a microbial inoculum into the biogas slurry with an inoculum size of 10% (v/v), performing static treatment in an anaerobic reactor at 10 ℃ for 40 days as a treatment group, and performing anaerobic digestion with the dairy manure without microbial inoculum as a control group to determine the methane yield.
After 20 days, compared with a control group, the acid yield is improved by 44%, and the gas yield is improved by 77%; after 40 days, compared with the control group, the acid yield is improved by 133 percent, and the gas yield is improved by 59 percent.
The preparation method of the microbial inoculum comprises the following steps: inoculating single colony into MRS culture medium, shake culturing at 10-20deg.C for 48-72 hr at 150-200 r/min. Obtaining OD by centrifugation and resuspension 600 5-6 acid-producing bacteria.
The method for measuring the acid yield (volatile fatty acid content) adopts a gas chromatography method. The assay is according to reference (Banel et al, 2011); the gas production amount is measured by adopting a water draining and gas collecting method, a measuring cylinder is filled with water and then is reversely buckled into a container filled with water, and a gas outlet of a gas collecting bag is inserted into the measuring cylinder, so that gas enters the measuring cylinder, and after the gas is exhausted, the liquid level scale is read to obtain the gas production amount.
Example 2 use of Low temperature acid-producing inoculants in anaerobic digestion
The solid content of the manure is adjusted to be 4wt%, the bottom mud is added into the reactor, the inoculation volume is 10% of the biogas slurry volume, the microbial inoculum is added into the biogas slurry in an inoculation amount of 20% (v/v), the mixture is used as a treatment group, the static treatment is carried out in an anaerobic reactor at 15 ℃ for 40 days, and the manure of a dairy farm without microbial inoculum is used as a control group for anaerobic digestion, so that the methane yield is measured.
After 20 days, compared with a control group, the acid yield is improved by 78%, and the gas yield is improved by 102%; after 40 days, compared with the control group, the acid yield is improved by 144%, and the gas yield is improved by 72%.
Example 3 use of Low temperature acid-producing inoculants in anaerobic digestion
The solid content of the manure is adjusted to be 4wt%, the bottom mud is added into the reactor, the inoculation volume is 10% of the biogas slurry volume, the microbial inoculum is added into the biogas slurry with the inoculation amount of 10% (v/v), the mixture is used as a treatment group, the static treatment is carried out in an anaerobic reactor at 15 ℃ for 40 days, and the manure of a dairy farm without the microbial inoculum is used as a control group for anaerobic digestion, so that the methane yield is measured.
After 20 days, compared with a control group, the acid yield is improved by 48 percent, and the gas yield is improved by 81 percent; after 40 days, compared with the control group, the acid yield is improved by 135%, and the gas yield is improved by 53%.
Example 4 use of Low temperature acid-producing inoculants in anaerobic digestion
Adjusting the solid content of the culture manure to be 4wt%, adding bottom mud into the reactor, inoculating 10% of the volume of the biogas slurry, adding a microbial inoculum into the biogas slurry with an inoculum size of 10% (v/v), performing static treatment in an anaerobic reactor at 20 ℃ for 40 days as a treatment group, and performing anaerobic digestion with the dairy manure without microbial inoculum as a control group to determine the methane yield.
After 20 days, compared with a control group, the acid yield is improved by 81 percent, and the gas yield is improved by 95 percent; after 40 days, compared with the control group, the acid yield is improved by 149 percent, and the gas yield is improved by 63 percent.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Chinese university of agriculture
<120> an acidogenic bacterium for treating livestock and poultry manure, an acidogenic bacterium agent and application thereof
<130> MP21022953
<160> 1
<170> SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial Sequence)
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ggttagccca ccggctttgg gtgttacaaa ctctcatggt gtgacgggcg gtgtgtacaa 60
gacccgggaa cgtattcacc gcggcatgct gatccgcgat tactagcgat tccgacttca 120
tgtagtcgag ttgcagacta caatccgaac tgagacgtac tttaagagat tagcttaccg 180
tcgccggttc gcaactcgtt gtatacgcca ttgtagcacg tgtgtagccc aggtcataag 240
gggcatgatg atctgacgtc atccccgcct tcctccggtt tgtcaccggc agtctgtcta 300
gagtgcccaa ctaaatgctg gcaactaaac ataagggttg cgctcgttgc gggacttaac 360
ccaacatctc acgacacgag ctgacgacga ccatgcacca cctgtcactt tgtctccgaa 420
gagaacaata ttatctctaa catcttcaaa ggatgtcaag acctggtaag gttcttcgcg 480
ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg tccccgtcaa ttcctttgag 540
tttcaacctt gcggtcgtac tccccaggcg gagtgcttaa tgcgttagct ccggcactaa 600
gaggcggaaa cctcctaaca cctagcactc atcgtttacg gtgtggacta ccagggtatc 660
taatcctgtt tgctacccac actttcgagc ctcaacgtca gttacagtcc agcaagccgc 720
cttcgccact ggtgttcttc catatatcta cgcattccac cgctacacat ggagttccac 780
ttgcctctac tgcactcaag ttcgccagtt tccaaagcca ttccacagtt gagctgtggg 840
ctttcacttc agacttaacg aaccgtctgc gctcgcttta cgcccaataa atccggataa 900
cgctcgggac atacgtatta ccgcggctgc tggcacgtat ttagccgtcc ctttctggta 960
tggtaccgtc acattaacat catttcctac attaactatt cttcccatac aacagtgctt 1020
tacgacccga aagccttcat cacacacgcg gcgttgctcc atcaggcttg cgcccattgt 1080
ggaagattcc ctactgcagc ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg 1140
ccgatcagtc tctcaactcg gctatgcatc atcgtcttgg taagccttta cctcaccaac 1200
taactaatgc accgcggatc catctctaag tgaggccgaa gcccctttta aacacacttc 1260
atgcgaaatg cgttgttata cggtattagc atctgtttcc aaatgttatc ccctgcttag 1320
aggcaggttg tccacgtgtt actcacccgt tcgccactct agccatatgc tttacgtctc 1380
accgaagtga tcaggtccgc gactagcgtt 1410

Claims (10)

1. Leuconostoc fram (leuconostoc cfalax) NX13 with a preservation number of CGMCC NO:21664.
2. the leuconostoc fram of claim 1, wherein the nucleotide sequence of 16S rDNA is shown in SEQ ID No. 1.
3. Use of a leuconostoc fram in accordance with claim 1 or 2 for treating livestock manure or for preparing an acidogenic agent for improving low-temperature anaerobic digestion of livestock manure.
4. The use according to claim 3, wherein the livestock manure comprises wastewater and sewage produced by livestock farms.
5. An acid-producing microbial agent for treating livestock and poultry manure, which is characterized by comprising the leuconostoc fram nrx 13 of claim 1 or 2.
6. The method for producing an acid-producing microbial agent according to claim 5, characterized in that,
the preservation number is CGMCC NO:21664 is inoculated with the leuconostoc fram NX13 in MRS culture medium for culture, and is centrifuged and resuspended to obtain OD 600 5-6 acid-producing bacteria.
7. The method according to claim 6, wherein the culturing is: shake culturing at constant temperature of 10-20deg.C and 150-200r/min for 48-72 hr.
8. A method for treating livestock and poultry manure, which is characterized in that the leuconostoc fram fraudum described in claim 1 or 2, the acid-producing microbial agent described in claim 5 or the acid-producing microbial agent prepared by the preparation method described in claim 6 or 7 is put into high-salt livestock and poultry manure for anaerobic digestion treatment.
9. The method of claim 8, wherein the acid-producing microbial inoculum is added in an amount of 10-20% of the livestock and poultry manure volume.
10. The method of claim 8, wherein the anaerobic digestion treatment is carried out at a temperature of 10-20 ℃ for a period of 20-40 days.
CN202111222495.9A 2021-10-20 2021-10-20 Acid-producing bacteria for treating livestock and poultry manure, acid-producing bacterial agent and application thereof Active CN113789284B (en)

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