CN113789284A

CN113789284A - Acid-producing bacteria and acid-producing microbial inoculum for treating livestock and poultry manure and application thereof

Info

Publication number: CN113789284A
Application number: CN202111222495.9A
Authority: CN
Inventors: 孙英; 马艳雯; 袁博; 张月; 杜贤; 陈俊豪; 王婷婷; 张轶欣; 冯绍轩
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2021-12-14
Anticipated expiration: 2041-10-20
Also published as: CN113789284B

Abstract

The invention relates to the technical field of microorganisms, in particular to an acid-producing bacterium and an acid-producing microbial inoculum for treating livestock and poultry manure and application thereof. The preservation number of the acid-producing bacteria in China general microbiological culture Collection center is CGMCC NO: 21664 is a fraudulent Ming Chun Strain (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 start time of anaerobic fermentation, improves the gas production efficiency, is beneficial to the popularization of the methane anaerobic fermentation technology, promotes the resource utilization of the livestock and poultry feces, and has wide market prospect.

Description

Acid-producing bacteria and acid-producing microbial inoculum for treating livestock and poultry manure and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to an acid-producing bacterium and an acid-producing microbial inoculum for treating livestock and poultry manure and application thereof.

Background

Leuconostoc is a gram-positive lactic acid bacteria for carrying out heterotype fermentation and is a microorganism playing a main role in the fermentation process of pickled vegetables. The leuconostoc has a wide living environment, mainly exists on the surface and the root of a plant, can be metabolized and generate various aromatic compounds such as organic acid, alcohol, diacetyl and the like through fermentation, can adjust intestinal balance, promote nutrient absorption, improve the flavor of a product, and has antioxidant and anti-bacterial capabilities, so the leuconostoc is widely applied to the food industry, particularly the production of probiotic products.

In recent years, large-scale and intensive livestock and poultry breeding has become the most active growth point and the main supporting industry in rural economy in China, and meanwhile, livestock and poultry manure has become one of the 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 ℃, acid-producing bacteria such as Anaerolineceae and Erysipelotrix, which are key microorganisms in the anaerobic digestion process, and methanogenic bacteria such as Methanosaeta and Methanosarcina, which are key microorganisms, are reduced in growth and metabolism capabilities, so that the anaerobic digestion reactor is long in starting time and insufficient in methane production, and the methane technology is difficult to popularize.

Biogas fermentation is a process of the joint coordination of various microorganisms, wherein the degradation of macromolecular organic matters is the rate-limiting step of the whole anaerobic fermentation process, and acid-producing bacteria play an important role in the process. The acid-producing bacteria decompose organic matters to produce volatile fatty acid, and the volatile fatty acid is supplied to the methanogen for producing methane. Therefore, the separation and identification of the low-temperature acid-producing bacteria in the anaerobic reactor are of great 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 acid-producing bacterium and an acid-producing microbial inoculum for treating livestock and poultry manure and application thereof. The acid-producing bacteria are fraudulent Ming-Chuan-Mian strain (Leuconostoc cfallix) NX13, can improve the low-temperature anaerobic fermentation efficiency,

in order to achieve the above object, the present invention provides the following technical solutions:

a Leuconostoc fraudulenta (Leuconostoc cfallix) NX13 with the preservation number of CGMCC NO: 21664.

the invention screens out a microbial strain from the cattle farm wastewater domesticated at low temperature, and the separation method comprises the following steps: sucking 1ml of cattle farm wastewater under aseptic condition, diluting with normal saline by 10-fold dilution method, selecting single colony, inoculating to MRS culture medium, and performing inverted culture at 15 deg.C for 48 h. Two drops of methyl red indicator are dripped, red colonies are picked, streaked and cultured on a solid culture medium containing 30ug/ml vancomycin for three times, separation and purification are carried out, and a single pure culture is confirmed to be obtained, wherein the morphological characteristics of the bacterial strain include smooth surface, roundness, milky white, opacity and regular edge, as shown in figure 1. The nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO.1 through sequencing, the sequence is subjected to Blast comparison, the similarity with the 16SrDNA gene sequence of Leuconostoc decemlinella (Leuconostoc cfallix) AB023239.1 is highest, and therefore the strain is identified as Leuconostoc decemlinella (Leuconostoc cfallix) and named as NX 13.

The invention also provides application of the fraudulent leuconostoc NX13 in treating the livestock and poultry manure or preparing an acid-producing microbial inoculum for improving the low-temperature anaerobic digestion of the livestock and poultry manure. The livestock and poultry manure includes, but is not limited to, wastewater and sewage generated by livestock and poultry farms.

In some embodiments, the livestock manure has a solid content of 4 wt%.

In some embodiments, the salt concentration of the wastewater is preferably 1-4% (w/w) of high-salt wastewater, and specifically may be 1% to 4%.

The invention also provides an acid-producing microbial inoculum for treating livestock and poultry manure, which comprises leuconostoc fraudulentum NX 13.

The invention also provides a preparation method of the acid-producing microbial inoculum, which comprises the following steps of: 21664 the fraudulent Mingchuan strain NX13 is inoculated in MRS culture medium for culture, centrifuged and resuspended to obtain OD₆₀₀5-6 acid-producing bacteria agent.

Wherein the culturing is as follows: shaking culture at constant temperature of 150-.

The invention also provides a method for treating livestock and poultry manure, which comprises the step of putting the leuconostoc fraudulentum NX13 and the acid-producing microbial inoculum prepared by the same into the high-salt livestock and poultry manure for anaerobic digestion treatment.

In some embodiments, the amount of the acid-producing microbial inoculum is preferably 10-20% of the volume of the livestock and poultry manure, and specifically may be 10% or 20%.

In some embodiments, the anaerobic digestion treatment is at a temperature of 10 to 20 ℃ for a period of 20 to 40 days.

The preservation number of the acid-producing bacteria in China general microbiological culture Collection center is CGMCC NO: 21664, it is Leuconostoc strain NX 13. 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 starting time of anaerobic fermentation, improves the gas production efficiency, is beneficial to the popularization of the methane anaerobic fermentation technology, promotes the resource utilization of livestock and poultry feces, and has wide market prospect.

Biological preservation Instructions

Biological material: NX13, classification name: leuconostoc leuconostoc cfallix, deposited in China general microbiological culture Collection center at 18 months 1 and 2021, with the addresses of: the collection number of the microbial research institute of Chinese academy of sciences, No. 3 of Xilu No.1 of Beijing, Chaoyang, and the institute of microbiology is CGMCC No. 21664.

Drawings

FIG. 1 is a morphological diagram of a colony isolated on MRS medium by the strain NX13 of the present invention;

FIG. 2 is a phylogenetic tree of strain NX 13.

Detailed Description

The invention provides an acid-producing bacterium for treating livestock and poultry manure, an acid-producing microbial inoculum and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The reagents and starting 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 Agents in anaerobic digestion

Adjusting the solid content of the culture manure to be 4 wt%, adding bottom mud into a reactor, inoculating the bottom mud to the reactor with the volume of 10% of the volume of the biogas slurry, adding a microbial inoculum into the biogas slurry with the inoculation amount of 10% (v/v) to serve as a treatment group, carrying out static treatment in an anaerobic reactor at 10 ℃ for 40 days, carrying out anaerobic digestion by taking the manure of a dairy farm without the microbial inoculum as a control group, and measuring the methane production.

After 20 days, compared with a control group, the acid yield is improved by 44 percent, and the gas yield is improved by 77 percent; after 40 days, compared with a control group, the acid production is improved by 133%, and the gas production is improved by 59%.

The preparation method of the microbial inoculum comprises the following steps: inoculating a single colony in an MRS culture medium, and performing shake culture at constant temperature for 48-72h at the temperature of 10-20 ℃ and at the speed of 150-. Obtaining OD by centrifuging and resuspending₆₀₀5-6 acid-producing bacteria agent.

The acid yield (volatile fatty acid content) was determined by gas chromatography. Assay methods were according to the reference (Banel et al, 2011); the gas production is measured by a water and gas discharging method, the measuring cylinder is filled with water and then is reversely buckled into a container filled with water, the exhaust port of the collecting bag is inserted into the measuring cylinder, so that gas enters the measuring cylinder, and after the exhaust is finished, the liquid level scale is read, namely the gas production.

Example 2 application of Low temperature acid-producing microbial Agents in anaerobic digestion

Adjusting the solid content of the culture manure to be 4 wt%, adding bottom mud into a reactor, inoculating the bottom mud to the reactor with the volume of 10% of the volume of the biogas slurry, adding a microbial inoculum into the biogas slurry with the inoculation amount of 20% (v/v) to serve as a treatment group, carrying out static treatment in an anaerobic reactor at 15 ℃ for 40 days, carrying out anaerobic digestion by taking the manure of a dairy farm without the microbial inoculum as a control group, and measuring the methane production.

After 20 days, compared with a control group, the acid production is improved by 78 percent, and the gas production is improved by 102 percent; after 40 days, compared with a control group, the acid production is improved by 144 percent, and the gas production is improved by 72 percent.

Example 3 application of Low temperature acid-producing microbial Agents in anaerobic digestion

Adjusting the solid content of the culture manure to be 4 wt%, adding bottom mud into a reactor, inoculating the bottom mud to the reactor with the volume of 10% of the volume of the biogas slurry, adding a microbial inoculum into the biogas slurry with the inoculation amount of 10% (v/v) to serve as a treatment group, carrying out static treatment in an anaerobic reactor at 15 ℃ for 40 days, carrying out anaerobic digestion by taking the manure of a dairy farm without the microbial inoculum as a control group, and measuring the methane production.

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 a control group, the acid production is improved by 135%, and the gas production is improved by 53%.

Example 4 application of Low temperature acid-producing microbial Agents in anaerobic digestion

Adjusting the solid content of the culture manure to be 4 wt%, adding bottom mud into a reactor, inoculating the bottom mud to the reactor with the volume of 10% of the volume of the biogas slurry, adding a microbial inoculum into the biogas slurry with the inoculation amount of 10% (v/v) to serve as a treatment group, carrying out static treatment in an anaerobic reactor at 20 ℃ for 40 days, carrying out anaerobic digestion by taking the manure of a dairy farm without the microbial inoculum as a control group, and measuring the methane production.

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 a control group, the acid production is improved by 149 percent, and the gas production is improved by 63 percent.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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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

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ccgatcagtc tctcaactcg gctatgcatc atcgtcttgg taagccttta cctcaccaac 1200

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accgaagtga tcaggtccgc gactagcgtt 1410

Claims

1. A Leuconostoc fraudulenta (Leuconostoc cfallix) NX13 with the preservation number of CGMCC NO: 21664.

2. the Leuconostoc fraudulentum according to claim 1, wherein the nucleotide sequence of the 16S rDNA is represented by SEQ ID No. 1.

3. Use of leuconostoc fraud strain of claim 1 or 2 in treating livestock and poultry manure or in preparing an acid-producing microbial agent for improving low-temperature anaerobic digestion of livestock and poultry manure.

4. The use of 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 comprises the leuconostoc fraudulentum NX13 as set forth in claim 1 or 2.

6. The process for producing an acid-producing microbial agent according to claim 5,

the preservation number is CGMCC NO: 21664 the fraudulent Mingchuan strain NX13 is inoculated in MRS culture medium for culture, centrifuged and resuspended to obtain OD₆₀₀5-6 acid-producing bacteria agent.

7. The method according to claim 6, wherein the culturing is: shaking culture at constant temperature of 150-.

8. A method for treating livestock and poultry manure is characterized in that leuconostoc fraudulentum as defined in claim 1 or 2, an acid-producing microbial inoculum as defined in claim 5 or an acid-producing microbial inoculum prepared by the preparation method as defined in claim 6 or 7 is added into high-salt livestock and poultry manure for anaerobic digestion treatment.

9. The method as claimed in claim 8, wherein the amount of the acid-producing microbial inoculum is 10-20% of the volume of the livestock and poultry manure.

10. The method according to claim 8, wherein the anaerobic digestion treatment is carried out at a temperature of 10-20 ℃ for a period of 20-40 days.