CN110452852B - Farmer market biomass waste fermentation inoculant and preparation method thereof - Google Patents
Farmer market biomass waste fermentation inoculant and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F9/00—Fertilisers from household or town refuse
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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
- C12N1/20—Bacteria; Culture media therefor
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a farmer market biomass waste fermentation inoculant, which is named as bacillus methylotrophicus, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.15555, and the preservation date is 2018, 4 months and 4 days. The microbial inoculum prepared by the strain is inoculated into biomass waste of farmer markets, so that the quantity of cellulose degrading bacteria in the biomass waste is increased, the fermentation and decomposition of the biomass waste of the farmer markets are promoted, the composting time is shortened, the treatment efficiency is improved, and the harmlessness is realized.
Description
Technical Field
The invention relates to the field of agricultural microorganisms, in particular to a fermentation microbial inoculum of biomass waste in farmer markets and a preparation method thereof.
Background
Farmer market biomass waste is one of the main sources of municipal domestic waste, and accounts for more than 5% of municipal domestic waste according to statistics. At present, biomass waste in farmer markets is mainly mixed with other household garbage for collection and transportation, and landfill treatment and a small amount of incineration treatment are adopted. However, compared with municipal solid waste, biomass waste in farmer markets generally has higher water content, more than 85%, large occupation of landfill space and production of a large amount of leachate, and incineration treatment increases the cost of auxiliary fuel, is easy to generate secondary pollution and wastes organic resources contained in the leachate. Because the biomass waste in farmer markets is mainly fragments such as vegetable leaves, roots and partial defective fish meat which are easy to ferment and rot, the proportion of organic matters such as cellulose is as high as about 95 percent, is close to 2 times of urban domestic garbage, is easy to collect, has higher biodegradability and availability, contains a large amount of nutrient elements such as nitrogen, phosphorus and potassium required by plant growth and various trace raw materials such as magnesium, copper and zinc, through land utilization, not only can soil organic matters be increased, the soil structure be improved, the greenhouse gas emission is reduced, but also the fertilizer can be partially replaced, nutrient elements such as nitrogen, phosphorus and potassium can be supplemented, the application of the fertilizer is reduced, the environmental pollution is reduced, organic resources and organic nutrients are effectively utilized, and the full circulation of resources is realized.
Composting is one of the current urban organic domestic garbage treatment methods, and is an important way for realizing garbage reduction, harmlessness and recycling. However, the conventional compost depends on natural microorganism inoculation, so that the starting time is long, the fermentation efficiency is low, and particularly, the fermentation efficiency is lower for substances which are rich in cellulose and are difficult to decompose, so that the composting treatment of biomass wastes in farmer markets cannot be widely applied. Microorganisms are the most key and active factors in compost fermentation, and the utilization of specific functional microorganisms to develop compost fermentation bacteria agents to accelerate the compost fermentation is a hot spot of current research and compost application. Aiming at the difficult decomposition substances such as cellulose in the biomass waste in the farmer market, the high-efficiency cellulose degrading microorganisms are separated and screened, and the microbial fermentation inoculant is developed, so that the decomposition of the cellulose can be promoted, the composting fermentation is accelerated, the composting period is shortened, the composting quality is improved, the treatment cost of the biomass waste in the farmer market is reduced, the treatment capacity of the biomass waste in the farmer market is increased, the resource utilization rate of the biomass waste in the farmer market is improved, and the quality of resource products is ensured.
Disclosure of Invention
The invention provides a farmer market biomass waste fermentation inoculant and a preparation method thereof.
The invention aims to solve the problems that the conventional compost adopted in the prior farmer market biomass waste treatment depends on natural microorganism inoculation, the starting time is long, the fermentation efficiency is low, and the fermentation efficiency is lower particularly for substances which are rich in cellulose and are difficult to decompose.
The technical scheme adopted by the invention for solving the technical problems is as follows: the farmer market biomass waste fermentation inoculum is prepared from Bacillus methylotrophicus (Bacillus methylotrophicus) PGJ-D (P), wherein the PGJ-D (P) is preserved in the China general microbiological culture Collection center (CGMCC) in 2018, 4 months and 4 days, and the preservation number is CGMCC No. 15555.
The farmer market biomass waste fermentation inoculant is applied to composting.
The preserved strain is cultured on a cellulose enrichment medium at 30 ℃ for 1 day, the bacterial colony is white and wrinkled and gram-positive, the cell is long-rod-shaped, and the diameter of the periphytic flagellum is 1.00-3.49 mu m; preparing the cellulose enrichment medium: 6.0g of NaCl, 0.1g of MgSO4.7H2O, 0.1g of KH2PO 41 g, 20.1g of CaCl, 10.0g of CMC-Na, (NH4)2 SO42.0 g of distilled water, 1000mL of pH 7.0-7.4, and autoclaving at 121 ℃ for 20 min.
The preparation method of the fermentation inoculum of the biomass wastes of the farmer market at least comprises the following steps,
A. sampling: collecting samples from humus and compost rich in cellulose in Jinhua and Wuyi Zhejiang, wherein the sampling time is 2016 years;
B. strain separation: separating cellulose degrading bacteria by using a cellulose selective medium, wherein the cellulose selective medium separation method comprises the following steps: (1) weighing 1g of soil sample, adding the soil sample into a 500ml triangular flask with 100ml of cellulose enrichment medium, and carrying out shake culture for 5 days; (2) sucking 5ml from the step (1), adding the 5ml into a 250ml triangular flask with 45ml of sterile water to obtain 10-1 dilution, continuously diluting the 10-1 dilution, 10-2 dilution, 10-3 dilution, 10-4 dilution, 10-5 dilution and 10-6 dilution in the way, and coating 100 mu l of each 10-4 dilution, 10-5 dilution and 10-6 dilution on a Congo red cellulose culture medium plate; (3) selecting a culture dish with 20-200 colony numbers to select a single colony for slant culture at 28-30 ℃ for 2-3 days; (4) taking 5 soil samples of 1g in total, and repeating the steps (1) - (3) for each soil sample; finally separating to obtain 12 cellulose degrading bacteria strains;
preparing the cellulose enrichment medium: 6.0g of NaCl, 0.1g of MgSO4.7H2O, 0.1g of KH2PO 41 g, 20.1g of CaCl, 10.0g of CMC-Na, (NH4)2 SO42.0g of distilled water, 1000mL of pH 7.0-7.4, and autoclaving at 121 ℃ for 20 min;
C. selecting a strain: comparing the cellulase activity of the obtained strains by adopting an FPA enzyme activity determination method and a CMC enzyme activity determination method to obtain the strains with the highest cellulase activity;
D. re-screening strains: extracting the genome of the obtained strain, identifying the strain by using a 16S universal primer, confirming that the strain is Bacillus methylotrophicus PGJ-D (P), and separating a soil sample of the strain from Jinhua in Zhejiang;
E. strain PGJ-D (P) activation culture: inoculating the isolated PGJ-D (P) strain slant stock to a slant culture medium of a bacterial seed slant, culturing for 2 days in an incubator at 30 ℃, then inoculating the stock as an inoculum to a liquid culture medium of the bacterial seed, and culturing the liquid culture medium of the bacterial seed in a shaking incubator at 30 ℃ for 2 days after inoculation;
preparing a bacterial seed slant culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15-20g of agar and 1000mL of distilled water, wherein the pH value is 7.0-7.4, and the mixture is sterilized at 121 ℃ for 20min under high pressure;
preparing a bacterial seed liquid culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 1000mL of distilled water, pH 7.0-7.4, and autoclaving at 121 ℃ for 20 min;
F. preparing a microbial agent: washing thallus in a bacterial seed liquid culture medium with sterile water, taking the thallus as an inoculation liquid, inoculating the thallus into a fermentation culture medium, and culturing for 2 days at 30 ℃ to obtain a microbial agent;
preparing the fermentation medium: 10.0g of peptone, 10.0g of yeast extract, 5.0g of CMC, 5.0g of KH2PO41.0 g of NaCl, 1000mL of distilled water, pH 7.0-7.2, autoclaving at 121 ℃ for 20min, and using the cooled culture medium as a culture of PGJ-D (P).
The invention has the beneficial effects that: the microbial inoculum prepared by the strain is inoculated into biomass waste of farmer markets, so that the quantity of cellulose degrading bacteria in the biomass waste is increased, the fermentation and decomposition of the biomass waste of the farmer markets are promoted, the composting time is shortened, the treatment efficiency is improved, and the harmlessness is realized.
Drawings
FIG. 1 is a photograph of colony morphology and an electron microscope photograph of the strain.
Detailed Description
The invention is further illustrated with reference to the following figures and examples:
the farmer market biomass waste fermentation inoculant is named as Bacillus methylotrophicus PGJ-D (P), the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.15555, and the preservation date is 2018, 4 months and 4 days.
The farmer market biomass waste fermentation inoculant is applied to composting.
The preparation method of the zymophyte agent at least comprises the following steps,
A. sampling: 5 samples are collected from humus soil and compost rich in cellulose in Jinhua and Wuyi Zhejiang, and the sampling time is 2016 years.
B. Strain separation: separating cellulose degrading bacteria by using a cellulose selective medium, wherein the cellulose selective medium separation method comprises the following steps: (1) weighing 1g of soil sample, adding the soil sample into a 500ml triangular flask with 100ml of cellulose enrichment medium, and carrying out shake culture for 5 days; (2) sucking 5ml from the step (1), adding the 5ml into a 250ml triangular flask with 45ml of sterile water to obtain 10-1 dilution, continuously diluting the 10-1 dilution, 10-2 dilution, 10-3 dilution, 10-4 dilution, 10-5 dilution and 10-6 dilution in the way, and coating 100 mu l of each 10-4 dilution, 10-5 dilution and 10-6 dilution on a Congo red cellulose culture medium plate; (3) selecting a culture dish with 20-200 colony numbers to select a single colony for slant culture at 28-30 ℃ for 2-3 days; (4) taking 5 soil samples of 1g in total, and repeating the steps (1) - (3) for each soil sample; finally, 12 cellulose-degrading strains were isolated. Preparing the cellulose enrichment medium: 6.0g of NaCl, 0.1g of MgSO4.7H2O, 0.1g of KH2PO 41 g, 20.1g of CaCl, 10.0g of CMC-Na, (NH4)2 SO42.0 g of distilled water, 1000mL of pH 7.0-7.4, and autoclaving at 121 ℃ for 20 min.
C. Selecting a strain: the cellulase activity was determined for the screened cellulose-degrading bacteria in order to compare their cellulose-degrading activities. The principle of the cellulase activity determination is that reducing sugar such as cellobiose, glucose and the like generated by cellulose degradation by cellulase can be reduced into a brownish red amino compound after being co-heated with 3, 5-dinitrosalicylic acid (DNS) under an alkaline condition, the amount of the reducing sugar and the color intensity of a reaction solution are in a proportional relationship within a certain range, and the level of the cellulase activity can be determined by determining the generation amount of the reducing sugar by a colorimetric method. The FPA enzyme activity determination method and the CMC enzyme activity determination method are adopted to compare the cellulase activity of the obtained strains to obtain the strains with the highest cellulase activity.
(1) FPA enzyme activity assay
Adding 1mL of crude enzyme solution into 1mL of acetic acid-sodium acetate buffer solution with the pH value of 1mL and 4.8, shaking uniformly, adding 50mg of filter paper, keeping the temperature at 50 ℃ for 30min, adding 2.5mL of LDNS reagent, boiling in boiling water for 5min, cooling, fixing the volume to 25mL, carrying out color comparison at 520nm, and measuring the OD value.
(2) CMC enzyme activity assay
Adding 1mL of 1% CMC-containing acetic acid-sodium acetate buffer solution with pH of 4.8 into 1mL of crude enzyme solution, mixing, performing thermostatic water bath at 50 deg.C for 30min, adding 2.5mL of LDNS reagent, boiling in boiling water for 5min, cooling, diluting to 25mL, and performing color comparison at 520 nm.
The above crude enzyme solution inactivated in 100 deg.C boiling water bath for 5min is used as blank control. The enzyme activity units are all International Units (IU). That is, 1 unit of enzyme activity was defined as the amount of enzyme required to catalyze a substrate to 1. mu. mol of glucose per minute in 1mL of cellulase solution, expressed in IU.mL-1.
(3) The cellulase activity calculation formulas of the two enzyme activity determination methods are as follows:
(4) the reagent is prepared as follows:
1mg/mL glucose standard solution: 100 mg of analytically pure glucose dried to a constant weight at 105 ℃ is weighed, dissolved in distilled water, and the volume is determined to be 100 mL. The glucose standard solution was used as a standard solution for reducing sugars in cellulase activity assay.
DNS reagent (dinitrosalicylic acid reagent): weighing 200g of sodium potassium tartrate, dissolving the sodium potassium tartrate in a certain amount of distilled water, heating the solution to 50 ℃, sequentially adding 10.0g of 3, 5-dinitrosalicylic acid, 10.0g of sodium hydroxide, 2.0g of phenol and 0.5g of anhydrous sodium sulfite, and stirring the solution until the sodium potassium tartrate is completely dissolved. After cooling, the volume is adjusted to 1000mL by distilled water. The solution was stored in a brown bottle and used after 1 week.
Acetic acid-sodium acetate buffer (ph 4.8): solution A: 1.2mL of glacial acetic acid, and adding distilled water to 100 mL; and B, liquid B: sodium acetate (NaAc/3H 2O), 2.72g, was dissolved in 100mL of distilled water in a 2: 3, mixing and using;
1% CMC-Na solution: weighing 0.5g of CMC-Na into a beaker, adding about 20mL of acetic acid-sodium acetate buffer solution with pH of 4.8, heating until the solution is completely dissolved, and transferring the solution into a 50mL volumetric flask for constant volume.
(5) Comparison of enzyme Activity of different strains
Wherein the strain with the highest cellulase activity is PGJ-D (P).
| Bacteria numbering | CMCase（IU/mL） | FPAase（IU/mL） |
| PGJ-D(P) | 3.622 | 2.580 |
| BFHT-A(R) | 1.470 | 1.379 |
| BFHT-PA(R) | 0.280 | 0.761 |
| BFHT-A(R)P | 0.292 | 0.589 |
| BFHT-B(R) | 1.619 | 1.613 |
| BFHT-C(R) | 0.114 | 0.378 |
| SFB-A(R) | 1.436 | 1.562 |
| SFB-B(R) | 1.499 | 1.573 |
| SFB-E(R) | 1.310 | 1.425 |
| FSJ-A(R) | 1.385 | 1.510 |
| FSJ-B(R) | 0.229 | 0.509 |
| FGJ-A(R) | 1.207 | 1.608 |
D. Re-screening strains: extracting the genome of the obtained strain, identifying the strain by using a 16S universal primer, confirming that the strain is Bacillus methylotrophicus PGJ-D (P), and separating a soil sample of the strain from Jinhua in Zhejiang;
E. strain PGJ-D (P) activation culture: inoculating the isolated PGJ-D (P) strain slant stock to a slant culture medium of a bacterial seed slant, culturing for 2 days in an incubator at 30 ℃, then inoculating the stock as an inoculum to a liquid culture medium of the bacterial seed, and culturing the liquid culture medium of the bacterial seed in a shaking incubator at 30 ℃ for 2 days after inoculation;
preparing a bacterial seed slant culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15-20g of agar and 1000mL of distilled water, wherein the pH value is 7.0-7.4, and the mixture is sterilized at 121 ℃ for 20min under high pressure;
preparing a bacterial seed liquid culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 1000mL of distilled water, pH 7.0-7.4, and autoclaving at 121 ℃ for 20 min;
F. preparing a microbial agent: washing thallus in a bacterial seed liquid culture medium with sterile water, taking the thallus as an inoculation liquid, inoculating the thallus into a fermentation culture medium, and culturing for 2 days at 30 ℃ to obtain a microbial agent;
preparing the fermentation medium: 10.0g of peptone, 10.0g of yeast extract, 5.0g of CMC5.0g of K2HPO41.0 g of NaCl5.0g of distilled water, 1000mL of pH7.0-7.2, autoclaving at 121 ℃ for 20min, and using the cooled culture medium as a culture of PGJ-D (P).
The preserved strain is cultured on a cellulose enrichment medium at 30 ℃ for 1 day, the bacterial colony is white and wrinkled and gram-positive, the cell is long-rod-shaped, and the diameter of the periphytic flagellum is 1.00-3.49 mu m; preparing the cellulose enrichment medium: 6.0g of NaCl, 0.1g of MgSO4.7H2O, 0.1g of KH2PO 41 g, 20.1g of CaCl, 10.0g of CMC-Na, (NH4)2 SO42.0 g of distilled water, 1000mL of pH 7.0-7.4, and autoclaving at 121 ℃ for 20 min.
The 16S rDNA sequence of the deposited strain PGJ-D (P) is shown in a sequence table, and the sequence alignment result is as follows: the strain can be classified as bacillus methylotrophicus.
Sequence listing
<110> Zhejiang province academy of agricultural sciences; zhejiang Jinhuafeng agriculture science and technology limited
<120> fermentation inoculum of biomass waste in farmer market and preparation method thereof
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1453
<212> DNA
<213> Bacillus methylotrophicus (Bacillus methylotrophicus)
<400> 1
aatctggtca ccttcggcgg ctggctccta aaggttacct caccgacttc gggtgttaca 60
aactctcgtg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg 120
ctgatccgcg attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga 180
actgagaaca gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc 240
cattgtagca cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac 300
cttcctccgg tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa 360
gatcaagggt tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac 420
aaccatgcac cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag 480
aggatgtcaa gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc 540
gcttgtgcgg gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc 600
ggagtgctta atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact 660
catcgtttac ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct 720
cctcagcgtc agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct 780
acgcatttca ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt 840
ttccaatgac cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg 900
cgagcccttt acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg 960
ctggcacgta gttagccgtg gctttctggt taggtaccgt caaggtgccg ccctatttga 1020
acggcacttg ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg 1080
cggcgttgct ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta 1140
ggagtctggg ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca 1200
tcgtcgcctt ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta 1260
agtggtagcc gaagccacct tttatgtctg aaccatgcgg ttcaaacaac catccggtat 1320
tagccccggt ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac 1380
ccgtccgccg ctaacatcag ggagcaagct cccatctgtc cgctcgactt gcattatagc 1440
tggccccaat tgt 1453
Claims (2)
1. The farmer market biomass waste fermentation inoculum is characterized in that the farmer market biomass waste fermentation inoculum is prepared from Bacillus methylotrophicus (Bacillus methylotrophicus) PGJ-D (P), wherein the PGJ-D (P) has been deposited in China general microbiological culture Collection center (CGMCC) in 2018, 4.4.s..
2. Use of a farmer market biomass waste fermentation inoculum according to claim 1 in composting.
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| CN105670958A (en) * | 2015-11-10 | 2016-06-15 | 黑龙江省科学院微生物研究所 | Low-temperature biocontrol bacterial strain and application thereof |
| CN105950495A (en) * | 2016-01-31 | 2016-09-21 | 中国热带农业科学院热带生物技术研究所 | Bacillus methyltrophicus and application thereof in livestock and poultry breeding wastewater treatment |
| KR20160144639A (en) * | 2015-06-09 | 2016-12-19 | 대한민국(농촌진흥청장) | Bacillus methylotrophicus strain MH-2 and uses thereof |
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| KR20160144639A (en) * | 2015-06-09 | 2016-12-19 | 대한민국(농촌진흥청장) | Bacillus methylotrophicus strain MH-2 and uses thereof |
| CN105670958A (en) * | 2015-11-10 | 2016-06-15 | 黑龙江省科学院微生物研究所 | Low-temperature biocontrol bacterial strain and application thereof |
| CN105950495A (en) * | 2016-01-31 | 2016-09-21 | 中国热带农业科学院热带生物技术研究所 | Bacillus methyltrophicus and application thereof in livestock and poultry breeding wastewater treatment |
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| 高产纤维素酶菌株的筛选及所产酶的应用研究;唐乐丽;《中国优秀硕士学位论文全文数据库 基础科学辑》;20190115;第14页第2.2.1.2节，第20页表2-4、第2.3.3节 * |
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