CN112195134B - Cellulose degradation composite microbial inoculant, and preparation method and application thereof - Google Patents

Cellulose degradation composite microbial inoculant, and preparation method and application thereof Download PDF

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CN112195134B
CN112195134B CN202011218361.5A CN202011218361A CN112195134B CN 112195134 B CN112195134 B CN 112195134B CN 202011218361 A CN202011218361 A CN 202011218361A CN 112195134 B CN112195134 B CN 112195134B
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composite microbial
serratia marcescens
acinetobacter calcoaceticus
cellulose
mushroom
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CN112195134A (en
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杨兴明
胡康
陈雪
刘艳霞
卢亚洲
胡贲
李�瑞
喻奇伟
孙光军
刘燕翔
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Nanjing Agricultural University
Guizhou Tobacco Co Ltd Bijie Branch
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Nanjing Agricultural University
Guizhou Tobacco Co Ltd Bijie Branch
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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
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • 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
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
    • 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
    • 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 cellulose degradation composite microbial inoculum, a preparation method and application thereof, wherein the cellulose degradation composite microbial inoculum comprises Serratia marcescens (Serratia marcescens) with the preservation number of CGMCC No.20557 and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) with the preservation number of CGMCC No. 20558. The cellulase secreted by the two strains is used as a main active ingredient, mushroom residues are used as degradation substances, and the cellulase is applied to mushroom residue compost and has the characteristics of quick temperature rise, good mushroom residue degradation effect, no secondary pollution and the like. The culture in CMC-NA culture medium has obvious hydrolysis circle, and in mushroom residue fermentation culture medium, the degradation effect on mushroom residue is obvious, and the germination rate and the decomposition degree of compost seeds are obviously improved.

Description

Cellulose degradation composite microbial inoculant, and preparation method and application thereof
Technical Field
The invention relates to a cellulose degradation composite microbial agent, a preparation method and application thereof, and belongs to the technical field of microorganisms.
Background
With the rapid development of the edible fungus industry in China, the mushroom residue waste generated by the production activity is increased year by year. The main component of the mushroom residue is cellulose, the mushroom residue is not easy to degrade in a short period under natural conditions, and the treatment of mushroom residue waste becomes an important link of the edible fungus industry. Therefore, the microorganisms for efficiently degrading the mushroom residues are screened, the mushroom residue degrading flora is built, the resource utilization of mushroom residue waste can be accelerated, and theoretical basis and technical support are provided for the production of the mushroom residue organic fertilizer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a cellulose degradation composite microbial inoculum, a preparation method and application thereof, wherein cellulase produced by fermentation of a strain has a promoting effect on mushroom residue decomposition fermentation under high temperature conditions.
In order to solve the technical problems, the invention provides a cellulose degradation composite microbial inoculum, which comprises Serratia marcescens (Serratia marcescens) with the preservation number of CGMCC No.20557 and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) with the preservation number of CGMCC No. 20558.
Preferably, the mass ratio of Serratia marcescens (Serratia marcescens) to Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) is 1:1.
Preferably, the microbial inoculum also comprises rice chaff, and the microbial inoculum contains 1×10 bacteria 8 CFU/g。
The invention also provides a preparation method of the cellulose degradation composite microbial inoculum, which comprises the following steps:
inoculating Serratia marcescens (Serratia marcescens) and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) into a fermentation culture solution according to a mass ratio of 1:1 in an inoculation amount of 3%, and culturing for 48 hours in a constant-temperature shake incubator at a temperature of 28 ℃ and a rotating speed of 180r/min to prepare a seed solution;
mixing the cultured seed liquid with rice chaff to prepare the solid microbial inoculum.
Preferably, the fermentation broth is: beef extract 5g, tryptone 10g, sodium chloride 5g, distilled water 1000ml, initial pH7.0, and sterilization at 121 ℃ for 20min.
The invention also provides application of the cellulose degradation composite microbial inoculum, which is used for preparing the mushroom residue organic fertilizer in the mushroom residue composting fermentation process, so that the composting is accelerated to be nontoxic and harmless, the decomposition degree is improved, and no secondary pollution is caused.
Preferably, the composite microbial inoculum is added into mushroom residue compost according to the inoculation amount of 0.15% by mass percent.
Preferably, the C/N of the mushroom residues and the cow dung in the mushroom residue compost is regulated to be 7:3, and the pH is 7.
The invention has the beneficial effects that:
(1) The two strains screened by the invention can be suitable and grow in the environment with the high temperature of 60-70 ℃, and the secreted cellulase has higher activity and has promotion effect on the mushroom residue decomposition fermentation under the high temperature condition.
(2) The cellulose secreted by Serratia marcescens and Acinetobacter calcoaceticus provided by the invention is used as a main active ingredient, and mushroom residues are used as degradation substances, so that the cellulose is applied to mushroom residue compost, and has the characteristics of quick temperature rise, good mushroom residue degradation effect, no secondary pollution and the like. The culture in CMC-NA culture medium has obvious hydrolysis circle, and in mushroom residue fermentation culture medium, the degradation effect on mushroom residue is obvious, and the germination rate and the decomposition degree of compost seeds are obviously improved.
Drawings
FIG. 1 is a graph of compost temperature variation for different treatments;
FIG. 2 is the effect of different treatments on the compost maturity (seed germination rate) of the mushroom residue;
FIG. 3 is the effect of different treatments on the degradation rate of compost mushroom residue.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Preservation of biological material samples:
serratia marcescens (Serratia marcescens) and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) have been deposited in Beijing, china general microbiological culture Collection center, accession numbers: the collection number of Serratia marcescens is CGMCC No.20557, the classification name is Serratia marcescens, and the collection date is 2020, 8 months and 27 days; the preservation number of the Acinetobacter calcoaceticus is CGMCC NO.20558, the classification name is Acinetobacter calcoaceticus, and the preservation date is 2020, 8, 27. Hereinafter, strain 1 and strain 2 will be abbreviated.
The strain 1 and the strain 2 are cultured at different temperatures, and the specific method is as follows:
(1) Cellulose degrading bacteria selected from sodium carboxymethyl cellulose culture medium: and (3) selecting single colony strains from the strain 1 and the strain 2, inoculating the single colony strains into 15mL of sterilized fermentation culture solution at an inoculum size of 3%, respectively placing the single colony strains into an incubator at the temperature of 20, 30, 40, 50, 60 and 70 ℃ for culturing for 48 hours, measuring the absorbance value of the bacterial solution, and researching the influence of the initial temperature on the bacterial concentration of the fermentation solution.
(2) Single colonies were picked and cultured at 30℃for 48 hours to prepare seed solutions. Inoculated into a 250mL triangular flask containing 30mL of fermentation culture solution according to an inoculum size of 1.0%, and shake-cultured at 30 ℃. Centrifuging at 4000r/min for 10min to obtain supernatant as crude enzyme solution. Each strain was replicated 3 times.
(3) Mixing seed solutions of the strain 1 and the strain 2 according to a mass ratio of 1:1, and mixing with rice chaff to prepare a solid microbial inoculum, which is named as HK strain.
(4) The verification test adopts a heap fermentation process, three treatments are set, each fermentation heap is 3t, the heap height is about 1.5m, the initial pH value is 7, the initial water content is 60%, and the treatments are set as follows:
treatment one: 2.6 tons of mushroom residue, 0.3 ton of cow dung and 4.5kg of HK strain (the bacterial content is 1 multiplied by 10) 8 CFU/g)。
And (2) treatment II: 2.6 tons of mushroom residue, 0.3 tons of cow dung and 4.5kg of market purchased strain (the bacterial content is 1 multiplied by 10) 8 CFU/g)。
Control (CK): 1.5 tons of mushroom residues and 1.5 tons of cow dung.
According to the test design (1), tests are carried out, and according to the characteristics of high temperature resistance and enzyme production capacity, the passing results of the target strain are shown as follows:
table 1 physiological and biochemical characteristics of each strain
Figure BDA0002761181220000031
As shown in Table 1, it was found that the cellulase activity of strain 1 was 19.80U/ml and the cellulase activity of strain 2 was 17.12U/ml after 48 hours of cultivation. The two strains can grow at the high temperature of 60-70 ℃, and after 48 hours, the concentration OD of bacterial solution of the strain 1 and the strain 2 600 Respectively reaching 1.12 and 1.25.
As shown in fig. 1, the first treatment (inoculation of the mixed microbial inoculum) reaches 50.1 ℃ at 3d, the second treatment (inoculation of market strain) reaches 50.9 ℃ at 7 d, and the contrast reaches 50 ℃ at 11d, which shows that the inoculation of the mixed microbial inoculum can quickly start the mushroom residue composting fermentation process; the highest temperature in the first high temperature period reaches 70 ℃, the highest temperature in the second high temperature period reaches 68 ℃, and the highest temperature in the contrast CK reaches 62 ℃, so that the C/N is higher than the contrast CK, and a sufficient carbon source is provided for the growth of microorganisms, which indicates that the addition of the proper C/N and the decomposed microbial inoculum promotes the temperature rise of the compost; treatment one was maintained at 60 ℃ for 14 days, treatment two was maintained for 10 days, and control CK was maintained for 9 days.
After about 24d composting fermentation, the germination rate results before and after the composting fermentation of the mushroom residue are shown in fig. 2, the germination rate index of the first treatment is increased from 48% to 93%, the germination rate index of the second treatment is increased from 43% to 80%, and the germination rate index of the control group is increased from 39% to 79%. Indicating that all three treatments had been thoroughly decomposed at 24d of composting. The effect of treatment one on germination rate was significantly affected compared to treatment two and the control, and the effect of treatment two on germination rate was not significantly affected compared to the control. Whether or not the microbial inoculum and the material formula are added, the composting body can be decomposed, but the microbial inoculum, the proper material formula and the fermentation environment can accelerate the decomposition process and improve the quality of the compost.
As shown in FIG. 3, the degradation rate of the mushroom residues after the decomposition of the pile is completed is 70.3% for the first treatment, 63.0% for the second treatment and 49.9% for the control. The degradation efficiency of the efficient degradation cellulose microbial inoculum is better due to the first treatment, the degradation rate of mushroom residues is higher than that of the second treatment, and the degradation effect of the control group without the microbial inoculum is worst. The degradation effect of the prepared composite microbial inoculum mushroom residue is improved by 7% compared with the cellulose degradation rate of the common microbial inoculum, and the degradation rate of the prepared composite microbial inoculum mushroom residue is improved by 20% compared with the cellulose degradation rate of the control cellulose without cellulose degradation bacteria.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Claims (8)

1. The cellulose degradation composite microbial inoculum is characterized by comprising Serratia marcescens (Serratia marcescens) with the preservation number of CGMCC No.20557 and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) with the preservation number of CGMCC No. 20558.
2. The cellulose degrading composite bacterial agent according to claim 1, wherein the mass ratio of Serratia marcescens (Serratia marcescens) to Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) is 1:1.
3. The cellulose degradation composite microbial inoculant according to claim 1, further comprising rice chaff, wherein the microbial inoculant has a bacterial content of 1×10 8 CFU/g。
4. The method for preparing the cellulose degradation composite microbial agent according to any one of claims 1 to 3, comprising the steps of:
inoculating Serratia marcescens (Serratia marcescens) and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) into a fermentation culture solution according to a mass ratio of 1:1 in an inoculation amount of 3%, and culturing for 48 hours in a constant-temperature shake incubator at a temperature of 28 ℃ and a rotating speed of 180r/min to prepare a seed solution;
mixing the seed liquid after culture with rice chaff.
5. The method for preparing the cellulose degradation composite microbial inoculant according to claim 4, wherein the fermentation broth is: beef extract 5g, tryptone 10g, sodium chloride 5g, distilled water 1000ml, initial pH7.0, and sterilization at 121 ℃ for 20min.
6. The use of a cellulose degrading composite bacterial agent according to any one of claims 1-3, for a mushroom dreg composting fermentation process.
7. The application of the cellulose degradation composite microbial inoculant according to claim 6, wherein the composite microbial inoculant is added into mushroom residue compost according to an inoculum size of 0.15% by mass.
8. The application of the cellulose degrading composite microbial inoculant according to claim 6, wherein the raw materials of mushroom residues and cow dung are adjusted to be 30 in C/N and 7 in pH in the mushroom residue composting.
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