CN114317381A - T-level microbial composite inoculant and preparation method and application thereof - Google Patents
T-level microbial composite inoculant and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a T-level microbial composite inoculant, and a preparation method and application thereof, and belongs to the technical field of preparation of efficient lignocellulose composite inoculants. The preparation method of the T-level microbial composite inoculant provided by the invention has the effective viable count of 1 multiplied by 107～1×109Equal volume mixing of CFU/mL microbial culture solution with different temperature characteristics, and culturing in gradient heating mode to make total effective viable count 1 × 107～1×109And (5) CFU/mL, subculturing, carrying out fermentation culture on the compound microbial inoculum obtained by the subculturing according to the compost temperature, and screening to obtain the compound microbial inoculum. The composite microbial inoculum prepared by the preparation method has the function of efficiently degrading lignocellulose, ensures that the dominant bacterial strain can exert the maximized activity and generate the degradation capability at the corresponding composting temperature, ensures that the effect generated by the dominant bacterial strain at different temperatures is just like the T level when a rocket is ignited, not only avoids the competitive exclusion effect among microorganisms, but also can rapidly degrade organic components in various periods of composting.
Description
Technical Field
The invention belongs to the technical field of preparation of high-efficiency lignocellulose composite microbial inoculants, and particularly relates to a T-level microbial composite microbial inoculants and a preparation method and application thereof.
Background
Along with the increase of population and the development of social economy, the living standard of people in China is continuously improved, and the yield of agricultural wastes is greatly increased. If the agricultural wastes are not properly treated, not only the resources are lost, but also the environment is greatly influenced. Aerobic fermentation is a main mode for treating agricultural wastes for resource utilization, but the degradation and high-quality resource utilization of the agricultural wastes are seriously influenced due to the problems of weak activity, low enzyme yield of microorganisms and the like.
Agricultural wastes typically comprise higher lignocellulosic biomass, consisting mainly of cellulose, lignin and hemicellulose linkages. The lignin is wrapped on the outermost layer of the lignocellulose biomass, so that the utilization of internal cellulose and hemicellulose is limited, and the lignocellulose with higher molecular weight is difficult to enter the cellulose for utilization. Aerobic fermentation is a process in which lignocellulosic components are decomposed under the action of microorganisms and humic acid is formed. Microorganisms play an important role in the degradation process of aerobically fermented lignocellulose. The screened microbes with special functions can break the stubborn lignocellulose structure and depolymerize the outer lignin, and are favorable for the combination of enzyme molecules and substrates. However, the prior art does not relate to researches on how to solve the problems of difficult degradation, low degradation efficiency and the like of the lignocellulose waste in the composting process by using microorganisms.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for preparing a T-level microbial composite inoculant, which has an effect of efficiently degrading lignocellulose, can ensure that an advantageous strain exerts maximized activity at a corresponding composting temperature and generates a degrading capability, not only avoids a competitive exclusion effect between microorganisms, but also can rapidly degrade organic components at various composting periods.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preparation method of a composite microbial inoculum for efficiently degrading lignocellulose, which comprises the following steps: the effective viable count is 1 × 107～1×109Equal volume mixing of CFU/mL microbial culture solution with different temperature characteristics, and culturing in gradient heating mode to make total effective viable count 1 × 107～1×109And (5) CFU/mL, subculturing, carrying out fermentation culture on the compound microbial inoculum obtained by the subculturing according to the compost temperature, and screening to obtain the compound microbial inoculum.
Preferably, the gradient heating mode culture is co-culture for 5-8 days at 20-35-45-50-55-60 ℃.
Preferably, the microorganisms with different temperature characteristics comprise low temperature resistant microorganisms, normal temperature microorganisms, mesophilic microorganisms and high temperature resistant microorganisms, wherein the low temperature is below 20 ℃, the normal temperature is 30-35 ℃, the medium temperature is 40-50 ℃, and the high temperature is above 55 ℃.
Preferably, the microorganisms with low temperature resistance characteristics comprise psychrobacter and psychrophilum, the microorganisms with normal temperature characteristics comprise aspergillus fumigatus and bacillus amyloliquefaciens, the microorganisms with mesophilic characteristics comprise bacillus halodurans and penicillium chrysogenum, and the microorganisms with high temperature resistance characteristics comprise bacillus subtilis and bacillus halodurans.
Preferably, the subculture is performed for 10-20 generations in a gradient temperature rise mode at 20-35-45-50-55-60 ℃.
Preferably, the screening comprises enzyme activity screening and degradation rate screening.
Preferably, the fermentation culture is carried out by using a fermentation culture medium with rice straws as a carbon source.
Preferably, the fermentation culture at the composting temperature is specifically a culture at 30 ℃ for 1 day, a culture at 35 ℃ for 1 day, a culture at 40 ℃ for 2 days, a culture at 45 ℃ for 2 days, a culture at 50 ℃ for 2 days, a culture at 55 ℃ for 2 days, a culture at 60 ℃ for 7 days, a culture at 55 ℃ for 5 days, a culture at 50 ℃ for 2 days, a culture at 45 ℃ for 5 days, a culture at 40 ℃ for 5 days, and a culture at 35 ℃ for 7 days.
The invention also provides the composite microbial inoculum prepared by the preparation method.
The invention also provides an application of the composite microbial inoculum in agricultural waste degradation.
The invention has the beneficial effects that:
the composite microbial inoculum prepared by the preparation method has the effect of efficiently degrading lignocellulose, can ensure that the dominant bacterial strains in each period of composting are different when the composting degradation treatment of agricultural wastes is carried out, can exert the maximized activity and generate the degradation capability at the corresponding temperature, further ensure that the effect generated by the dominant bacterial strains at different temperatures is just like the 'T' level when a rocket is ignited, maximally promote the generation of microbial enzymes, avoid the competitive exclusion effect among microorganisms, quickly degrade organic components in each period of composting and finally achieve the effects of degrading and converting the lignocellulose wastes.
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FIG. 1 shows the results of the final lignocelluloses and cellulase activities of the advantageous complex microbial agents in LB medium after 15 generations, wherein the horizontal coordinate FPase represents the filter paper enzyme activity, CMCase represents the cellulase activity, EG represents beta-glucoronidase, Xylanase represents the Xylanase activity, Lac represents the laccase activity, MnP represents the manganese peroxidase activity, and LiP represents the lignin peroxidase activity;
FIG. 2 shows the activity of four kinds of complex bacteria cultured in a rice straw fermentation medium on lignocelluloses, and the enzyme activity corresponding to the abscissa code is the same as that in FIG. 1;
FIG. 3 shows the results of the contents of cellulose, hemicellulose and lignin after the culture in the rice straw fermentation medium.
Detailed Description
The invention provides a preparation method of a composite microbial inoculum for efficiently degrading lignocellulose, which comprises the following steps: the effective viable count is 1 × 107～1×109Equal volume mixing of CFU/mL microbial culture solution with different temperature characteristics, and culturing in gradient heating mode to make total effective viable count 1 × 107～1×109And (5) CFU/mL, subculturing, carrying out fermentation culture on the compound microbial inoculum obtained by the subculturing according to the compost temperature, and screening to obtain the compound microbial inoculum.
In the present invention, the microorganisms having different temperature characteristics preferably include microorganisms having low temperature resistance, normal temperature resistance, mesophilic resistance, and high temperature resistance, and the microorganisms having different temperature characteristics do not have competitive exclusion, and the low temperature is preferably 20 ℃ or less, the normal temperature is preferably 30 ℃ to 35 ℃, the medium temperature is preferably 40 ℃ to 50 ℃, and the high temperature is preferably 55 ℃ or more. The present invention is not particularly limited with respect to the specific types of microorganisms having different temperature characteristics, and any types of microorganisms that meet the temperature characteristics conventionally used in the art may be used. In a specific embodiment of the present invention, the microorganisms having low temperature resistance characteristics preferably include psychrobacter (psychrobacterp) and psychrobacter (psychrobacter pulmonistrain), and more preferably include psychrobacter and psychrobacter; the microorganisms of the normal temperature characteristics preferably include aspergillus fumigatus (aspergillus fumigatus) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and more preferably include aspergillus fumigatus; the mesophilic characteristic microorganisms preferably include Bacillus halolerans (Bacillus halolerans), Penicillium chrysogenum (Penicillium chrysogenum); the microorganisms with high temperature resistance preferably comprise Bacillus subtilis and Bacillus halotolerans.
In the present invention, it is preferable that microorganisms having different temperature characteristics are respectively cultured in LB medium at a corresponding suitable temperature to make the total effective viable count of each functional microorganism to be 1X 107～1×109CFU/mL, and then carrying out equal-volume mixed culture on the microbial culture solution with different temperature characteristics. The purpose of the invention to limit the number of initial viable bacteria is to ensure that the strains growing in the culture medium are able to function more efficiently, ensuring that the concentration of all initial strains performs a consistent function. The specific source of the LB culture medium is not specially limited, and the LB culture medium can be prepared according to the formula of the LB culture medium, and the conventional commercial product can be selected.
In the invention, the gradient heating mode culture is preferably a 20-35-45-50-55-60 ℃ gradient heating mode for 5-8 days, and the gradient heating mode is more preferably a 20 ℃ culture for 1 day, a 35 ℃ culture for 1 day, a 45 ℃ culture for 1 day, a 50 ℃ culture for 2 days, a 55 ℃ culture for 2 days, and a 60 ℃ culture for 1 day. The effective viable count of the bacterial liquid obtained by the gradient temperature-rising culture is 1 multiplied by 107～1×109At CFU/mL, subculture was performed. The subculture aims to enable target strains to reach a stabilized state at respective dominant temperatures and enable the dominant strains to play a role, and finally obtains a composite microbial inoculum which can stably exist in compost and can play the best effect of a 'T' level. In the invention, the subculture is preferably performed for 10-20 generations in a gradient temperature rise manner of 20-35-45-50-55-60 ℃, and more preferably for 15-20 generations in the gradient temperature rise manner. In the process of passage, preferably after each passage, the compound microbial inoculum with strong decomposition capacity is detected by enzyme activity or screened according to a molecular biological method, wherein the enzyme activity detection preferably comprises protease, cellulase, ligninase, hemicellulase and peroxidationDetecting catalase and the like; the molecular biological method refers to DNA extraction, identification of lignocellulose degradation related genes by real-time quantitative analysis, and morphological identification methods such as electron microscopy and the like.
After the subculture is finished, carrying out fermentation culture on the compound microbial inoculum obtained by the subculture according to the composting temperature, wherein the fermentation culture medium used for the fermentation culture preferably takes rice straws as a carbon source so as to ensure that the compound microbial inoculum has corresponding decomposition capacity when used for subsequent agricultural waste composting fermentation. The other specific components and the dosage of the fermentation medium are not particularly limited in the invention, and the conventional fermentation medium in the field can be adopted. In the present invention, the fermentation culture at the composting temperature is preferably culture at 30 ℃ for 1 day, at 35 ℃ for 1 day, at 40 ℃ for 2 days, at 45 ℃ for 2 days, at 50 ℃ for 2 days, at 55 ℃ for 2 days, at 60 ℃ for 7 days, at 55 ℃ for 5 days, at 50 ℃ for 2 days, at 45 ℃ for 5 days, at 40 ℃ for 5 days, and at 35 ℃ for 7 days. And after the fermentation culture is finished, screening, wherein screening is preferably carried out according to enzyme activity screening and lignocellulose degradation rate. The enzyme activity preferably comprises cellulose endonuclease, filter paper enzyme, beta-glucoronidase, xylanase, lignin peroxidase, manganese peroxidase, laccase and the like, and the degradation rate preferably comprises cellulose, hemicellulose, lignin and organic matter degradation rate. The composite microbial inoculum with the function of efficiently degrading lignocellulose is determined by measuring various indexes such as the activity of the lignocellulose, the degradation rate of the lignocellulose and the like in the fermentation culture process.
The invention also provides the composite microbial inoculum prepared by the preparation method and application of the composite microbial inoculum in agricultural waste degradation.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Four mesophilic characteristic bacteria respectively numbered as Z1, B4, H41 and D3, three high temperature resistant characteristic bacteria respectively numbered as D2, D8 and A1 and normal temperature characteristic bacteria respectively numbered as B4, H61 and H41 (wherein two bacteria of B4 and H41 are usedHaving both mesophilic and normal temperature characteristics) are respectively cultured in LB culture medium at 18 ℃, 32 ℃, 45 ℃ and 58 ℃ for 48 hours, so that the effective viable count of each bacterium is 1 multiplied by 107～1×109CFU/mL, then according to Z1D 3D 2A 1B 1, Z1D 1H 1, Z1H 1A 1D 1, Z1B 1H 1, A1B 1, D1A 1, D1A 1B 1, D1H 1A 1, A1B 1, D1, D1A 1, H1A 1B 1, the above mentioned bacteria are inoculated into LB medium according to the same volume for 1 day at 20 ℃, 1 day at 35 ℃, 1 day at 45 ℃, 2 days at 60 ℃, 2 days at 1 ℃, and 10 ℃ for total days (the culture time)7～1×109And (3) carrying out subculturing on the CFU/mL according to a subculturing mode of culturing for 1 day at 20 ℃, culturing for 1 day at 35 ℃, culturing for 1 day at 45 ℃, culturing for 2 days at 50 ℃, culturing for 2 days at 55 ℃ and culturing for 1 day at 60 ℃, and carrying out enzyme activity detection on cellulase, ligninase and hemicellulase once every passage, wherein the result shows that after 15 passages, the enzyme activities of cellulase, ligninase and hemicellulase of the compound microbial inoculum tend to be stable and the enzyme activities are highest. The results of the activity of each dominant complex microbial inoculum final lignocellulose and cellulase in LB medium after 15 generations are shown in figure 1.
Selecting four composite microbial agents with better effects in the figure 1, inoculating the four composite microbial agents into a fermentation culture medium with rice straw powder as a carbon source for culture, wherein the inoculation amount is 10% of the dry weight of the straw powder, the culture temperature is changed according to the typical composting temperature, the culture is carried out for 1 day at 30 ℃, the culture is carried out for 1 day at 35 ℃, the culture is carried out for 2 days at 40 ℃, the culture is carried out for 2 days at 45 ℃, the culture is carried out for 2 days at 50 ℃, the culture is carried out for 2 days at 55 ℃, the culture is carried out for 7 days at 60 ℃, the culture is carried out for 5 days at 55 ℃, the culture is carried out for 2 days at 50 ℃, the culture is carried out for 5 days at 45 ℃, the culture is carried out for 5 days at 40 ℃, the culture is carried out for 7 days at 35 ℃, the liquid in the fermentation culture medium and the rice straw residue are respectively sampled at 45 ℃, 55 ℃, 40 ℃ and 35 ℃, and detecting the content of lignocellulose and the content of cellulose, hemicellulose and lignin after the fermentation culture medium is cultured. The results are shown in FIGS. 2 and 3. Inoculating the finally determined composite microbial inoculum into rice straws for composting, wherein the inoculation amount is 3% of dry weight of mycelia, and the results show that: the method can rapidly degrade lignocellulose in a short time to generate higher activity of extracellular enzyme, and the degradation rate of the lignocellulose in the prepared composite microbial inoculum is obviously improved by 24 percent compared with that of single bacteria, the cellulase activity is improved by 20 percent, the hemicellulase activity is improved by 25 percent, and the ligninase activity is improved by 15 percent.
Example 2
The difference from example 1 is that the psychrophilic bacillus is added as a cold-resistant functional strain on the basis of each group of composite strains in example 1, and the other steps are the same as example 1.
The result shows that the method can rapidly degrade the lignocellulose in a short time, and the degradation rate of the lignocellulose of the prepared composite microbial inoculum is obviously improved by 20 percent compared with that of the lignocellulose of a single bacterium, the activity of the cellulase is improved by 25 percent, the activity of hemicellulase is improved by 20 percent, and the activity of the ligninase is improved by 20 percent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of a T-level microbial compound inoculant is characterized by comprising the following steps: the effective viable count is 1 × 107～1×109Equal volume mixing of CFU/mL microbial culture solution with different temperature characteristics, and culturing in gradient heating mode to make total effective viable count 1 × 107～1×109And (5) CFU/mL, subculturing, carrying out fermentation culture on the compound microbial inoculum obtained by the subculturing according to the compost temperature, and screening to obtain the compound microbial inoculum.
2. The method according to claim 1, wherein the gradient temperature-raising culture is a co-culture at 20 ℃ -35 ℃ -45 ℃ -50 ℃ -55 ℃ -60 ℃ for 5-8 days.
3. The method according to claim 1, wherein the microorganisms having different temperature characteristics include low temperature resistant microorganisms, normal temperature microorganisms, mesophilic microorganisms, and high temperature resistant microorganisms, the low temperature is 20 ℃ or less, the normal temperature is 30 ℃ to 35 ℃, the medium temperature is 40 ℃ to 50 ℃, and the high temperature is 55 ℃ or more.
4. The method according to claim 3, wherein the low temperature resistant microorganisms include psychrobacter and psychrophilum, the normothermic microorganisms include Aspergillus fumigatus and Bacillus amyloliquefaciens, the mesophilic microorganisms include Bacillus halodurans and Penicillium chrysogenum, and the high temperature resistant microorganisms include Bacillus subtilis and Bacillus halodurans.
5. The preparation method according to claim 1, wherein the subculture is subcultured for 10-20 generations in a manner of gradient temperature rise from 20 ℃ to 35 ℃ to 45 ℃ to 50 ℃ to 55 ℃ to 60 ℃.
6. The method of claim 1, wherein the screening includes an enzyme activity screening and a degradation rate screening.
7. The method according to claim 1, wherein the fermentation culture is carried out in a fermentation medium containing rice straw as a carbon source.
8. The method according to claim 1, wherein the fermentation culture is carried out at a composting temperature of 30 ℃ for 1 day, 35 ℃ for 1 day, 40 ℃ for 2 days, 45 ℃ for 2 days, 50 ℃ for 2 days, 55 ℃ for 2 days, 60 ℃ for 7 days, 55 ℃ for 5 days, 50 ℃ for 2 days, 45 ℃ for 5 days, 40 ℃ for 5 days, and 35 ℃ for 7 days.
9. The complex microbial inoculum prepared by the preparation method of any one of claims 1 to 8.
10. The use of the complex microbial inoculum of claim 9 in the degradation of agricultural wastes.
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