CN114933986B - Cellulose degrading bacterium and application thereof - Google Patents
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Abstract
The invention discloses a cellulose degrading bacterium and application thereof, and belongs to the technical field of agricultural microorganisms. The invention screens out a bacillus bacterium with cellulose degradation capability from agaricus bisporus cultivation material, and tests the cellulase activity, the filter paper enzyme activity and the xylanase activity of the bacillus bacterium, and finds that the average enzyme activity of the xylanase is 14.32U/mL; cellulase times, 2.84U/mL; the filter paper enzyme was the lowest, 1.88U/mL. In the tieback test of cellulose degrading bacteria, the time for the agaricus bisporus of the treated group cultivation material added with the cellulose degrading bacteria liquid to grow into a mushroom bed is 16.33d, which is 2d less than that of a control group; the fruiting time is 21.33d, which is 3 days shorter than that of the control group; the biological efficiency of the cultivation material is 78.10 percent, which is obviously higher than that of a control group, and provides theoretical basis and guidance for developing the agaricus bisporus growth-promoting microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a cellulose degrading bacterium and application thereof.
Background
The agaricus bisporus is a straw-saprophytic fungus, the cultivation substrate takes crop straws such as straws, wheat straws and the like as main materials, and after prewetting, cow dung, chicken dung or other livestock manure and mineral substances are added in a certain proportion to ferment, so that the cultivation material suitable for growth of agaricus bisporus hyphae is formed.
The compost fermentation of the agaricus bisporus culture medium to form a culture medium is a key link for determining the industrial development of the agaricus bisporus industry, and determines the production level of the agaricus bisporus. How to effectively improve the degradation and conversion efficiency of crop straws and decompose cellulose, hemicellulose and lignin contained in the crop straws for the agaricus bisporus hyphae to be utilized becomes a current research hot spot. The microorganism plays a key role in the fermentation process, and the change of the microbial community structure, the type of the microbial community structure and the composting decomposition related enzyme system directly affects the decomposition degree of the straw in terms of different fermentation periods of the cultivation material. Therefore, the strain which can efficiently degrade cellulose and has no inhibition effect on the growth of the agaricus bisporus mycelium is screened from the agaricus bisporus cultivation material sample, and the growth and development of the agaricus bisporus are promoted indirectly or directly through the degradation of the strain on crop straws.
In the previous study, chen Shan and the like (2014) are screened to a bacillus licheniformis (Bacillus licheniformis) with cellulose degradation activity by a unique carbon source method, and the bacillus licheniformis is cultured for 48 hours under the optimal condition to reach the peak of enzyme production, and the enzyme activity can reach 1.82U/mL. Gao Fengju et al (2006) selected a strain of cellulase-producing Bacillus subtilis (Bacillus subilius) with sodium carboxymethylcellulose as the sole carbon source, and had an enzyme activity of 1.28U/mL under the optimal growth conditions. Wang Xianfeng et al (2015) screened out a strain of Bacillus licheniformis (Bacillus licheniformis) capable of degrading bagasse cellulose, and the enzyme activity of the strain is 2.80U/mL under the optimal condition. But the cellulase has low enzymatic activity, and further needs to screen strains with strong cellulose degradation capability, thereby providing theoretical basis and technical support for improving the utilization of cellulose substances.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a cellulose degrading bacterium and application thereof in promoting the growth of agaricus bisporus.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a cellulose degrading bacterium, classified under the name Bacillus sp (C4), which has been preserved in the China center for type culture collection, and has a preservation number of CCTCC No: m2022477, date of preservation: 2022, 4, 25, deposit address: university of martial arts in chinese.
A microbial preparation containing the cellulose degrading bacteria.
Preparing a microbial preparation containing the cellulose degrading bacteria and the agaricus bisporus culture material.
The cellulose degrading bacteria or the microbial preparation is applied to efficiently degrading cellulose and/or preparing cellulose degradation products.
The cellulose degrading bacteria or the microbial preparation are applied to composting fermentation and/or preparation of composting fermentation products.
Further, the main material of the compost is crop straws.
The cellulose degrading bacteria or the microbial preparation is applied to the promotion of the growth of the agaricus bisporus and/or the preparation of a product for promoting the growth of the agaricus bisporus.
Compared with the prior art, the invention has the beneficial effects that:
the invention screens out a Bacillus bacterium with cellulose degradation capability from agaricus bisporus cultivation material, and tests the cellulase activity, the filter paper enzyme activity and the xylanase activity of the Bacillus bacterium, and finds that the average enzyme activity of the xylanase is 14.32U/mL; cellulase times, 2.84U/mL; the filter paper enzyme was the lowest, 1.88U/mL. In the tieback test of cellulose degrading bacteria, the time for the agaricus bisporus of the treated group cultivation material added with the cellulose degrading bacteria liquid to grow into a mushroom bed is 16.33d, which is 2d less than that of a control group; the fruiting time is 21.33d, which is 3 days shorter than that of the control group; the biological efficiency of the cultivation material is 78.10 percent, which is obviously higher than that of a control group, and provides theoretical basis and guidance for developing the agaricus bisporus growth-promoting microbial inoculum.
Drawings
FIG. 1 is a graph showing the results of a transparent circle test and a plate challenge test of cellulose degrading bacterium C4; in the figure, a is a transparent ring of cellulose degrading bacteria C4 on a discrimination medium; b is the front surface of a dish of cellulose degrading bacteria C4 and agaricus bisporus mycelium against an experiment culture medium; c is the reverse side of the dish of cellulose degradation bacteria C4 and agaricus bisporus mycelium resisting experiment culture medium;
FIG. 2 is a graph showing the results of a disintegration test of a filter paper strip of cellulose degrading bacterium C4;
FIG. 3 is a flow chart of the strain identification of cellulose degrading bacteria;
FIG. 4 is a colony morphology and bacterial staining pattern of cellulose degrading bacterium C4; in the figure, a is a colony morphology diagram of cellulose degrading bacteria C4; b is a gram stain of cellulose degrading bacterium C4; c is a spore staining chart of cellulose degrading bacteria C4;
FIG. 5 is a molecular biological identification chart of cellulose degrading bacterium C4; in the figure, a is a gel diagram of a 16S rRNA PCR product of cellulose degrading bacteria C4; b is a phylogenetic tree diagram;
FIG. 6 is a graph showing the change of the cellulose-related enzyme activity of cellulose-degrading bacterium C4; in the figure, a is a graph of the maximum enzyme activity of different cellulose degrading enzymes; b is a graph showing the change of enzyme activities of different cellulose degrading enzymes.
Detailed Description
The screening materials used in the following examples were: culture materials of agaricus bisporus in different fermentation periods.
The test media, main reagents and laboratory instruments used in the following examples were:
test medium: CMC-Na medium, NA solid medium, NB liquid medium, PDA solid medium, PDA liquid medium, identification medium, seed medium and filter paper disintegration medium.
The main reagent comprises: 1g/L Congo red solution, 1mol/L sodium chloride solution, 0.1mol/L sodium acetate solution, acetic acid-sodium acetate buffer solution, 0.8% (w/v) sodium carboxymethylcellulose solution and DNS reagent.
Experimental instrument: electronic balance Quintix224-CN, vertical sterilization pot MLS-3781-PC ultraviolet visible spectrophotometry, nucleic acid electrophoresis apparatus Mini-SubCEL, gel imager Geldoc XR+, electrothermal constant temperature blast drying oven, full temperature oscillation incubator, ultra clean bench, pH meter, positive microscope, mixer, biochemical incubator, digital display constant temperature water bath, PCR instrument C1000 Touch, high speed refrigerated centrifuge J-25, refrigerator BCD-315TNGS, ultra low temperature refrigerator DW-86L578J, central pure water system ROEZ-150, and heating magnetic stirrer 79-1.
The invention is further described below in connection with specific embodiments.
Example 1
1. Sample collection
The cultivation material sample mainly comprises wheat straw, mixed chicken manure and gypsum, and is produced by adopting a tunnel type three-time fermentation method. Sampling is carried out from the cultivation material in the stacking period, the cultivation material after the primary fermentation is finished, the cultivation material after the secondary fermentation is finished and the cultivation material after the tertiary fermentation (concentrated fermentation) is finished respectively. The five-point sampling method is adopted, a sterile polypropylene bag is used for preserving samples, the samples are respectively sampled from the upper layer, the middle layer and the lower layer of the cultivation material, 500g of the samples are sampled from the cultivation material after the samples are uniformly mixed, the samples are divided into 10 parts, liquid nitrogen is quickly frozen, part of the samples are subjected to experimental operation, and the rest of the samples are placed in a refrigerator at the temperature of minus 80 ℃ for later study and use.
Pre-wetting wheat straw in water for 3-4 days, performing pre-fermentation, adding mixed chicken manure and gypsum, and uniformly mixing, wherein the process is a stacking period, and the number of collected samples is A (A1, A2 and A3); the cultivation materials in the stacking period are uniformly mixed after being overturned and loaded, and enter a fermentation tank for fermentation, the process lasts for about 12 days, 3 times of warehouse turning are carried out in the middle according to a control standard, the warehouse turning is used for enabling the cultivation materials to be uniformly mixed and supplementing water, the process is first fermentation, and the collected samples are numbered as B (B1, B2 and B3); then, the cultivation material after the primary fermentation is sent into a tunnel for secondary fermentation, the process lasts for about 6 days, and the collected samples are numbered as C (C1, C2 and C3); sowing agaricus bisporus strain on the cultivation material after the secondary fermentation is completed, and then carrying out tertiary fermentation in a tunnel, namely intensively growing the agaricus bisporus strain, wherein the process lasts for about 17 days, and the collected samples are numbered as D (D1, D2 and D3).
2. Primary screening of cellulose degrading bacteria
(1) The method comprises the steps of cleaning a crushing box of a crusher with tap water, washing the crushing box for a plurality of times with distilled water, soaking the crushing box with 75% alcohol for sterilization, washing the crushing box with sterile water for a plurality of times, and placing the crushing box into an ultra-clean bench to be sterilized together with an operation bench by ultraviolet rays.
(2) Pulverizing the agaricus bisporus cultivation material in a sterilizing pulverizer, slightly crushing, and accurately weighing 10.0g of the crushed cultivation material without long wheat straw, adding into a 250mL conical flask containing 90mL sterile normal saline, and vibrating at a constant speed of 150r/min for 30min to prepare a bacterial suspension diluted by 10 times;
(3) Preparing sample bacterial suspension with ten times of gradient concentration, wherein the minimum time is 10 -7 ；
(4) Respectively sucking 0.2mL at 10 -4 ～10 -7 Uniformly coating the bacterial liquid in an NA solid culture medium prepared in advance, and setting 3 bacterial liquids in parallel in each group;
(5) Culturing microorganisms: coating a sealing film on the coated plate, inverting the plate in a biochemical incubator, and culturing for 24 hours at 37 ℃;
(6) Single colony is picked from the flat plate, and the partition streak separation culture is continuously carried out for a plurality of times until single colony bacteria are separated. Single colonies were stored in NA solid slant medium for later experiments.
(7) The isolated bacteria were inoculated in CMC-Na solid medium and cultured at 37℃for 3d.
(8) The CMC-Na solid culture medium with vigorous bacterial growth in (7) is dyed by 1g/L Congo red solution, the dyeing liquid is poured off after 20min, 1mol/L sodium chloride solution is added for washing, and the solution is poured off after 1-2min, and the transparent ring is provided to indicate that the solution can degrade cellulose.
During the initial screening, 34 strains of bacteria had transparent circles on CMC-Na solid medium, i.e. these strains had cellulose degrading function. Wherein 14 strains are screened from the cultivation materials in the group A, 8 strains are screened from the cultivation materials in the group B, 7 strains are screened from the cultivation materials in the group C, and 5 strains are screened from the cultivation materials in the group D.
3. Compound sieve for cellulose degrading bacteria
(1) Inoculating bisporus mycelium on PDA solid culture medium, activating, culturing at 24deg.C for about two weeks until mycelium grows on the culture dish, perforating with 7mm puncher, and taking agar block with mycelium as strain.
(2) Inoculating agaricus bisporus mycelium agar block in the center of the plate, dibbling the strain obtained by primary screening in four directions at a position 1.5-2.0cm away from the center, culturing at 24 ℃ for 14-20d until the agaricus bisporus mycelium grows beyond the outer edge of the diameter of a bacterial colony, observing whether the bacteria obtained by primary screening have an inhibition effect on the growth of the agaricus bisporus mycelium, and retaining the bacterial strain which has no antagonism on the growth of the agaricus bisporus mycelium.
(3) Selecting strains which have no antagonism with agaricus bisporus mycelium, inoculating the strains on a discrimination culture medium by a dibbling method, and performing three biological parallel repetition on each strain, and performing inversion culture for 2-7d at normal temperature. After dyeing with 1g/L Congo red for 15-20min, decolorizing with 1mol/L sodium chloride solution. The diameter (D) of the transparent ring and the diameter (D) of the colony were measured, and the strain having a larger D/D value was screened for the filter paper disintegration test.
(4) Inoculating the strain to NB liquid culture medium, culturing at 37deg.C and 180r/min, and culturing for 24 hr to obtain seed bacterial suspension. 1mL of the bacterial suspension is respectively inoculated into a conical flask with 100mL of filter paper strip disintegration culture medium and the capacity of 250mL, 1mL of sterile water is used for control group connection, three parallel repetitions are arranged for each group, the culture is carried out at 37 ℃ and 120r/min, and the disintegration condition of the filter paper strip is observed regularly. The degree of disintegration of the filter paper is as shown in Table 1.
TABLE 1 Standard of degree of disintegration of Filter papers
The 34 strains of bacteria screened initially were co-cultivated with agaricus bisporus mycelium during the re-screening process, and it was found that there was no antagonistic relationship between 5 strains of bacteria and agaricus bisporus mycelium. Table 2 shows the sizes of the transparent circle diameter and colony diameter of these 5 bacteria, and strain C4 (see FIG. 1 a) having the largest ratio of transparent circle diameter to colony diameter was selected as the experimental strain for the subsequent experiments. Fig. 1b and C are graphs of the results of the experiment of the cellulose degrading bacteria C4 and the agaricus bisporus mycelium in the dish, showing that the growth of the agaricus bisporus mycelium is more vigorous and robustly at and around the place where the colony of the C4 bacteria exists, indicating that the growth of the agaricus bisporus mycelium by C4 has no antagonism and even can make the growth of the agaricus bisporus mycelium more vigorous.
TABLE 2 screening results for cellulose degrading bacteria
Note that: the results in the table are expressed as mean ± standard error, n=3.
The filter paper strip disintegration test is adopted to verify the filter paper enzyme degradation characteristic of cellulose degrading bacteria C4, as shown in figure 2, under the condition of 4d culture, compared with a CK group, the strain C4 can completely degrade the filter paper strip into paste, so that the effect of the strain C4 on disintegration of the filter paper is quite remarkable.
4. The strain of cellulose degrading bacteria is determined, and the specific identification flow is shown in figure 3.
1. Morphological and physiological biochemical identification
A photograph of the morphological identification of the bacteria is shown in FIG. 4. It was observed that strain C4 was round on a plate (FIG. 4 a), smooth at the edges, convex, milky white in colony color, opaque, and wet in the cells. Gram staining showed that strain C4 was a gram positive bacterium (see fig. 4 b). The spore stain malachite green stain (as in fig. 4 c) shows sporulation. The physiological and biochemical identification results are shown in Table 3.
TABLE 3 physiological and biochemical characterization of Strain C4
Description: "+" represents positive response and "-" represents negative response.
2. Molecular biological identification
As shown in FIG. 5a, which shows a 1% gel electrophoresis pattern of the PCR product of the 16S rRNA of strain C4, the C4 band is clear and bright, which indicates that the PCR product has good quality and can be sent for measurement. And assembling the obtained sequencing result, performing blast comparison on NCBI, downloading the species gene sequences with higher similarity according to the comparison result, performing multiple sequence comparison on the sequences of the obtained genes by using MEGA 7.0, and constructing a phylogenetic tree (as shown in figure 5 b). The 16S rRNA sequence analysis was compared with NCBI database to find that strain C4 was closest to strain Bacillus nakamurai (NR 151897.1), and reached 97.21%, and strain C4 was identified as Bacillus bacterium (GenBank accession number: OM 918750) in combination with morphological and physiological biochemical identification.
5. Measurement of cellulase Activity
The enzyme activity is defined in terms of international units: the amount of enzyme catalyzing hydrolysis to generate 1 mu mol of glucose per minute is one enzyme activity unit U.
(1) Inoculating the strain obtained by re-screening to NB liquid culture medium, culturing at 37deg.C for 2d under constant temperature and shaking at 180r/min, centrifuging at 4deg.C for 15min at 5000r/min, and collecting supernatant to obtain crude enzyme solution.
(2) Carboxymethyl cellulase activity assay: taking 1mL of crude enzyme solution to a 25mL test tube with a plug, adding 1mL of sodium carboxymethylcellulose solution, fully mixing, placing into a water bath kettle at 37 ℃ for warm bath saccharification for 30min, then adding 2mL of DNS color development liquid, immediately developing color in a boiling water bath for 5min after mixing, taking out flowing water, showering and cooling, fixing the volume to 25mL, and colorizing at 540nm by using a spectrophotometer. The inactivated crude enzyme solution after boiling at 100deg.C for 10min was used as control, and three replicates were set for each group.
(3) Filter paper enzyme activity assay: the procedure was as in (2), and the sodium carboxymethyl cellulose solution was changed to 1mL of distilled water and two pieces of 1 cm. Times.1 cm filter paper.
(4) Xylanase activity assay: the step (2) is the same as that of the step (2), and the sodium carboxymethyl cellulose solution is changed into xylan solution.
(5) And (3) taking the OD values measured in (2), (3) and (4) into a regression equation and a standard enzyme activity formula by using a standard curve, and calculating to obtain the enzyme activity formula of each strain as follows:
this example performed quantitative measurements of cellulase activity, filter paper enzyme activity and xylanase enzyme activity on the C4 strain for 5 consecutive days. The results show that the cellulose degrading bacteria have 3 cellulose degrading enzyme activities at the same time, but the maximum enzyme activities of different enzymes are different. As shown in FIG. 6a, the maximum enzyme activity was xylanase, 25.16U/mL was reached, followed by cellulase, 4.63U/mL was reached, the maximum enzyme activity was minimal was filter paper enzyme, 3.14U/mL.
The xylanase activity, filter paper enzyme activity and cellulase activity of the cellulose degrading bacterium C4 were measured by the DNS method. As shown in FIG. 6b, among the 3 cellulose-related enzymes of the C4 strain, the xylanase activity was highest in average enzyme activity as compared with the other two enzymes, and was 14.32U/mL; cellulase times, 2.84U/mL; the lowest amount was 1.88U/mL of filter paper enzyme. The cellulase activity of C4 and the filter paper enzyme activity are slightly decreased after rising and then decreased, and generally show a trend of rising and then decreasing, and the enzyme activities of the two enzymes reach the maximum on the fourth day. The xylanase enzyme activity change of C4 shows a trend of ascending and then descending, the ascending and descending amplitude is larger, and the descending starts after the maximum enzyme activity is reached in the third day.
6. Strain tieback
The test was developed at the edible fungus industry base of the sampling site. Inoculating C4 strain into seed culture medium, shake culturing at 37deg.C and 180r/min for 2 days to obtain seed solution. In the later period of the second fermentation, the cultivation material is subjected to a cooling stage, seed liquid is sprayed into the cultivation material according to the inoculation amount of 10L/t, and the cultivation material is uniformly mixed. Laying cultivation materials in the same fruiting room, performing subsequent fermentation and fruiting management according to factory standardized operation flow, and setting 10m in each cell 2 3 replicates were set to give a control group of the cultivation material without seed fluid. Recording the time of mycelium growing over the mushroom bed and the fruiting time, harvesting the mature agaricus bisporus at proper time, and recording the yield.
Data processing was performed using Excel and Duncan multiple comparison analysis was performed on SPSS 22.0, with P < 0.05 being statistically significant for the differences between the groups.
As shown in Table 4, the treatment group was a treatment group to which a C4 bacterial liquid was addedThe time for the agaricus bisporus mycelium to grow into the mushroom bed is 16.33d, and the time is 2d earlier than that of a control group (normal cultivation material without adding C4 bacterial liquid); in the fruiting time, the fruiting time of the treatment group is shortened by 3 days compared with that of the control group and is 21.33d. In terms of yield of the first crop mushrooms, the treatment group was 23.43kg/m 2 Is extremely higher than that of the control group (P < 0.01). In the factory for standardized production of agaricus bisporus for the test, the most advanced technology and equipment for producing agaricus bisporus in Europe are introduced, and the standard of dry matter weight per unit area is controlled at 30kg/m 2 Left and right. Biological efficiency refers to the percentage of weight of fresh mushroom fruiting bodies to the dry base of the culture medium. From this, it can be calculated that the biological efficiency of the control group cultivation material is 72.87%, and the treatment group is extremely higher than the control group by 78.10%.
TABLE 4 Effect of different cultivation materials on yield of agaricus bisporus
Note that: * p < 0.05, p < 0.01.
Claims (6)
1. A cellulose degrading bacterium, classified under the name Bacillus sp (C4), which has been preserved in the China center for type culture collection, and has a preservation number of CCTCC No: m2022477, date of preservation: 2022, 4, 25, deposit address: university of martial arts in chinese.
2. A microbial preparation comprising the cellulose-degrading bacterium according to claim 1.
3. Use of a cellulose degrading bacterium according to claim 1 or a microbial preparation according to claim 2 for efficient degradation of cellulose and/or for the preparation of cellulose degradation products.
4. Use of a cellulose degrading bacterium according to claim 1 or a microbial preparation according to claim 2 in composting fermentation and/or in the preparation of a composting fermentation product.
5. The use according to claim 4, wherein the main material of the compost is crop straw.
6. Use of a cellulose degrading bacterium according to claim 1 or a microbial preparation according to claim 2 for promoting the growth of agaricus bisporus and/or for the preparation of a product promoting the growth of agaricus bisporus.
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|---|---|---|---|---|
| CN101974436A (en) * | 2010-09-21 | 2011-02-16 | 中国农业科学院农业资源与农业区划研究所 | Lignocellulose degrading bacteria and application thereof |
| WO2015180624A1 (en) * | 2014-05-27 | 2015-12-03 | Novozymes A/S | Methods for mushroom cultivation |
| CN105602874A (en) * | 2016-03-29 | 2016-05-25 | 中国农业科学院农业资源与农业区划研究所 | Bacillus amyloliquefaciens strain and application thereof in edible fungus residue composting degradation |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974436A (en) * | 2010-09-21 | 2011-02-16 | 中国农业科学院农业资源与农业区划研究所 | Lignocellulose degrading bacteria and application thereof |
| WO2015180624A1 (en) * | 2014-05-27 | 2015-12-03 | Novozymes A/S | Methods for mushroom cultivation |
| CN105602874A (en) * | 2016-03-29 | 2016-05-25 | 中国农业科学院农业资源与农业区划研究所 | Bacillus amyloliquefaciens strain and application thereof in edible fungus residue composting degradation |
Non-Patent Citations (1)
| Title |
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| 高温纤维素酶产生菌的筛选鉴定及其酶性质研究;颜霞;柳晓东;杨俊杰;;太阳能学报(第06期);全文 * |
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