CN108841743B - Cold region straw rotten bacterial strain and preparation method and application thereof - Google Patents
Cold region straw rotten bacterial strain and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cold-region straw-decomposing bacterium strain and a preparation method and application thereof, and belongs to the field of separation, preparation and application of cold-region straw-decomposing bacteria. The invention firstly discloses a separated cold-region straw decomposing bacterial strain, and the microbial preservation number is as follows: CGMCC No. 15178. The invention further discloses a preparation method of the cold region straw rotten bacterial strain. The invention further discloses application of the cold-region straw decomposing bacterium strain in a straw decomposing process. The cold region straw decomposing bacteria strain separated by the invention is bacillus amyloliquefaciens with strong cold resistance, has strong cellulose decomposition capacity and also has good glycolysis effect on rice straws under the condition of low temperature. The cold-region straw decomposing bacterial strain separated by the invention has good application prospect in preparing a cold-region straw decomposing agent, especially in preparing a cold-region rice straw decomposing agent.
Description
Technical Field
The invention relates to a separated cold-region straw-decomposing bacterial strain, and also relates to application of the cold-region straw-decomposing bacterial strain in efficient glycolysis of rice straws at low temperature, belonging to the field of separation and application of cold-region straw-decomposing bacteria.
Background
The rice straw contains rich nutrient substances such as cellulose, hemicellulose, lignin, protein, mineral elements and the like, and is a renewable biological resource. However, the utilization rate of the straws is less than 33 percent in China, and because the content of cellulose in the straws is highest, the screening of microorganisms with high cellulase enzyme production characteristics becomes one of important directions for researching straw decomposition agents.
The EM is a beneficial flora composed of various microorganisms such as photosynthetic flora, lactic acid bacteria flora, yeast flora, fermentation filamentous fungi, actinomycetes and the like, has the advantages of low cost, simple and convenient use method and the like when being applied, and is widely applied in the fields of agriculture, animal husbandry, aquatic product, water pollution and the like. According to the research, the research laboratory shows that the EM bacteria fermentation liquor is adopted to ferment the rice straws under the condition that the outdoor temperature of the northeast is between 12 ℃ below zero and 25 ℃, the effective glycolysis of the straws can be realized within 45d, the test takes decomposed substances of the EM bacteria glycolysis of the rice straws in winter in alpine regions as research objects, the decomposed bacterial strains of the rice straws in the cold regions are separated, high-efficiency strains capable of decomposing the straws under the low-temperature condition are screened out, and the identification is carried out through morphology and molecular biology; the cold resistance and fermentation influence factors of the strain are discussed, a glycolysis test is carried out, the capability of the strain for glycolyzing the rice straws at low temperature is determined, and a practical basis and a theoretical basis are provided for increasing the strain composition of the rice straw decomposing inoculant in the cold region and the application of the straw decomposing inoculant through the research.
Disclosure of Invention
The invention aims to solve the first technical problem of providing a separated cold-region straw decomposing bacterial strain;
the second technical problem to be solved by the invention is to provide the application of the cold-region straw-decomposing bacterial strain in preparing a rice straw decomposing agent and fermenting rice straws.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention separates and purifies 23 strains of cold-region rice straw decomposing bacteria from microorganisms for fermenting rice straw decomposing substances by using cold-region EM bacterial liquid. The invention further utilizes culture conditions of different temperatures to carry out primary screening on the separated low-temperature straw rotten bacterial strains, and 7 bacterial strains which can normally grow at the low temperature of 15 ℃ are screened out.
The invention further uses the capacity of decomposing cellulose of 7 strains of bacteria obtained by primary screening as a basis to re-screen the straw rotten bacteria in cold regions, and judges the capacity of the strains to generate the cellulase by observing the ratio (D/D) of the diameter of a transparent ring of the obtained bacteria in a CMC-Congo red solid culture medium to the diameter of bacterial colonies. As a result, it was found that the ratio (D/D) of the diameter of the clearing zone of the C4 strain to the diameter of the colony was 2.86, indicating that the strain C4 has a strong ability to produce cellulase and a strong ability to decompose cellulose.
The straw rotten bacteria C4 were cultured on LB solid and liquid medium, and the colony and cell morphology were observed. Morphological observation results show that the bacteria can form a single colony on a solid culture medium. The bacterial colony of the strain C4 is white, semitransparent, smooth, moist and slightly raised round and neat bacterial colony, bacilli with blunt circles at two ends and different lengths can be seen under a light lens, gram-positive bacilli are shown, and through the observation, the screened straw rotten bacterial strains are preliminarily deduced to be bacillus amyloliquefaciens. The invention designates the strain asBacillus amyloliquefaciens-C4。
The cold resistance identification result shows that,Bacillus amyloliquefaciens-C4the bacterial strains can grow at different temperatures and still have vigorous growth at a culture temperature of 5 ℃, which shows that the decomposed bacterial strains screened by the test are cold-resistant microorganisms, have strong cold resistance and can normally grow in low-temperature areas in the north.
The invention separates the cold area straw rotten bacterial strainBacillus amyloliquefaciens-C4The patent is filed with an agency for deposit with the microorganism deposit numbers: CGMCC number 15178; the classification is named as: bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens). The preservation unit: china general microbiological culture Collection center; the preservation time is 1 month and 11 days 2018; and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The invention further discloses the cold region straw rotten bacterial strainBacillus amyloliquefaciens-C4The application in glycolysis of straw, especially in glycolysis of rice straw.
The invention further discloses the cold region straw rotten bacterial strainBacillus amyloliquefaciens-C4Application in preparing straw decomposition agent.
The invention also discloses the cold region straw decomposing bacterial strainBacillus amyloliquefaciens-C4The application in preparing the straw decomposition agent for the rice in the cold region. The cold area is a low-temperature area, and the temperature range of the cold area is 0-35 ℃.
The invention also discloses a rice straw decomposing agent, which comprises: the invention relates to a cold region straw decomposing bacterial strainBacillus amyloliquefaciens-C4The fermentation broth of (1).
The invention also discloses a rice straw decomposing agent, which comprises: the invention relates to a cold region straw decomposing bacterial strainBacillus amyloliquefaciens-C4The fermentation liquid and EM bacterial liquid. Wherein the cold region straw decomposing bacterial strainBacillus amyloliquefaciens-C4The volume ratio of the fermentation liquid to the EM bacterial liquid is 1:1 and mixing.
The preparation method of the fermentation liquor of the cold-region straw decomposing bacterial strain comprises the following steps: decomposing the cold area straw into bacterial strainsBacillus amyloliquefaciens-C4Inoculating into LB liquid culture medium, culturing, and collecting bacterial liquid.
The invention adopts a nitrogen-free Hedyson inorganic salt culture medium as a basic culture medium. Wherein, the nitrogen source of the liquid culture medium is peptone, yeast extract, ammonium sulfate or urea respectively, and the adding amount of the nitrogen source is 0.2% (mass percentage); according to the volume percentage, the inoculation amount of the inoculation is 1-9%; the culture temperature is 10-40 ℃; the initial pH value of the liquid culture medium is 4-10; the culture time is 1-7 days. Preferably, the nitrogen source of the liquid culture medium is peptone, and the addition amount of the nitrogen source is 0.2% (mass percentage); the inoculation amount of the inoculation is 5 percent (volume percentage); the temperature of the culture is 25 ℃, the initial pH value of the liquid culture medium is 7, and the culture time is 3 d.
The EM bacterial liquid is not particularly limited, and commercially available EM bacterial liquids are all suitable for the invention.
The invention relates to a bacterial strain for decomposing straws in cold regionsBacillus amyloliquefaciens-C4The fermentation conditions of (a) are optimizedAnd (4) transforming. The nitrogen source optimization result shows that the activity of the strain for producing the cellulase on a culture medium with ammonium sulfate or urea as a nitrogen source is low, the activity of the strain for producing the cellulase on a culture medium with peptone or yeast extract as the nitrogen source is high, the activity of the strain for producing the cellulase on the culture medium with peptone as the nitrogen source is highest, and the activity difference of the strain for producing the cellulase on different nitrogen source culture media is obvious. The invention determines that the best nitrogen source suitable for fermentation culture of the cold-region straw decomposing bacteria is peptone. The result of the inoculation amount optimization shows that the strain has higher cellulase activity within the range of 1-3%, the highest enzyme production activity is achieved when the inoculation amount is 5%, and the enzyme activity begins to decrease after the inoculation amount exceeds 5%; and comprehensively comparing, and determining that the optimal inoculation amount of the cold area straw rotten bacterial fermentation is 5%. The culture temperature optimization result shows that the enzyme activity of the strain is gradually increased at the temperature of 10-25 ℃ and is highest at the temperature of 25 ℃; as the temperature continued to rise, the enzyme production activity gradually decreased, and the enzyme production activity of the strain at 40 ℃ decreased to the minimum. The invention determines that the optimal temperature for fermenting the straw rotten bacteria in the cold area is 25 ℃. The initial pH value optimization result of the culture medium shows that the initial pH value is the bacteria for decomposing the straws in the cold areaBacillus amyloliquefaciens-C4The influence of the activity of the strain for producing the cellulase is large; peracid or overbase, the enzyme activity is very low, and the cellulase activity can hardly be detected; the cellulase production activity of the strain is shown to be higher when the initial pH of the culture medium is in the range of 5-8, the cellulase activity is highest when the initial pH is 7, and the cellulase production activity of the strain is in a descending trend when the initial pH of the culture medium is increased to 8. Therefore, the invention determines that the acidic and neutral solution environment is favorable for producing enzyme, and the optimal initial pH value of the fermentation of the cold-region straw decomposing bacteria is 7. The result of the optimization of the culture time shows that the cold area straw decomposing bacteriaBacillus amyloliquefaciens-C4The enzyme production activity of the strain shows a trend of rapidly increasing and then slowly decreasing along with the extension of the culture time, the strain has the best enzyme production activity when cultured for 3d, and the CMC enzyme activity is gradually reduced after 3d, but still has stronger enzyme activity. The invention determines that the optimal culture time for producing the cellulase by the cold region straw rotten bacteria is 3 d.
Straw for cold regionsThe result of the analysis of the glycolysis effect of the decomposing bacteria on the filter paper shows that,Bacillus amyloliquefaciens-C4the strain can effectively ferment the filter paper, the weight loss rate of the filter paper is obviously increased 1-3 days after treatment, the decomposition rate of the filter paper is in a slow rising trend after 5 days, and the decomposition rate of the straw filter paper reaches over 81.8% at 15 days. The results show that the straw decomposing bacteria screened by the inventionBacillus amyloliquefaciens-C4Has strong filter paper glycolysis capability.
The invention adopts bacteria under the condition of low temperatureBacillus amyloliquefaciens-C4The straw glycolysis test of the rice straw is carried out by mixing the composite fermentation liquid with the EM (effective microorganism) fermentation liquid (mixing according to the volume ratio of 1: 1), and the rice straw is glycolyzed at a low temperature for 30d, so that the result shows that the straw glycolysis efficiency of the composite fermentation liquid is improved by 48.8 percent compared with the treatment only with the EM fermentation liquid.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the high-efficiency bacterial strain obtained by the invention can be used for fermenting decomposed rice straws under the condition of low temperatureBacillus amyloliquefaciens-C4The bacillus amyloliquefaciens is identified as the psychrophile and has better rice straw glycolysis effect. The invention provides practical basis and theoretical basis for increasing the strain composition of the straw decomposition inoculant in the cold area and the application of the straw decomposition inoculant.
Drawings
FIG. 1 shows cold-region straw decomposing bacteria on CMC-Congo red mediumBacillus amyloliquefaciens-A transparent ring of C4;
FIG. 2 shows bacteria for decomposing straw in cold regionBacillus amyloliquefaciens-Morphological identification of C4; wherein A is the colony morphology of the C4 strain; b is the microscopic form of the C4 strain;
FIG. 3 shows the strain of straw in cold areaBacillus amyloliquefaciens-C4 phylogenetic tree constructed based on 16SrDNA sequence homology;
FIG. 4 shows the bacteria for decomposing straw in cold area by different nitrogen sourcesBacillus amyloliquefaciens-C4Influence of the strain on enzyme production;
FIG. 5 shows the bacteria with different inoculation amounts for straw decomposition in cold regionsBacillus amyloliquefaciens-C4The effect of enzyme production;
FIG. 6 shows straw decomposing bacteria at different temperatures in cold regionsBacillus amyloliquefaciens-C4The effect of enzyme production;
FIG. 7 shows the initial pH of the medium decomposing the straw in cold regionsBacillus amyloliquefaciens-C4The effect of enzyme production;
FIG. 8 shows bacteria for decomposing straw in cold area during cultivationBacillus amyloliquefaciens -C4The effect of enzyme production;
FIG. 9 shows bacteria for decomposing straw in cold regionBacillus amyloliquefaciens-C4Influence on the decomposition rate of filter paper.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 separation and identification of straw-decomposing bacteria in Cold region
1. Test materials and methods
1.1 test materials
The EM bacterial liquid used in the invention is provided by agricultural academy of Heilongjiang province. The rice straw is collected from the experimental field of northeast agriculture university.
Test method
2.2.1 Cold region stalk EM fungus rotten
In 2016, 12 months, in a test field of northeast agriculture university of Harbin, Heilongjiang province, the outdoor temperature is between minus 12 ℃ and 25 ℃, the mixture of the crushed rice straws and the EM bacterial liquid is buried in a deep groove, and the mixture is covered by untreated straws and plastic cloth and decomposed for 60 days. The taken-out straw rotten matter is in a rotten state, and strip-shaped or small flake-shaped straw fragments which are not completely rotten are occasionally seen.
Separation of cold region straw decomposing bacteria
2.2.2.1 isolation, Primary screening and preservation of strains from straw-decomposing Agents
(1) Separation of bacterial strains in the decomposed product: weighing 5 g of straw rotten matter, adding 50 mL of sterile water into a conical flask, and culturing for 24h on a shaking table at 37 ℃. And dipping the suspension supernatant of the decomposed product by using a strain inoculating ring, streaking on a strain isolation culture medium, and inoculating the strain isolation culture medium after single bacteria of the bacteria are separated on an LB solid culture medium.
(2) Primary screening of strains in the decomposed product: the cold-region straw decomposing bacteria can grow under the low-temperature condition, and the enriched and separated strains are primarily screened at different temperatures. Culturing the separated strains under the temperature conditions of 4 ℃, 10 ℃, 15 ℃, 25 ℃, 37 ℃ and the like, observing the growth condition of the strains, and screening out the strains which can normally grow under the low temperature condition.
(3) And (3) preservation of the strain: and (3) inoculating the separated bacteria in an LB liquid culture medium, culturing for 24h at 37 ℃, and placing the bacterial liquid in 50% glycerol which is sterilized by high pressure and stored at-80 ℃.
Double-screening of cold-region straw rotten fungi
And re-screening the straw rotting fungi in the cold area based on whether the microorganisms obtained by low-temperature screening have the capacity of decomposing cellulose. Inoculating the bacteria obtained by primary screening and capable of growing under the condition of low temperature on a CMC-Congo red culture medium, observing the size of a transparent ring on the culture medium, and calculating the ratio (D/D) of the transparent ring to the diameter of the strain. The size of a transparent ring of the strain on a CMC-Congo red solid culture medium can reflect the capacity of the strain to decompose cellulose to a certain extent, and the larger the transparent ring is, the stronger the capacity of the strain to decompose cellulose is.
Identification of straw decomposing bacteria in cold region
2.2.3.1 morphological identification of straw rotten bacteria in cold region
And respectively inoculating the re-screened bacterial strains into LB solid and LB liquid culture media, observing the appearance and the morphology of the bacterial colonies after 24 hours, and classifying and identifying the bacteria by adopting a gram staining method.
Molecular identification of straw decomposing bacteria in cold region
Inoculating the purified single colony from an LB plate culture medium into a 30 mL LB liquid culture medium, culturing at 37 ℃ for 24h, carrying out PCR amplification by adopting DNA of SDS bacteria and using 16SrDNA sequence universal primers 27F/541R identified by the bacteria as molecular identification primers of the cold-region straw-rotten bacteria, and sequencing the PCR product to Harbin Boshi biotechnology Limited. And (3) adopting MEGA5.0 software to construct a phylogenetic tree and carrying out molecular identification on the bacterial species.
Cold resistance identification of straw rotten bacteria in cold region
Respectively inoculating the straw rotten bacteria obtained by early screening and identification to LB solid and LB liquid culture media, recording the growth conditions of the strains under the culture conditions of 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃, and determining the cold resistance of the screened straw rotten bacteria.
Fermentation condition optimization of cold region straw decomposing fungi
2.2.5.1 influence of different nitrogen sources of culture medium on fermentation of decomposing bacteria
In order to study the influence of different nitrogen sources of the culture medium on the fermentation of the mature bacteria, the experiment adopts a nitrogen-free Hedyson inorganic salt culture medium as a basic culture medium, different nitrogen sources are added on the basis, the added nitrogen sources are peptone, yeast extract, ammonium sulfate and urea respectively, the adding amount is 0.2 percent (mass percent), and 3 times of treatment are carried out for 4 bottles. When the cells were cultured at 25 ℃ for 3 days, 0.2 mL of the cell suspension was taken and used for the cellulase activity measurement, and the measurement method was the DNS method.
Influence of strain inoculation amount on fermentation of decomposed bacteria
In order to study the influence of different strain inoculum sizes on fermentation of the decomposing bacteria, different strain inoculum sizes were designed in the experiment. Five inoculum (volume percent) treatments of 1%, 3%, 5%, 7% and 9% were set. Inoculating into liquid fermentation medium with peptone as nitrogen source, wherein the initial pH value of the medium is 7.0, culturing at 25 ℃, treating 5 bottles each, and repeating for 3 times. When the cells were cultured for 7 days, 0.2 mL of each cell suspension was used for cellulase activity measurement.
Influence of culture temperature on fermentation of decomposing bacteria
In order to study the influence of different culture temperatures on the cellulase activity of the decomposed bacteria, different strain culture temperatures were designed in the experiment. Set up 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ total 7 culture temperature treatment. Inoculating the culture medium with 5% inoculum size in liquid fermentation medium with peptone as nitrogen source, wherein the initial pH value of the culture medium is 7.0, and each 7 bottles are treated for 3 times. When the cells were cultured for 7 days, 0.2 mL of each cell suspension was used for cellulase activity measurement.
Influence of initial pH value of culture medium on fermentation of decomposing bacteria
In order to study the influence of the initial pH values of different culture media on the fermentation of the mature bacteria, different initial pH values of the culture media are designed in the experiment. 4, 5, 6, 7, 8, 9 and 10 different culture medium initial pH value treatments are set. Taking a liquid fermentation culture medium with peptone as a nitrogen source as a basic culture medium, adjusting the initial pH of the culture medium, inoculating with an inoculum size of 5%, culturing at 25 ℃, treating 7 bottles each, and setting 3 times of repetition. When the cells were cultured for 7 days, 0.2 mL of each cell suspension was used for cellulase activity measurement.
Influence of culture time on fermentation of decomposing bacteria
In order to study the effect of different culture times on fermentation of the decomposing bacteria, different culture times were designed in this experiment. The treatment time of 1d, 2d, 3d, 4 d, 5d, 6 d and 7d is set to be 7 different. Liquid fermentation medium with peptone as nitrogen source, initial pH 7, inoculation with 5% inoculum size, 25 ℃ culture, 7 flasks per treatment, 3 replicates. After culturing according to the culture time designed by the experiment, respectively taking 0.2 mL of bacterial liquid for determining the activity of the cellulase.
Fermentation test of straw decomposition bacteria in cold region
2.2.6.1 Filter paper glycolysis test of straw-decomposing bacteria
Inoculating the screened straw rotten bacteria into LB liquid culture containing filter paper with the initial pH of a culture medium of 7 according to the inoculum size of 5%, and culturing at 15 ℃. After inoculation, the weight loss rate of the filter paper was measured every 24h, and 8 d was continuously measured. Each treatment was 8 vials, with 3 replicates. The method for measuring the weight loss rate of the filter paper comprises the steps of filtering the fermentation liquor, drying the residual substances in an oven at 80 ℃ to constant weight, and measuring the weight loss rate of the filter paper.
Low-temperature efficient glycolysis straw bacterium and EM bacterium compound glycolysis straw test
Mixing fermented low-temperature efficient glycolysis straw bacterium fermentation liquor with EM bacterium fermentation liquor in a ratio of 1:1, and glycolysis rice straws at the temperature of 4 ℃. The rice straw treatment method comprises the following steps: soaking the rice straw sections in 2 mol/L NaOH solution for 24h, washing with water until the pH value is =7, drying at 80 ℃ in a drying box, crushing the dried straw sections into 100 meshes, and screening for later use. According to the fermentation composition, 5% of EM (effective microorganisms) fermentation liquor, 5% of low-temperature high-efficiency glycolysis straw bacteria fermentation liquor and 1% of brown sugar are added according to the amount of fermented rice straws, the humidity is controlled to be 35% -65%, and the fermentation is carried out for 30d by taking the EM fermentation liquor as a reference. And (3) after the fermentation is finished, determining the weight loss rate of the straw by the same method as the method for determining the weight loss rate of the filter paper.
Test results
3.1 separation of straw-decomposing bacteria in Cold regions
3.1.1 Primary Screen of straw rotten fungus in Cold region
The microorganisms in the cold area straw decomposed substances are separated and purified for 6 times, and 23 bacterial strains are obtained in total. Culturing the separated strain under different temperature culture conditions, observing the growth condition of the strain, and primarily screening the low-temperature straw rotting fungi. The results show that the selected bacteria capable of normally growing under the low temperature condition of 15 ℃ are 7 strains, namely A3, C4, C1, C4, D7, E5 and E14.
Double-screening of cold-region straw decomposing bacteria
And (3) rescreening 7 strains of primarily screened bacterial strains capable of normally growing under a low-temperature condition, wherein the rescreening is mainly performed through the diameter of a transparent ring on the CMC-Congo red solid culture medium. The ratio (D/D) of the diameter of the transparent ring to the diameter of the colony in the CMC-Congo red solid culture medium of the bacteria obtained by screening is observed, wherein C4 has a larger ratio (D/D) of the diameter of the transparent ring to the diameter of the colony, which is 2.86, and the size of the transparent ring indicates the capability of the strain to produce cellulase to a certain extent, and indicates that the strain C4 has stronger glycolysis capability (see figure 1).
Morphological observation of cold region straw rotten fungi
Culturing the screened straw rotten bacteria C4 on LB solid and liquid culture medium, and observing colony and thallus morphology. The results show that the bacteria can form a single colony on the solid culture medium. The bacterial colony of the strain C4 is white, semitransparent, smooth and moist, slightly raised round and neat bacterial colony, and bacilli with blunt ends and different lengths can be seen under a light lens (see figure 2-A). The bacterial strains are presented as gram positive (see figure 2-B), and through the observation, the screened straw rotting bacterial strains are preliminarily deduced to be bacillus.
Molecular identification of straw decomposing bacteria in cold region
And (3) carrying out molecular biological identification on the screened mature bacterium C4 by bacterial 16SrDNA sequence analysis, wherein the length of the amplified 16SrDNA sequence fragment is 1449 bp. BLAST alignment and phylogenetic tree construction were performed on the obtained sequences, and the results are shown in FIG. 3. According to the result of phylogenetic tree, C4 is determined to be Bacillus pumilus (B.) (Bacillus amyloliquefaciens) This result, consistent with the morphological identification described above, was namedBacillus amyloliquefaciens-C4。
The invention will separateBacillus amyloliquefaciens-C4The strain is submitted to China general microbiological culture Collection center for preservation, and the preservation number of the microorganism is as follows: CGMCC number 15178.
Cold resistance identification of straw rotten bacteria in cold region
Obtained by screeningBacillus amyloliquefaciens-C4And (5) carrying out cold resistance identification on the strain. The screened decomposed bacteria are inoculated in a LB culture medium and cultured at different culture temperatures, and the result shows that the strain can grow at different temperatures, and still has vigorous growth force under the condition of 5 ℃, which shows that the decomposed bacteria have stronger cold resistance. The decomposed strain obtained by screening in the test is a cold-resistant microorganism and can normally grow in the low-temperature area in the north.
Fermentation condition optimization of straw decomposing bacteria in cold region
3.5.1 optimal Nitrogen Source selection
Decomposing bacteria for screened cold area strawBacillus amyloliquefaciens-C4And (4) screening the optimal nitrogen source for fermentation. The results are shown in FIG. 4, and it can be seen from the results that eggs are usedThe activity of producing cellulase on a nitrogen source culture medium with the white peptone and the yeast extract is high, while the activity of producing cellulase on a nitrogen source culture medium with the added ammonium sulfate and urea is low, and the difference is obvious. And (3) according to the comprehensive comparison result, the optimal nitrogen source suitable for culturing the cellulase produced by the straw rotten bacteria in the cold region is peptone.
Effect of inoculum size on fermentation
The optimal inoculation amount of the screened cold-region straw rotten bacteria for fermentation is screened, and the result is shown in fig. 5. The results show that the activity of producing the cellulase by liquid fermentation of the strain can be influenced by different inoculation amounts, the activity of producing the cellulase by straw-decomposing bacteria tends to increase firstly and then decrease with the increase of the inoculation amounts, and the bacteria have higher cellulase activity. When the inoculation amount is 1% and 3%, the enzyme production and the enzyme activity are gradually increased. When the inoculation amount exceeds 5%, the enzyme activity begins to decrease, probably because the bacterium content is too much, the nutrient substances in the environment are limited, and the enzyme production capacity is reduced because the nutrient substances are consumed by the growth of the thalli in large quantity. The results of the overall comparison show that the optimal inoculation amount is 5%.
Influence of temperature on the fermentation
As can be seen in FIG. 6, the strainsBacillus amyloliquefaciens-C4The enzyme activity is gradually increased at the temperature of 10-30 ℃, the enzyme activity is highest at the temperature of 30 ℃, but the enzyme production activity is not obviously different from that at the temperature of 25 ℃. The enzyme production activity began to decrease with increasing temperature, and at 40 ℃, the enzyme production activity of the strain decreased to the lowest. From these results, it can be seen that the strains selected in this test have a wide enzyme-producing activity range, and the enzyme production is more facilitated at low temperature. And (4) combining the comparison results to determine that the optimal fermentation temperature of the strain is 25 ℃.
Effect of initial pH of the Medium on fermentation
As can be seen from FIG. 7, the straw-decomposing bacteria in the peracid and over-alkali environmentBacillus amyloliquefaciens-C4The CMC has very low enzyme activity and even does not produce enzyme. The mature bacteria have enzyme-producing activity at pH 5 to 8, have the highest CMC enzyme activity at initial pH 7 of the culture medium, and have significantly reduced enzyme-producing activity when pH is increased to 8,the strain produces no enzyme at all. These results indicate that the low temperature straw-decomposing strain screened in this test is more suitable for producing enzyme under acidic and neutral conditions, and the optimal culture medium initial pH value is determined to be 7.
3.5.5 Effect of incubation time on enzyme production
As can be seen from figure 8 of the drawings,Bacillus amyloliquefaciens-C4the enzyme-producing activity of the strain shows a trend of increasing firstly and then decreasing with the extension of the culture time. When the cells are cultured for 3d, the cells have the best enzyme production activity, and the CMC enzyme activity is gradually reduced after 3d, but still has stronger enzyme activity. And determining the optimal culture time of the cold-region straw decomposing bacteria fermentation to be 3 d.
Analysis of glycolysis effect of straw decomposing bacteria in cold region
3.6.1 glycolysis Effect of bacteria on Filter paper
In order to verify the glycolysis effect of the screened straw decomposing bacteria in the cold region, a filter paper glycolysis test is carried out by adopting the decomposing bacteria. The decomposition rate of the filter paper after the bacterial treatment is shown in FIG. 9. As can be seen from the results, the use ofBacillus amyloliquefaciens-C4After the strain is treated for 1-3 days, the weight loss rate of the filter paper is obviously increased along with the prolonging of the treatment time, the decomposition rate of the filter paper after 5 days is in a slow rising trend, the maximum value is reached at 15 days, the decomposition rate is 81.8%, and the results show that the straw decomposing bacteria screened by the test have strong filter paper glycolysis effect.
Low-temperature efficient glycolysis straw bacterium and EM bacterium combined glycolysis straw test
Under the condition of low temperature, the straw is efficiently fermented and decomposed at low temperatureBacillus amyloliquefaciens-C4The glycolysis test of the rice straw is carried out by matching with EM (effective microorganisms) bacteria, the straw has weight loss and blackening phenomena in two treatments within 30d, but the weight loss and blackening degrees of the straw are larger in the combination with the low-temperature high-efficiency glycolysis straw bacteria, the weight loss rate of the rice straw is measured, the weight loss rate of the combination with the low-temperature high-efficiency glycolysis straw bacteria is 61%, while the weight loss rate of the straw is 41% in the treatment with only EM (effective microorganisms) fermentation liquor, which indicates that the low-temperature high-efficiency glycolysis straw bacteria is subjected to glycolysisBacillus amyloliquefaciens-C4Mixing with EM bacteria, and fermenting with strawThe decomposition efficiency is improved by 48.8 percent, and low-temperature efficient zymolysis straw bacteria are added in the straw decomposition process, so that the straw is better in rotten degree and easier to break when fermented for 30 days.
Claims (18)
1. Separated bacillus amyloliquefaciens for low-temperature glycolysis of strawsBacillus amyloliquefaciens) The strain is characterized in that the microorganism preservation number is as follows: CGMCC No. 15178.
2. A method of preparing a fermentation broth of the bacillus amyloliquefaciens strain of claim 1, comprising: inoculating the strain of claim 1 into a fermentation culture medium for fermentation culture, and collecting the fermentation liquid.
3. The method of claim 2, wherein: the nitrogen source of the fermentation medium is peptone, yeast extract, ammonium sulfate or urea, and the addition amount of the nitrogen source is 0.2 wt%.
4. A method according to claim 3, characterized by: the nitrogen source is peptone.
5. The method of claim 2, wherein: inoculating the strain of claim 1 in an amount of 1-9% by volume into a fermentation medium for culturing.
6. The method of claim 5, wherein: the strain of claim 1 is inoculated into a fermentation medium at an inoculum size of 3-9% for culture.
7. The method of claim 6, wherein: the strain of claim 1 is inoculated into a fermentation medium at an inoculum size of 5% for culture.
8. The method of claim 2, wherein: the fermentation culture temperature is 10-40 ℃.
9. The method of claim 8, wherein: the fermentation culture temperature is 20-30 ℃.
10. The method of claim 9, wherein: the fermentation culture temperature is 25 ℃.
11. The method of claim 2, wherein: the initial pH of the fermentation medium is 4-10.
12. The method of claim 11, wherein: the initial pH of the fermentation medium is 5-8.
13. The method of claim 12, wherein: the initial pH of the fermentation medium was 7.
14. The method of claim 2, wherein: the time of fermentation culture is 1-7 days.
15. The method of claim 14, wherein: the time of fermentation culture is 3 d.
16. The decomposition agent for glycolysis of straws is characterized by comprising: the bacterial agent of the bacillus amyloliquefaciens strain or the fermentation liquid thereof and the EM bacterial solution of the bacillus amyloliquefaciens strain of claim 1; wherein the volume ratio of the two is 1: 1.
17. The use of the bacillus amyloliquefaciens strain or the fermentation liquid thereof as claimed in claim 1 in the glycolysis of straws.
18. The use of the decomposing agent of claim 16 in the glycolysis of straw.
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