CN108865927B - Bacterial strain for low-temperature glycolysis of corn straw and fermentation culture method and application thereof - Google Patents

Bacterial strain for low-temperature glycolysis of corn straw and fermentation culture method and application thereof Download PDF

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CN108865927B
CN108865927B CN201810591553.7A CN201810591553A CN108865927B CN 108865927 B CN108865927 B CN 108865927B CN 201810591553 A CN201810591553 A CN 201810591553A CN 108865927 B CN108865927 B CN 108865927B
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straw
fermentation
bacillus pumilus
bacteria
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CN108865927A (en
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李凤兰
徐媛媛
徐永清
冯艳忠
杨秀梅
袁强
王丽娟
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Northeast Agricultural University
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Abstract

The invention discloses a bacterial strain for low-temperature glycolysis of corn straw, a fermentation culture method and application thereof. The invention firstly discloses an isolated bacillus pumilus strain, which has the following microorganism preservation number: CGMCC No. 15177. The invention optimizes the relevant parameters of the fermentation preparation method of the separated bacillus pumilus strain. The bacillus pumilus separated by the invention can grow at different temperatures and still has vigorous growth power at a low temperature of 4 ℃. The separated bacterial strain can ferment straw efficiently under low temperature condition, and can be applied to the fermented straw independently or in combination with EM (effective microorganisms). After the fermentation liquid of the strain is mixed with the EM bacterial liquid, the corn straw is fermented at low temperature, the straw fermentation effect is remarkably improved, and the fermentation rate is improved by 47.4%. The bacillus pumilus strain separated by the invention has wide application prospect in preparing straw decomposition agents, in particular corn straw decomposition agents.

Description

Bacterial strain for low-temperature glycolysis of corn straw and fermentation culture method and application thereof
Technical Field
The invention relates to a separated cold region corn straw decomposing bacteria strain, and also relates to application of the cold region corn straw decomposing bacteria strain in efficient glycolysis of corn straws at low temperature, belonging to the field of separation and application of cold region straw decomposing bacteria.
Background
The crop straw contains rich nutrient substances such as cellulose, hemicellulose, lignin, protein, mineral elements and the like, and is a renewable biological resource. But the utilization rate of the Chinese straws is less than 33 percent, mainly because the straws are difficult to collect in the field and inconvenient to transport, and most of the straws are incinerated or discarded. The Heilongjiang province is a big province of agriculture in China, the quantity of straws produced every year is huge, and because the straws are burnt or abandoned due to low environmental temperature and low returning condition and popularization degree, not only are precious crop resources wasted, but also huge pollution is caused to the environment. The straws are mainly rich in cellulose, hemicellulose and lignin, and a plurality of fungi, bacteria, actinomycetes and the like can secrete cellulase, hemicellulase and lignin enzyme to decompose the straws. Because the content of cellulose in the straws is highest, screening 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. Many test results prove that the EM bacteria have the microorganisms capable of efficiently decomposing the straw cellulose, and the microorganisms can also normally play a role under the low-temperature condition, so that the EM bacteria are utilized to screen the cold-region straw decomposing bacteria to screen the efficient strains capable of decomposing the straw under the low-temperature condition, and practical basis and theoretical basis are provided for increasing the strain composition of the cold-region straw decomposing bacteria and the application of the straw decomposing agent.
Disclosure of Invention
The invention aims to solve the first technical problem of providing a separated bacterial strain for efficiently fermenting corn straws at low temperature;
the second technical problem to be solved by the invention is to provide the application of the cold-region straw-decomposing bacterial strain in efficient glycolysis of the corn straw at low temperature.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention separates and purifies the microorganism in the straw decomposed material in the cold area for 6 times to obtain 23 bacterial strains in total; culturing 26 separated strains under different temperature culture conditions, observing the growth conditions, performing primary screening on low-temperature straw rotting bacteria, and screening 7 strains of bacteria capable of normally growing at the low temperature of 15 ℃, namely A3, B7, C1, C4, D7, E5 and E14. The invention further carries out secondary screening on 7 strains of bacteria which are preliminarily screened and can normally grow under the low-temperature condition, the ratio (D/D) of the diameter of the transparent ring to the diameter of the bacterial colony in the CMC-Congo red solid culture medium of the bacteria obtained by observation and screening is observed, and the result shows that B7 strain has a larger ratio (D/D) of the diameter of the transparent ring to the diameter of the bacterial colony, and is 2.68; as the size of the transparent ring can reflect the capability of the strain to produce cellulase to a certain extent, the screened strain B7 has stronger capability of producing cellulase.
The invention observes the bacterial colony and the thallus form of the screened straw rotten bacteria B7. As a result, it was found that the bacterium was able to form a single colony on a solid medium. The bacterial colony of the bacterial strain B7 is a round bacterial colony which is yellow, semitransparent, smooth and moist, irregular in edge and slightly raised, the thallus is in the shape of a thin rod, the tail end of the circle is single or short-chain arranged, the thallus is gram positive, and the screened straw rotten bacterial strain B7 is preliminarily deduced to be bacillus through the observation. According to the result of the phylogenetic tree, B7 was determined to be Bacillus pumilus (Bacillus pumilus), and the strain was named Bacillus pumilus-B7.
According to the invention, the screened Bacillus pumilus-B7 strain is subjected to cold resistance identification, and the result shows that the strain can grow at different temperatures and still has vigorous growth force at 4 ℃, which indicates that the decomposed strain has strong cold resistance. Morita states that cold-tolerant microorganisms are characterized by the ability to grow and reproduce at temperatures between 0 ℃ and 5 ℃ with optimum growth temperatures above 15 ℃ and maximum growth temperatures above 20 ℃. Therefore, the strain Bacillus pumilus-B7 screened by the invention is a cold-resistant microorganism and can normally grow in northern low-temperature areas.
The invention submits the separated Bacillus pumilus-B7 strain to an approved patent organization for preservation, and the microorganism preservation numbers are as follows: CGMCC No. 15177; the classification is named as: bacillus pumilus strain. 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 preparation of the fermentation liquor of the Bacillus pumilus-B7 strain comprises the following steps: inoculating the Bacillus pumilus-B7 strain into a liquid culture medium for culture, and collecting a bacterial liquid to obtain the Bacillus pumilus-B7 strain.
Wherein, the liquid culture medium can be a Hexon's inorganic salt culture medium as a basic culture medium, the nitrogen source can be peptone, yeast extract, ammonium sulfate or urea, and the addition amount is 0.2 wt%; preferably, the nitrogen source is peptone; the inoculation amount of the strain can be 1-9% by volume percentage, and preferably, the inoculation amount of the strain is 5%; the culture temperature may be 10-40 ℃, preferably 25 ℃; the initial pH of the liquid medium may be 4 to 10, preferably 7; the time of the fermentation culture can be 1-7d, and is preferably 3 d.
The optimal nitrogen source screening experiment of the fermentation medium shows that the activity of producing the cellulase on the culture medium taking peptone and yeast extract as nitrogen sources is higher, while the activity of producing the cellulase on the crop nitrogen source culture medium added with ammonium sulfate and urea is lower, and the difference is obvious. And (3) according to the comprehensive comparison result, the best nitrogen source suitable for the culture of cellulase produced by the cold-region straw decomposing bacterium Bacillus pumilus-B7 strain is peptone.
The screening result of the optimal inoculation amount for fermentation shows that the activity of cellulase produced by liquid fermentation of the strain can be influenced by the difference of the inoculation amounts, the activity of cellulase produced 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 bacteria content is too much, nutrient substances in the environment are limited, and the enzyme production capacity is reduced because the bacteria grow to consume a large amount of nutrient substances. The results of the overall comparison show that the optimal inoculation amount is 5%.
The fermentation temperature screening experiment results show that the enzyme activity of the strain Bacillus pumilus-B7 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 ℃. As the temperature increased, the enzyme production activity began to decrease, and at 40 ℃, the enzyme production activity of the strain decreased to the minimum. 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) comprehensively comparing results, and determining that the optimal fermentation temperature of the strain is 25 ℃.
The screening experiment result of the optimal initial pH value of the culture medium shows that the CMC enzyme activity of the straw-decomposing bacteria in the peracid and over-alkali environment is very low, even no enzyme is produced. The rotten bacteria have enzyme-producing activity at a pH of 5 to 8, have the highest CMC enzyme activity at an initial pH of 7 of the culture medium, and show a significant decrease in enzyme-producing activity when the pH is increased to 8. These results indicate that the low temperature straw-decomposing strain screened in this experiment is more suitable for producing enzyme under acidic and neutral conditions, and the optimal initial pH of the culture medium is determined to be 7.
The screening experiment result of the optimal culture time shows that the enzyme production activity of the strain is in a trend of increasing firstly and then decreasing along with the extension of the culture time. When the CMC is cultured for 3d, the CMC has the best enzyme activity, and the CMC has stronger enzyme activity after 3 d. And determining the optimal culture time of the cold region straw rotten bacteria fermentation to be 3 d.
In order to verify the glycolysis effect of the screened low-temperature efficient glycolysis straw bacterial strain Bacillus pumilus-B7, the filter paper glycolysis test is carried out by adopting decomposed bacteria. According to the filter paper glycolysis test result, the screened straw decomposing bacteria strain Bacillus pumilus-B7 has a strong filter paper glycolysis effect.
According to the invention, low-temperature efficient glycolysis straw bacteria Bacillus pumilus-B7 are further matched with EM bacteria for carrying out glycolysis straw tests, in 30d, the straw has weight loss and blackening phenomena, but in the combination of low-temperature efficient glycolysis straw bacteria Bacillus pumilus-B7, the degree of straw weight loss and blackening is larger, the weight loss rate of the corn straw is measured, the weight loss rate of the combination of low-temperature efficient glycolysis straw bacteria Bacillus pumilus-B7 is 56%, and only the EM bacteria fermentation liquor is used for processing, the weight loss rate of the straw is 38%, the glycolysis rate is increased by 47.4%, and the low-temperature efficient glycolysis straw bacteria Bacillus pumilus-B7 is added in the straw rotting process, so that the straw has better rotting degree and is easy to break when fermented for 30 d. Therefore, the Bacillus pumilus-B7 strain separated by the method can ferment straw efficiently at low temperature, and can be applied to fermenting crop straws, especially corn straws.
The invention further discloses application of the Bacillus pumilus-B7 strain in preparation of a straw decomposing inoculant.
The invention also discloses a rice straw decomposing agent, which comprises: the invention relates to fermentation liquor of a Bacillus pumilus-B7 strain.
The fermentation liquor of the Bacillus pumilus-B7 strain separated by the method can be independently applied to fermenting straws, and can also be applied to glycolysis by matching with EM (effective microorganisms) bacteria liquid, so that the glycolysis rate of the straws is remarkably improved.
The invention also discloses a corn straw decomposing inoculant, which comprises the following components: the invention relates to fermentation liquor of a Bacillus pumilus-B7 strain and EM bacterial liquid. Preferably, the fermentation liquor of the Bacillus pumilus-B7 strain and the EM bacterial liquor are mixed according to the volume ratio of 1: 1.
The EM bacterial liquid is not particularly limited, and commercially available EM bacterial liquids are all suitable for the invention.
The separated Bacillus pumilus-B7 bacterial strain can grow at different temperatures, still has vigorous growth force and strong cold resistance at the temperature of 4 ℃, and can efficiently ferment and decompose straws at low temperature; can be applied to glycolysis of straws independently or in combination with EM bacteria; the fermentation liquid of the Bacillus pumilus-B7 strain is mixed with the EM fermentation liquid and then the corn straw is glycolyzed at low temperature, the glycolysis effect of the straw is obviously improved, and the glycolysis rate is improved by 47.4 percent.
Drawings
FIG. 1 is a transparent circle of decomposed bacteria on CMC-Congo red medium;
FIG. 2 shows colony morphology (A) and microscopic morphology (B) of decomposed bacteria;
FIG. 3 is a phylogenetic tree constructed based on 16SrDNA sequence homology for strain B7;
FIG. 4 shows the effect of different nitrogen sources on the enzyme production of a strain;
FIG. 5 is a graph of the effect of different inoculum sizes on strain fermentation;
FIG. 6 is a graph of the effect of different temperatures on the fermentation of a strain;
FIG. 7 is a graph showing the effect of initial pH of different media on enzyme production by 3 strains;
FIG. 8 is a graph showing the effect of fermentation at different incubation times;
FIG. 9 shows the decomposition rate of the strain on 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 intended as limitations on 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 isolation and identification of straw-decomposing fungi in Cold regions
1. Test method
1.1 test materials
1.1.1 strains and straws
The EM bacterial liquid used in the test is provided by farm academy of Heilongjiang province. Corn stalks were harvested from the experimental field of northeast university of agriculture.
1.2 test methods
1.2.1 Cold region stalk EM fungus rotten
In 2016, 12 months, in the experimental field of northeast agriculture university of Harbin, Heilongjiang province, the outdoor temperature is between-12 ℃ and 25 ℃, the mixture of the crushed straws and the EM bacterial liquid is buried in a deep groove, and 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.
1.2.2 separation of straw-decomposing bacteria in Cold regions
1.2.2.1 separation, Primary screening and preservation of bacterial strains in straw-decomposed products
(1) Separation of bacterial strains in the decomposed product: weighing 5g of straw rotten matter, adding 50mL of sterile water into a conical flask, and culturing for 24h on a shaking table at 37 ℃. And (3) dipping the decomposed material suspension supernatant by using a bacteria inoculating ring, streaking on a strain isolation culture medium, inoculating the bacteria on an LB agar culture medium after single bacteria are separated, and storing.
(2) Primary screening of bacterial strains in the decomposed product under low temperature conditions: 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 strain at 4 deg.C, 10 deg.C, 15 deg.C, 25 deg.C and 37 deg.C, observing the growth condition of the strain, and screening out the strain capable of normally growing at low temperature.
(3) And (3) preservation of the strain: and classifying the separated single colonies, culturing the bacteria in an LB liquid culture medium at 37 ℃ for 24h, taking a bacterial liquid, placing the bacterial liquid in 50% glycerol which is sterilized by high pressure, and storing at-80 ℃.
1.2.2.2 rescreening of straw rotten fungi in cold areas
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. And (3) inoculating the bacteria which are obtained by primary screening and can grow under the low-temperature condition on a CMC-Congo red culture medium, and culturing until the strains grow. The size of the transparent circle on the medium was observed, and the ratio (D/D) of the transparent circle to the diameter of the strain was calculated. 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.
1.2.3 identification of bacteria decomposing straw in Cold region
1.2.3.1 morphological identification of straw-decomposing bacteria in cold regions
And respectively inoculating the re-screened bacterial strains into LB solid culture medium and LB liquid culture medium, 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.
1.2.3.2 molecular identification of bacteria decomposing straw in Cold region
Inoculating the purified single colony from an LB plate culture medium into a 30mL LB 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. Inputting the sequence obtained by sequencing into GenBank, utilizing BLAST comparison to make sequence homology analysis, adopting MEGA5.0 software to construct phylogenetic tree and making molecular identification of bacterial species.
1.2.4 Cold resistance identification of straw-decomposing bacteria in Cold region
Respectively inoculating the straw-decomposing bacteria obtained by early screening and identification on an LB liquid culture medium and an LB solid culture medium, recording the growth conditions of the strains under the culture conditions of 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃, and determining the cold resistance of the screened straw-decomposing bacteria.
2. Test results
2.1. Primary screen for cold region straw decomposing bacteria
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 rotten bacteria. The results show that the selected bacteria capable of normally growing at the low temperature of 15 ℃ are 7 strains, namely A3, B7, C1, C4, D7, E5 and E14.
2.2 rescreening of straw decomposing bacteria in Cold regions
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 B7 has a larger ratio (D/D) of the diameter of the transparent ring to the diameter of the colony, which is 2.68, 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 B7 has stronger capability of producing cellulase (see figure 1).
2.3 morphological Observation of straw decomposing bacteria in Cold region
2.3.1 morphological Observation of decomposing bacteria
Culturing the screened straw rotten bacteria B7 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 bacterial strain B7 is a yellow, semitransparent, smooth and moist, irregular edge and slightly raised round bacterial colony, and the thallus is in a thin rod shape, round end and single or short chain arrangement (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.
2.4 molecular identification of straw-decomposing bacteria in Cold region
And (3) carrying out molecular biological identification on the screened mature bacteria B7 through 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. B7 was determined to be Bacillus pumilus (Bacillus pumilus) according to the phylogenetic tree results, which were consistent with the previous morphological identification, and was designated Bacillus pumilus-B7.
2.5 Cold resistance identification of straw-decomposing fungi in Cold region
And (4) carrying out cold tolerance identification on the screened Bacillus pumilus-B7 strain. The screened rotten ripe 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 at 4 ℃, which shows that the rotten ripe bacteria have strong cold resistance. Morita states that cold-tolerant microorganisms have the characteristic of being able to grow and reproduce at temperatures between 0 and 5 ℃ with an optimum growth temperature above 15 ℃ and a maximum growth temperature above 20 ℃. Therefore, 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.
Test example 1 optimization test of fermentation conditions of straw-decomposing fungi in cold area
1. Test method
1.1 Effect of different Nitrogen sources in the Medium on fermentation Performance of decomposing bacteria
In order to determine the influence of different nitrogen sources of the culture medium on the fermentation of the decomposing bacteria, the experiment adopts a nitrogen-free Hexon 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, the adding amount is 0.2%, 4 bottles are treated, and 3 times of treatment are repeated. Fermenting at 25 deg.C for 3d, collecting 0.2mL bacterial liquid, and determining cellulase activity by DNS method.
1.2 Effect of inoculum size on fermentation of decomposing bacteria
In order to observe the influence of different strain inoculation amounts on fermentation of the decomposed bacteria, different strain inoculation amounts are designed in the experiment. Five inoculum sizes of 1%, 3%, 5%, 7% and 9% are set for treatment. Inoculating into liquid fermentation medium with peptone as nitrogen source, wherein the initial pH value of the medium is 7.0, culturing at 25 deg.C, treating 5 bottles each, and repeating for 3 times. When the cells were cultured at 25 ℃ for 3 days, 0.2mL of the cell suspension was collected and used for the cellulase activity assay.
1.3 Effect of culture temperature on fermentation of decomposing bacteria
In order to observe the influence of different culture temperatures on fermentation of the decomposing bacteria, different strain culture temperatures are designed in the experiment. The culture temperature treatment is carried out at 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C for 7 times. The liquid fermentation media containing peptone as a nitrogen source were inoculated at 5% inoculum size, the initial pH of the media was 7.0, and 7 flasks were treated in 3 replicates per flask. When the culture is carried out for 3 days, 0.2mL of the bacterial liquid is taken and used for measuring the activity of the cellulase.
1.4 Effect of initial pH of Medium on fermentation of decomposing bacteria
In order to observe the influence of the initial pH values of different culture media on the fermentation of the decomposing 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. Liquid fermentation medium using peptone as nitrogen source is used as basic medium, initial pH of the medium is adjusted, inoculation amount of 5% is used for inoculation, culture is carried out at 25 ℃, 7 bottles are treated, and 3 times of treatment are set. When the culture is carried out for 3 days, 0.2mL of the bacterial liquid is taken and used for measuring the activity of the cellulase.
1.5 Effect of incubation time on Fulvox cellulase Activity
In order to observe the influence of different culture times on fermentation of the decomposing bacteria, different culture times are designed in the experiment. The bacteria are treated for 7 different culture times of 1d, 2d, 3d, 4d, 5d, 6d and 7d, a liquid fermentation culture medium with peptone as a nitrogen source is used, the initial pH value of the culture medium is 7, inoculation is carried out in a 5% inoculation amount, culture is carried out at 25 ℃, 7 bottles are treated in each bacterial test, and after culture is carried out for the culture time designed by the experiment, 0.2mL of bacterial liquid is respectively taken and used for measuring the activity of the cellulase.
2. Results of the experiment
2.1 optimal Nitrogen Source screening results
And (4) screening the optimum nitrogen source for fermenting the screened cold-region straw decomposing bacteria. As shown in FIG. 4, it can be seen that the cellulase production activity was high in the medium containing peptone and yeast extract as nitrogen sources, while the cellulase production activity was low in the medium containing ammonium sulfate and urea as nitrogen sources, and the difference was significant. And (3) according to the comprehensive comparison result, the optimal nitrogen source suitable for culturing the cellulase produced by the cold-region straw rotten bacteria is peptone.
2.2 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%.
2.3 Effect of temperature on fermentation
As can be seen from FIG. 6, the activity of the strain Bacillus pumilus-B7 is gradually increased at a temperature of 10-30 ℃ and is highest at a temperature of 30 ℃, but the enzyme production activity is not significantly different from that at 25 ℃. As the temperature increased, the enzyme production activity began to decrease, and at 40 ℃, the enzyme production activity of the strain decreased to the minimum. From these results, it can be seen that the strains selected in this test have a wide range of enzyme-producing activity, and low temperature is more favorable for enzyme production. And (4) comprehensively comparing results, and determining that the optimal fermentation temperature of the strain is 25 ℃.
2.4 Effect of initial pH of the Medium on fermentation
As can be seen from FIG. 7, the CMC enzyme activity of the straw-decomposing bacteria is very low in the peracid and alkali environment, even no enzyme is produced. The mature bacteria have enzyme-producing activity at pH 5 to 8, have the highest CMC enzyme activity at initial pH 7 of the medium, and show a significant decrease in enzyme-producing activity when pH is raised to 8. These results indicate that the low-temperature straw-decomposing strains screened in this test are more suitable for producing enzymes under acidic and neutral conditions, and the optimal initial pH value of the culture medium is determined to be 7.
2.5 Effect of incubation time on enzyme production
As can be seen from FIG. 8, the enzyme-producing activity of the strain tended to increase first and then decrease as the culture time was prolonged. 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.
Test example 2 application effect test of glycolysis straw of straw-decomposing bacteria in cold area
1. Test method
1.1 straw-decomposing bacteria Filter paper glycolysis test
Inoculating the screened straw rotten bacteria Bacillus pumilus-B7 on a filter paper liquid culture medium with the initial pH of 7 of the culture medium in an inoculation amount of 5%, and culturing at 15 ℃. After inoculation, the weight loss rate of the filter paper was measured every 24h, and 8d 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 ℃ until the weight of the residual substances is constant, and further measuring the weight loss rate of the filter paper.
1.2 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 corn straw at 4 ℃. The method for treating the corn straws comprises the following steps: soaking the corn straw sections in 2mol/L NaOH solution for 24 hours, washing with water until the pH value is 7, drying in a drying oven at 80 ℃, 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 corn, the humidity is controlled to be 35% -65%, and 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.
2. Test results
2.1 glycolysis Effect of bacteria on Filter paper
In order to verify the glycolysis effect of the screened cold-region straw decomposing bacteria Bacillus pumilus-B7, 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. According to the results, after 1-3 d treatment by Bacillus pumilus-B7, the weight loss rate of the filter paper is obviously increased along with the extension of the treatment time, the decomposition rate of the filter paper after 3d is in a slow rising trend, the maximum value is reached at 15d, and the decomposition rate is over 75 percent.
2.2 Low-temperature efficient glycolysis straw bacteria and EM bacteria combined glycolysis straw test result
Under the condition of low temperature, the low-temperature efficient glycolysis straw bacteria Bacillus pumilus-B7 is matched with EM bacteria to carry out glycolysis tests on corn straws, when 30d is carried out, the straw has weight loss and blackening phenomena in two treatments, but in the combination of the low-temperature efficient glycolysis straw bacteria, the weight loss and blackening degree of the straw is larger, the weight loss rate of the corn straw is measured, the weight loss rate of the combination of the low-temperature efficient glycolysis straw bacteria is 56%, and only the EM bacteria fermentation liquor is used for treatment, the weight loss rate of the straw is 38%, the glycolysis rate is improved by 47.4%, and in the process of straw decomposition, the low-temperature efficient glycolysis straw bacteria are added, so that the rotting degree of the straw is better and the straw is easy to break when the straw is fermented for 30 d.

Claims (17)

1. An isolated Bacillus pumilus strainbacillus pumilus) The strain is characterized in that the microorganism preservation number is as follows: CGMCC No. 15177.
2. A method of preparing a Bacillus pumilus strain of claim 1bacillus pumilus) A method for producing a fermentation broth of a strain, 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 is 0.2 wt%.
4. A method according to claim 3, characterized by: the nitrogen source of the fermentation medium is peptone.
5. The method of claim 2, wherein: inoculating the strain of claim 1 into a fermentation medium at an inoculum size of 1-9% by volume 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 5% for culture.
7. The method of claim 2, wherein: the fermentation culture temperature is 10-40 ℃.
8. The method of claim 7, wherein: the fermentation culture temperature is 25-30 ℃.
9. The method of claim 8, wherein: the fermentation culture temperature is 25 ℃.
10. The method of claim 2, wherein: the initial pH of the fermentation medium is 4-10.
11. The method of claim 10, wherein: the initial pH of the fermentation medium is 5-8.
12. The method of claim 11, wherein: the initial pH of the fermentation medium was 7.
13. The method of claim 2, wherein: the time of fermentation culture is 1-7 days.
14. The method of claim 13, wherein: the time of fermentation culture is 3 d.
15. The decomposition agent for glycolysis of straws is characterized by comprising: a Bacillus pumilus strain as claimed in claim 1 (A)bacillus pumilus) Bacterial agent of the strain or fermentation liquor thereof and EM bacterial solution; wherein the volume ratio of the two is 1: 1.
16. A Bacillus pumilus strain as claimed in claim 1 (A)bacillus pumilus) The application of the strain in glycolysis of straws.
17. The use of the decomposing agent of claim 15 in the glycolysis of straw.
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