CN108823103B - Penicillium lesinum strain of cold region corn straw rotten fungi as well as fermentation culture method and application thereof - Google Patents
Penicillium lesinum strain of cold region corn straw rotten fungi as well as fermentation culture method and application thereof Download PDFInfo
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Abstract
The invention discloses a cold region corn straw rotten fungal strain, a fermentation culture method and application thereof. The invention firstly discloses an isolated Penicillium raidrickii-A8 strain, the microorganism preservation number is: CGMCC No. 15099. The Penicillium raidrickii-A8 strain isolated by the invention can grow at different temperatures and still has vigorous growth under low temperature conditions. The invention optimizes the relevant parameters of the fermentation preparation method of the separated fungus strain. The separated fungus strain can efficiently ferment straws under the low-temperature condition, and can be applied to the fermented straws independently or in combination with EM (effective microorganisms); the fermentation liquid of the strain is mixed with the EM bacterial liquid and then the corn straw is glycolyzed at low temperature, the glycolysis effect of the straw is obviously improved, the glycolysis rate is improved by 46.2 percent, and the straw is better in rotting degree and is easier to break.
Description
Technical Field
The invention relates to a separated cold region corn straw decomposing fungus strain, and also relates to an application of the cold region corn straw decomposing fungus penicillium lesinum strain in efficient glycolysis of corn straws at low temperature, belonging to the field of separation and application of cold region straw decomposing fungi.
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 fungus 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 fungus 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 method comprises the steps of firstly, carrying out separation and purification on microorganisms in cold region straw rotten matters for 6 times to obtain 23 strains in total, culturing the 23 separated strains under different temperature culture conditions, observing the growth conditions of the strains, and carrying out primary screening on low-temperature straw rotten bacteria; as a result, 7 fungal strains capable of normally growing at a low temperature of 15 ℃ were selected, namely A5, A8, A12, B1, C3, E3 and F1. The invention further inoculates the primarily screened 7 strains of low-temperature fungi on a filter paper liquid culture medium, and cultures the fungi for 10 days at the temperature of 28 ℃. Re-screening the cold-region straw rotten fungi by checking the damage degree of filter paper in the liquid culture medium; as can be seen from the fungus filter paper decomposition effect, the liquid culture filter paper of the low-temperature fungus strain A8 has a large damage degree, and after the strain A8 is cultured for 10 days in a liquid culture mode, the filter paper is decomposed to be semi-clear, and the results show that the strain has strong decomposition capacity on cellulose. The re-screened A8 strain was cultured on a solid medium at 28 ℃ and the growth of the strain and the degree of damage to the filter paper were observed, indicating that after 10 days of culture, the strain grew well and the filter paper was decomposed as a nutrient for the growth of the strain. The middle of the filter paper decays to a greater extent, while the edges of the filter paper decay to a lesser extent. The results show that the screened fungus strain A8 has stronger cellulose decomposition capability.
The screened straw rotten fungi A8 are cultured on a solid culture medium, and the colony and the thallus morphology are observed. The results showed that a single fungal colony was formed on the medium. The bacterial colony of the strain A8 is white or light mycelium, has concentric ring veins and a velvet texture, the reverse side of the bacterial colony is light yellow, when the thallus is observed, conidiophores are generated on aerial hyphae, the tip broom-shaped conidiophores are observed, and the conidiophores are spherical. The A8 is preliminarily judged to be penicillium. And (3) performing molecular biological identification on the screened mature bacteria through fungal ITS sequence analysis, wherein ITS sequence fragments obtained by amplification of the strain A8 are 606bp respectively. BLAST comparison and phylogenetic tree construction are carried out on the obtained sequences. Based on the phylogenetic tree results, A8 was finally identified as Penicillium reinhardii (Penicillium raisterckii) and the A8 strain was designated Penicillium raisterckii-A8.
The invention further carries out cold resistance identification on the screened Penicillium raidrickii-A8 strain, and the result shows that the strain can normally grow at 5 ℃, the diameter of the strain reaches 73.94 percent of the maximum diameter after the strain is cultured for 7 days at 15 ℃, the strain grows better in a low-temperature environment, and the cold resistance is strong. Morita states that cold-tolerant microorganisms are characterized by the ability to grow and reproduce at temperatures between 0 ℃ and 5 ℃, with optimal growth temperatures above 15 ℃ and maximum growth temperatures above 20 ℃. Therefore, the decomposed strain Penicillium raidrickii-A8 obtained by screening is a cold-resistant microorganism and can normally grow in northern low-temperature areas.
The Penicillium raidrickii-A8 strain obtained by separation is submitted to a patent-approved organization for preservation, and the microorganism preservation numbers are as follows: CGMCC No. 15099; the classification is named as: penicillium reicheri raidrickii. The preservation unit: china Committee for culture Collection of microorganisms general microbiological 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 Penicillium raidrickii-A8 strain comprises the following steps: inoculating the Penicillium raidrickii-A8 strain in a liquid culture medium for culture, and collecting the bacterial solution.
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, preferably the inoculation amount of the strain is 3-9%, and most preferably the inoculation amount of the strain is 5%;
the culture temperature may be 10-40 ℃, preferably 10-30 ℃, and most preferably 30 ℃;
the initial pH of the liquid medium may be 4 to 10, preferably 6 to 8, most preferably 7;
the time of the fermentation culture can be 3-18d, preferably 9-12d, and most preferably 39 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. According to the comprehensive comparison result, the best nitrogen source suitable for the cultivation of cellulase produced by the Penicillium raidrickii-A8 strain in the cold area is peptone.
The screening result of the optimal inoculation amount for fermentation shows that the activity of cellulase produced by fungi obtained by screening can be influenced by the difference of the inoculation amounts, the activity of cellulase produced by straw-decomposed fungi is rapidly increased along with the increase of the inoculation amount, then the activity of cellulase produced by the fungi slowly decreases, the strain has higher cellulase activity within the range of 3-9% of the inoculation amount, 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%. According to the comprehensive comparison result, the optimal inoculation amount of the straw rotten fungi fermentation in the cold area is 5%.
The fermentation temperature screening experiment results show that the enzyme activity of the strain is gradually increased at the temperature of 10-30 ℃ and is highest at the temperature of 30 ℃. The enzyme production activity decreased significantly with increasing temperature, and at 40 ℃ the enzyme production activity of the strain decreased to a minimum. And (4) comprehensively comparing results, and determining that the optimal temperature for fermenting the cold-region straw decomposing fungi is 30 ℃.
The screening experiment result of the optimal initial pH value of the culture medium shows that the initial pH value of the culture medium in liquid culture has great influence on the activity of the CMC enzyme produced by the cold-region straw-decomposing fungus Penicillium raidrickii-A8. The CMC enzyme activity in the peracid or alkalescence environment is very low, and the enzyme activity is reduced to the minimum at the pH value of 3, so that the cellulase activity can hardly be detected. The initial pH of the culture medium is within the range of 6-8, the CMC enzyme activity of the strain is shown in a higher level, the cellulase activity is highest when the initial pH is 7, and the enzyme production activity of the strain is in a descending trend when the initial pH of the culture medium is increased to 8. These results show that the optimum initial pH value of fermentation of the cold zone straw rotten fungi screened by the test is 7, and the neutral solution environment is favorable for enzyme production.
The screening experiment result of the optimal culture time shows that the enzyme production activity of the Penicillium raidrickii-A8 strain of the straw-rotting fungus in the cold area shows a trend of firstly rapidly increasing and then slowly decreasing along with the extension of the culture time, the strain has the best enzyme production activity when being cultured for 9 days, and the CMC enzyme activity is gradually reduced after 12 days, but still has stronger enzyme activity. And (4) determining the optimal culture time for producing the cellulase by the cold region straw rotting fungi to be 9 days by integrating the comparison result.
In order to verify the straw glycolysis effect of the screened cold-region straw decomposing fungi Penicillium raidrickii-A8, the strain is inoculated into a straw liquid culture medium, the straw glycolysis effect of the strain is determined, test results show that the strain can effectively glycolyze straws along with the prolongation of glycolysis time, and the weight loss rate of the straws reaches over 49% in 15 days. Test results show that the straw-decomposing fungus Penicillium raidrickii-A8 screened by the method has good straw glycolysis effect.
The invention further combines the low-temperature efficient glycolysis straw fungus Penicillium raidrickii-A8 with EM bacteria to carry out glycolysis test of corn straw, at 30d, the straw has weight loss and blackening phenomena in two treatments, but in the combination with the low-temperature efficient glycolysis straw fungus, the straw has larger weight loss and blackening degrees, the weight loss rate of the corn straw is determined, the weight loss rate of the combination with the low-temperature efficient glycolysis straw fungus is 57%, and the weight loss rate of the straw is 39% only by the treatment with the EM bacteria fermentation liquor; experiments show that the Penicillium raidrickii-A8 is matched with EM to ferment the straws, the glycolysis effect is improved by 46.2%, and when the Penicillium raidrickii-A8 is matched with EM to ferment the straws, the straws are better in rotting degree and easier to break.
The invention further discloses application of the Penicillium raidrickii-A8 strain in preparation of a straw-decomposing inoculant.
The invention also discloses a straw decomposing inoculant, which comprises the following components: the invention relates to a fermentation liquor of Penicillium raidrickii-A8 strain.
The fermentation liquor of the Penicillium raidrickii-A8 strain separated by the method can be independently applied to glycolysis of straws, and can also be matched with EM (effective microorganisms) bacteria liquid to be applied to glycolysis, so that the glycolysis rate of the straws is remarkably improved.
The invention also discloses a straw decomposing inoculant, which comprises the following components: the invention relates to fermentation liquor of Penicillium raidrickii-A8 strain and EM bacterial liquid. Preferably, the fermentation liquor of the Penicillium raidrickii-A8 strain and the EM bacterial liquor are composed 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 Penicillium raidrickii-A8 strain separated by the method has good growth in low-temperature environment, vigorous growth capacity and strong cold resistance. The straws can be efficiently fermented under the condition of low temperature; can be applied to glycolysis of straws independently or in combination with EM bacteria; the fermentation liquid of the Penicillium raidrickii-A8 strain is mixed with the EM fermentation liquid and then the corn straw is fermented at low temperature, the straw fermentation effect is obviously improved, and the fermentation rate is improved by 46.2%.
Drawings
FIG. 1 shows the filter paper decomposition and solid culture filter paper decomposition tests of liquid culture of A8 strain; a: control B: liquid culture of the strain A8; c: strain solids;
FIG. 2 is a morphological characterization of the maturing fungus A8; a is colony morphology of A8 strain; b is the microscopic form of A8 strain;
FIG. 3 is a phylogenetic tree constructed by strain A8 based on ITS sequence homology;
FIG. 4 is the temperature growth characteristics of the A8 strain;
FIG. 5 shows the effect of different nitrogen sources on the production of enzymes by the Penicillium raidrickii-A8 strain in cold areas;
FIG. 6 shows the effect of different inoculum sizes on the enzyme production of the cold region straw-decomposing fungus Penicillium raidrickii-A8;
FIG. 7 shows the effect of different temperatures on the enzyme production by the fungus Penicillium raidrickii-A8 of straw maturity in cold regions;
FIG. 8 is a graph of the effect of initial pH on enzyme production by the cold region straw-decomposing fungus Penicillium raidrickii-A8;
FIG. 9 shows the effect of different cultivation times on the production of enzyme by the fungus Penicillium raidrickii-A8 which is decomposing in cold areas.
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 materials and methods
1.1 test materials
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 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.
1.2.2 separation of straw-decomposing fungi in Cold regions
1.2.2.1 isolation, Primary screening and preservation of strains from straw-decomposing Agents
(1) Separation of 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 dipping the suspension supernatant of the decomposed material by using a strain inoculating ring, streaking on a strain isolation culture medium, and inoculating the strain isolation culture medium after fungus single fungus is isolated on a PDA culture medium.
(2) Primary screening of strains in the decomposed material at low temperature: 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: culturing the separated fungus in potato glucose aqueous medium (PD) at 28 deg.C for 36-48 hr until mycelium grows out, and storing at 4 deg.C.
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. Inoculating the primarily screened fungi which can grow under the low-temperature condition into a liquid filter paper culture medium, culturing for 7d at 15 ℃, observing the condition that the strain decomposes the filter paper, and recording the curling and expansion of the edge of the filter paper as (+); the filter paper amorphous state was scored as (++); the filter paper made a paste (+++); the ability of the fungus to break down the cellulose was determined by the ability of the filter to become a semi-clear liquid (+++).
1.2.3 identification of straw-decomposing bacteria in cold regions
1.2.3.1 morphological identification of straw-decomposing fungi in cold regions
Respectively inoculating the rescreened fungus strains in a PD culture medium and a PDA culture medium, culturing at 28 deg.C for 3-4 days, observing the shapes and appearances of bacterial colonies and bacterial plaques after hypha grows out, and observing the shapes of the hypha and conidia by using an optical microscope.
1.2.3.2 molecular identification of straw-decomposing bacteria in cold regions
Inoculating purified single colony from PDA plate culture medium into 30mL potato glucose aqueous medium (PD), culturing at 28 deg.C for 3-4 days, collecting mycelium, and extracting DNA by CTAB method. The ITS sequence universal primer ITS1/ITS4 for fungus identification is used as the molecular identification primer of the cold region straw rotten fungus to carry out PCR amplification, and the PCR product is sequenced 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 system development tree and making molecular identification for fungus species.
1.2.4 Cold resistance identification of straw-decomposing bacteria in Cold region
Respectively inoculating the straw rotten fungi obtained by early screening and identification to a PDA 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 rotten fungi.
2. Test results
2.1 preliminary screening 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 rotten bacteria. The results showed that the screened fungal strains capable of growing normally at low temperature of 15 ℃ had a total of 7 strains, a5, A8, a12, B1, C3, E3 and F1, respectively.
2.2 rescreening of straw rotten fungi in Cold regions
Inoculating the primary screened low-temperature fungus on a filter paper liquid culture medium, and culturing at 28 ℃ for 10 days. The secondary screening of the cold-region straw-decomposing fungi is carried out by checking the damage degree of filter paper in the liquid culture medium, and the test results are shown in table 1. As can be further seen from the decomposition effect of the fungus filter paper, the liquid culture filter paper of the low-temperature fungus strain A8 has a large damage degree, and after the strain A8 is cultured in liquid for 10 days, the filter paper is decomposed to be semi-clear, and the results show that the strain has strong decomposition capability on cellulose (see figure 2-B). The re-screened A8 strain was cultured on a solid medium at 28 ℃ and the growth of the strain and the degree of damage to the filter paper were observed, indicating that after 10 days of culture, the strain grew well and the filter paper was decomposed as a nutrient for the growth of the strain. The middle of the filter paper decayed to a greater extent, while the edges of the filter paper decayed to a lesser extent. The results show that the screened fungus strain A8 has stronger cellulose decomposition capability.
TABLE 1 Filter paper decomposition Effect of the strains
Note: filter paper edge curl expansion is reported as (+); the filter paper amorphous state was scored as (++); the filter paper became a paste (+++); the filter paper turned to a semi-clear liquid (+++)
2.3 morphological Observation of straw decomposing bacteria in Cold region
Culturing the screened straw rotten fungi A8 on a solid culture medium, and observing bacterial colonies and bacterial morphology. The results showed that a single fungal colony was formed on the medium. The bacterial colony of the strain A8 is white or light mycelium with concentric ring veins and velvet texture, the reverse side of the bacterial colony is light yellow, when the thalli of the bacterial colony are observed, conidiophores are generated on aerial hyphae, the tip of the bacterial colony is broom-shaped, and the conidiophores are spherical (see figure 2). The A8 is preliminarily judged to be penicillium.
2.4 molecular identification of straw-decomposing bacteria in Cold region
Performing molecular biological identification on the screened mature bacteria through fungal ITS sequence analysis, wherein the ITS sequence fragments obtained by amplification of the strain A8 are 606bp respectively; BLAST alignment and phylogenetic tree construction were performed on the obtained sequences, and the results are shown in FIG. 3. A8 was Penicillium reinhardii (Penicillium raidrickii) as a result of phylogenetic trees, which was consistent with the previous morphological identification, and was designated Penicillium raidrickii-A8.
2.5 Cold resistance identification of straw-decomposing fungi in Cold region
And (5) carrying out cold resistance identification on the strains obtained by screening. The selected decomposed fungal strain A8 was inoculated on PDA medium, cultured at different temperatures, and the growth of the strain was observed as shown in FIG. 4. The result shows that the strain can normally grow at 5 ℃, the diameter of the strain reaches 73.94% of the maximum diameter after the strain is cultured for 7d at 15 ℃, the strain grows better in a low-temperature environment, and the cold resistance is strong. 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 ℃. The decomposed strains obtained by screening in the test are all cold-resistant microorganisms and can normally grow in low-temperature areas 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 of decomposing bacteria
In order to observe 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, the adding amount is 0.2%, 4 bottles are treated, and 3 times of treatment are repeated. When cultured at 25 ℃ for 7 days, 0.2mL of the bacterial suspension was taken and used for the measurement of the cellulase activity, and the measurement method was the 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 for 7 days, 0.2mL of each cell suspension was collected and used for the measurement of cellulase activity.
1.3 Effect of culture temperature on fermentation of decomposing bacteria
In order to observe the influence of different culture temperatures on the activity of the cellulose enzyme of the decomposed 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. 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, 0.2mL of each cell suspension was used for cellulase activity measurement.
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 cells were cultured for 7 days, 0.2mL of each cell suspension was used for cellulase activity measurement.
1.5 Effect of culture time on fermentation of decomposing bacteria
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 treatment was carried out for 6 different incubation times, 3d, 6d, 9d, 12d, 15d, 18 d. The liquid fermentation medium using peptone as nitrogen source, the initial pH value of the medium is 7, the inoculation amount is 5%, each bacterium test treats 7 bottles, each fungus test treats 6 bottles, and 3 times of the tests are respectively set. After culturing according to the culture time designed by the experiment, 0.2mL of bacterial liquid is respectively taken for the activity determination of the cellulase.
2. Results of the experiment
2.1 determination of optimal Nitrogen Source
Screening the optimal nitrogen source for fermentation of the screened cold-region straw-decomposing fungus Penicillium raidrickii-A8. As shown in FIG. 5, it can be seen that the activity of the strain for producing cellulase on a medium containing peptone and yeast extract as nitrogen sources was high, while the activity of the strain for producing cellulase on a crop nitrogen source medium supplemented with ammonium sulfate and urea was low and the difference was significant. And (3) according to the comprehensive comparison result, the optimal nitrogen source suitable for fermentation culture of the straw rotten fungi in the cold region is peptone.
2.2 Effect of inoculum size on fermentation
The screening of the optimal inoculation amount of the screened cold-region straw-decomposing fungus Penicillium raidrickii-A8 fermentation culture is carried out, and the result is shown in FIG. 6. The results show that the activity of the cellulase produced by the fungi obtained by screening can be influenced by the difference of the inoculation amounts, the activity of the cellulase produced by the straw rotten fungi is increased sharply with the increase of the inoculation amounts, then the activity of the cellulase produced by the straw rotten fungi is reduced slowly, the strain has high cellulase activity within the range of 3-9% of the inoculation amount, the highest enzyme production activity is achieved when the inoculation amount is 5%, and the enzyme activity begins to be reduced after the inoculation amount exceeds 5%. According to the comprehensive comparison result, the optimal inoculation amount of the straw rotten fungi fermentation in the cold area is 5%.
2.3 Effect of temperature on fermentation
The influence of different culture temperatures on the fermentation of the screened cold-region straw-decomposing fungus Penicillium raidrickii-A8 is researched, and the result is shown in FIG. 7. The results show that the enzyme activity of the strain is gradually increased at the temperature of 10-30 ℃, and the enzyme activity is highest at the temperature of 30 ℃. The enzyme production activity decreased significantly with increasing temperature, and at 40 ℃ the enzyme production activity of the strain decreased to a minimum. And (4) comprehensively comparing results, and determining that the optimal temperature for fermenting the cold-region straw decomposing fungi is 30 ℃.
2.4 Effect of initial pH on fermentation
As can be seen from FIG. 8, the initial pH of the culture medium in liquid culture has a greater effect on the activity of CMC enzyme produced by the Penicillium raidrickii-A8 strain which is a cold-region straw-decomposing fungus. The CMC enzyme activity in the peracid or alkalescence environment is very low, and the enzyme activity is reduced to the minimum at the pH value of 3, so that the cellulase activity can hardly be detected. The initial pH of the culture medium is within the range of 6-8, the CMC enzyme activity of the strain is shown in a higher level, the cellulase activity is highest when the initial pH is 7, and the enzyme production activity of the strain is in a descending trend when the initial pH of the culture medium is increased to 8. These results show that the optimum initial pH value of fermentation of the cold zone straw rotten fungi screened by the test is 7, and the neutral solution environment is favorable for enzyme production.
2.5 Effect of incubation time on fermentation
As can be seen from FIG. 9, the enzyme production activity of the Penicillium raidrickii-A8 strain in the cold region is shown as a trend of rapid increase and slow decrease along with the extension of the culture time, the strain has the best enzyme production activity when cultured for 9 days, and the CMC enzyme activity is gradually reduced after 12 days, but still has stronger enzyme activity. And (4) determining the optimal culture time for producing the cellulase by the cold region straw rotten fungi to be 9 days by integrating the comparison result.
Test example 2 application effect test of straw decomposition bacteria glycolysis of straw in cold region
1. Test method
1.1 straw glycolysis test
This experiment studies the glycolysis effect of straw decomposing bacteria on straw. Inoculating 5% of straw decomposing bacteria fermentation liquid into straw liquid culture medium (straw fermentation liquid culture medium: weighing KH2PO4 1.0 g，MgSO4 0.3g，NaNO3 2.5g，NaCl 0.1g，CaCl2 0.1g，FeCl3Adding 0.01g of the powder into 1000mL of distilled water in a conical flask, adjusting the pH value to about 7.2, adding the treated corn straw powder, sterilizing the powder by high-temperature steam for later use), culturing the powder at 15 ℃, and sampling and measuring the weight loss rate of the straws at 3d, 6d, 9d, 12d and 15d respectively. The method for measuring the weight loss rate of the straws is the same as the method for measuring the weight loss rate of the filter paper, the filter paper of the fermentation liquor is filtered, the residues are placed in an oven to be dried to constant weight at 80 ℃, and then the weight loss rate of the straws is measured.
1.2 Low-temperature efficient glycolysis straw fungus and EM bacterium compound glycolysis straw test
Mixing fermented low-temperature efficient glycolysis straw fungus fermentation liquor and EM (effective microorganisms) fermentation liquor according to the volume ratio of 1:1, and glycolysis corn straw at the temperature of 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 sieving for later use. The fermentation composition is characterized in that 5% of EM (effective microorganisms) fermentation liquor, 5% of the low-temperature high-efficiency glycolysis straw fungus fermentation liquor and 1% of brown sugar are added according to the amount of fermented corn straws, the humidity is controlled to be 35% -65%, and the EM fermentation liquor is used as a reference for fermentation for 30 d. 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 decomposition Effect of decomposing fungi on straw
The decomposed fungi screened in the test are inoculated in a straw liquid culture medium, and the straw glycolysis effect of the strain is determined. The results show that the fungus strain can effectively ferment the straws with the prolongation of the glycolysis time, and the weight loss rate of the straws reaches over 49 percent in 15 days. The results show that the straw-decomposing fungus Penicillium raidrickii-A8 screened by the invention has good straw fermenting effect.
2.2 Low-temperature efficient glycolysis straw fungus and EM bacteria combined glycolysis straw test result
Under the condition of low temperature, the fungus Penicillium raidrickii-A8 is matched with EM to carry out the glycolysis test of the corn straws, when 30d is reached, the straws have weight loss and blackening phenomena in two treatments, but in the combination added with the Penicillium raidrickii-A8, the degree of weight loss and blackening of the straws is higher, the weight loss rate of the corn straws is determined, the weight loss rate of the combination added with the Penicillium raidrickii-A8 fungus is 57%, and the weight loss rate of the straws is only 39% by the treatment with the EM fermentation liquor; the straw glycolysis effect is improved by 46.2% after the Penicillium raidrickii-A8 and EM fermentation liquid are used in a matching way, and the low-temperature efficient glycolysis straw fungi are added in the straw decomposition process, so that the straw is better in rotting degree and easier to break.
Claims (19)
1. An isolated Penicillium reissum (Penicillium reissui) A8 strain for low-temperature glycolysis of straws, wherein the microbial collection number is as follows: CGMCC No. 15099.
2. A process for preparing a fermentation broth of the Penicillium reinhardtii A8 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 10-30 ℃.
10. The method of claim 9, wherein: the fermentation culture temperature is 30 ℃.
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 6-8.
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 3-18 d.
15. The method of claim 14, wherein: the time of fermentation culture is 9-12 days.
16. The method of claim 15, wherein: the time of fermentation culture is 3 d.
17. The decomposition agent for glycolysis of straws is characterized by comprising: the microbial agent or fermentation liquid of the penicillium rethenicum A8 strain according to claim 1, and an EM bacterial solution; wherein the volume ratio of the two is 1: 1.
18. Use of the penicillium reissui A8 strain of claim 1 or a fermentation broth thereof for the glycolysis of straw.
19. The use of the decomposing agent of claim 17 in the glycolysis of straw.
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