CN115353994B - Bacterial strain for degrading cellulose and resisting salt and alkali and application thereof - Google Patents
Bacterial strain for degrading cellulose and resisting salt and alkali and application thereof Download PDFInfo
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Abstract
The invention discloses a bacterial strain for degrading cellulose and resisting salt and alkalinity, which is named as Microbacterium arborescens SSF12, is classified as Microbacterium arborescens (Microbacterium arborescens), is preserved in China general microbiological culture Collection center (CGMCC) No.25296 in the 7 th month 14 days of 2022, and has a preservation address of North Chen Silu No. 1 and 3 in the Chaoyang area of Beijing city. The microbacterium arborescens SSF12 is applied to fermented straw compost, so that the efficiency of the fermented compost is improved, and the strain has strong saline-alkali resistance and can be used for soil improvement of saline-alkali soil.
Description
Technical Field
The invention relates to the technical field of screening and application of cellulose degradation and salt and alkali tolerance bacteria, in particular to a bacterial strain for degrading cellulose and salt and alkali tolerance and application thereof
Background
The production of 6.3 hundred million tons of crop straws per year in China accounts for 20-30% of the total yield of the straws in the world. Straw is used as main agricultural waste, is rich in abundant cellulose resources, but has the problems of difficult degradation and difficult recycling. The traditional method mainly carries out incineration treatment on the waste water, and has low efficiency, high energy consumption and easy environmental pollution. Straw composting is an effective technical method for solving the problems of straw pollution and low straw utilization rate. The straw organic fertilizer can effectively fertilize soil, improve soil quality and improve crop quality. The straw organic fertilizer is prepared by utilizing various high-activity enzymes (such as cellulose degrading enzymes) secreted by lignocellulose degrading bacteria, and under proper conditions, substances such as lignocellulose and the like which are difficult to degrade in the straw are converted into nutrient elements which can be utilized by plants.
Salinization is one of the main reasons for causing the soil degradation of the cultivated land worldwide, and accelerating the soil improvement can utilize the salinized cultivated land to a certain extent and reduce the salinization degree of the soil. Biological improvement measures are recognized as relatively efficient and environmentally friendly methods. The microbial organic fertilizer can be used for remarkably improving the soil property of the saline-alkali soil, and the microbial agent can be used for effectively promoting the absorption and utilization of nutrient substances of crops in the saline-alkali soil so as to promote the rapid growth of the crops, so that the microbial agent in the organic fertilizer plays a role in resisting the probiotics of the saline-alkali. Therefore, the microorganism with good saline-alkali resistance obtained by separation has important significance for improving saline-alkali soil and developing novel microbial agents.
Therefore, how to provide a strain that degrades cellulose and resists salt and alkalinity is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides a strain that degrades cellulose and is resistant to salt alkalinity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a strain for degrading cellulose and resisting salt and alkali is named as Microbacterium arborescens SSF12, is classified and named as Microbacterium arborescens (Microbacterium arborescens), and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.25296 and the preservation address of North Chenxi Lu No. 1 and No. 3 in the Korean region of Beijing in 2022, 7 and 14 days
The application of the microbacterium arborescens SSF12 in cellulase production by fermentation.
The application of the microbacterium arborescens SSF12 in fermenting straw compost.
Preferably, the straw is fermented by producing cellulases including filter paper enzymes, endoglucanases, exoglucanases and beta-glucanases.
Preferably, the enzyme activities of the cellulose degrading bacteria for producing cellulase through fermentation are respectively as follows: the filter paper has an enzyme activity of 33.45U/mL, the endoglucanase has an activity of 24.92U/mL, the exoglucanase has an activity of 31.88U/mL, and the beta-glucanase has an activity of 29.11U/mL.
The application of the microbacterium arborescens SSF12 in improving saline-alkali soil.
Compared with the prior art, the invention respectively screens cellulose degrading bacteria from farmland soil by CMC-Na, and obtains SSF12 to identify as Microbacterium arborescens through strain morphological characteristics, physiological and biochemical characteristics and 16SrDNA sequence analysis results. The filter paper has the enzyme activity of 33.45U/mL, the endoglucanase activity of 24.92U/mL, the exoglucanase activity of 31.88U/mL, and the beta-glucanase activity of 29.11U/mL, and has stronger salt tolerance performance, and can be used for subsequent soil improvement and crop growth promotion.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing morphological identification of cellulolytic bacteria SSF 12; a: growth morphology of strain SSF 6; b: gram staining characteristics of strain SSF 6; c: degradation circle of strain SSF6 on LB+CMC-Na medium;
FIG. 2 is a graph showing the results of GenIII assay plates:
FIG. 3 is a schematic diagram showing the SSF12 strain electrophoresis;
FIG. 4 is a schematic diagram of a phylogenetic tree of strain SSF 12;
FIG. 5 is a graph of glucose calibration;
FIG. 6 is a diagram showing the measurement of the enzyme activity of the strain.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Soil sample:
soil samples are collected in farmland humus soil of the inner Mongolia autonomous region. The soil sample is sampled by using a five-point method, namely, the soil of about 1cm of the surface layer of the soil is scraped at the center of the diagonal line of a sampling area, about 50g of the soil sample is taken and placed in a sterile self-sealing bag, and 4 points with the same distance from the center sample point are selected on the diagonal line for sampling. Uniformly mixing 5 samples in equal quantity, and storing in a refrigerator at 4 ℃ in a laboratory for later use.
Experimental reagent and equipment
The main experimental reagents are shown in table 1, and the equipment is shown in table 2.
TABLE 1 summary of reagents and apparatus
TABLE 2 Main instruments and apparatus
Culture medium:
beef extract peptone enrichment medium: beef extract 3.00g, peptone 10.00g, naCl 5.0g, constant volume 1L, pH value=7.0, 121 ℃ 20min
Screening the culture medium: CMC-Na 20.00g, na 2 HPO 4 2.50g、KH 2 PO 4 1.50g, peptone 2.50g, yeast extract 0.50g, agar 20.00g, constant volume 1L, pH=7.0, 20min at 121 ℃.
Fermentation medium: CMC-Na10.00g, KH 2 PO 4 1.00g、MgSO 4 ·7H 2 O 0.50g、(N H4) 2 SO 4 2.00g of NaCl 2.50g, yeast extract 2.50g, peptone 5.00g, constant volume 1L, pH value=7.0, 20min at 121 ℃.
Filter paper strip disintegration medium: (NH 4) 2 SO 4 3.00g、KH 2 PO 4 1.00g、MgSO 4 ·7H 2 0.40g of O, 0.10g of Yeast extract, 1L of constant volume, pH=7.0 and 20min at 121 ℃.
CMC-Na medium (g/L): k (K) 2 HPO 4 1.00g，MgSO 4 ·7H 2 O0.25g,Yeast extract 2.00g,Agar 15.00g adding CMC-Na 2.00g, constant volume 1L, and 121 deg.C for 20min, and preparing microcrystalline cellulose by the same method until K 2 HPO 4 、MgSO 4 ·7H 2 After O and yeast extract are melted and boiled, CMC-Na is slowly added, which can be dissolved but has a slower speed, agarose is then added, and after all the dissolution, the mixture is put into an autoclave for sterilization.
Reagent preparation
DNS chromogenic reagent (3, 5 dinitrosalicylic acid): preparing according to the standard DNS reagent of the Ministry of agriculture; citric acid buffer solution, glucose standard solution: formulated according to QB 2583-2003 standard: 3, 5-dinitrosalicylic acid (10+/-0.1 g) is weighed, placed in about 600mL of water, 10g of sodium hydroxide is gradually added, dissolved in a water bath at 50 ℃ by magnetic stirring, and then 200g of potassium sodium tartrate, 2g of phenol (re-steaming) and 5g of anhydrous sodium sulfite are sequentially added. After complete dissolution and clarification, cool to room temperature, volume to 1000mL with water, filter, store in brown reagent bottle, and use after 7d in the dark.
Congo red dye liquor: weighing 0.10g Congo red, and fixing the volume to 100mL;
NaCl solution: weigh 5.85g NaCl to volume to 100mL.
Whatman filter strip: clean starch-free Whatman filter paper was cut into 1X 6cm strips.
1% glucose standard solution: accurately weighing dry 0.1g glucose, and fixing the volume to 10mL by deionized water, and preparing in situ.
0.1mol/L of citric acid buffer pH 4.5: 9.6g of anhydrous citric acid and 2.7g of sodium hydroxide are weighed, added to 900mL of distilled water while stirring, and finally distilled water is added to fix the volume to 1L. The PH was tested and adjusted to ph=4.5 with citric acid or sodium hydroxide dilution solutions.
EXAMPLE 1 screening of cellulose degrading bacteria
1) Enrichment culture
Shaking 1g of soil sample in 99mL of sterile water, taking 5mL of soil sample, inoculating into beef extract peptone enrichment medium, and culturing at 37 ℃ and 180r/min for 24-48 h;
2) Primary screen
Taking 1mL of enrichment culture solution, carrying out equal-ratio dilution by using PBS buffer solution, and respectively sucking the dilution degree to be 10 -4 ～10 -8 200 mu L of the multiplied bacterial liquid is smeared on a screening culture medium, 3 times of the multiplied bacterial liquid are repeated, and the multiplied bacterial liquid is cultured for 24 to 48 hours at 37 ℃. Single colonies with different forms in each dilution gradient culture medium are selected and cultured in a screening culture medium for 24-48 hours at 37 ℃, congo red solution is used for dyeing for 1 hour, dye liquor is discarded, and finally NaCl solution is used for washing for 1 hour. 1 strain with larger transparent circle diameter ratio (D/D) was selected.
3) Double screen
Inoculating the bacteria subjected to primary screening to CMC-Na culture medium respectively, culturing at 37 ℃ for 24 hours, dyeing the CMC-Na culture medium with Congo red for 1 hour, washing with sodium chloride solution for 1 hour, measuring the diameter (D, cm) of a transparent ring of a bacterial colony in the culture medium and the diameter (D, cm) of the bacterial colony by using a vernier caliper, calculating the ratio of the diameter (D/D) and the diameter (D, cm), and taking the size of the ratio as an index for judging the cellulose degrading capacity of bacteria, wherein the index is shown in Table 3;
TABLE 3 determination of degradation circle diameter D/D of colonies on CMC-Na medium
The SSF12 degradation circle has larger diameter, the strain with large degradation circle ratio is selected to be inoculated into a fermentation medium for culturing for 48 hours at 37 ℃ and 180r/min, 10mL of fermentation liquor is taken to be centrifuged for 20min at 4 ℃ and 7000r/min, and crude enzyme liquid is prepared.
4) Identification of cellulose degrading bacteria
Morphological identification
Gram staining was performed on overnight cultured cellulose degrading bacteria to identify staining characteristics.
1. The smear is fixed by flame, and is dyed for 1 minute by adding (crystal violet solution) solution A, and the dye liquor is washed by clean water.
2. Adding (iodine solution) solution B for dyeing for 1min, and washing with water.
3. Add (95% ethanol) solution C, shake the slide from time to time for about 10-30 seconds until no purple fall off, wash with water.
4. Adding (sallow counterstain) solution D, dyeing for 1min, and washing with water.
5. And (5) performing oil microscopic examination after drying. The positive bacteria are purple and the negative bacteria are light red.
After drying, the mixture was observed with an oil mirror. First low power mirror and then high power mirror. After the use of the oil mirror is finished, the lens is cleaned by the mirror cleaning paper in time, so that the cedar oil residue is avoided.
As a result, as shown in FIG. 1, SSF12 colonies were yellow and smooth in surface, were grown aerobically, were gram-positive, were irregularly rod-shaped under a microscope, and were singly present or arranged in pairs.
Physiological and biochemical identification of cellulolytic bacteria
The SSF6 strain was subjected to physiological and biochemical analysis using a Biolog GEN III MicroStation automated microorganism identification system. The GenIII identification plate has 96 wells, including 71 carbon sources, 23 chemosensitive substances, 1 negative and 1 positive control. The color development change of SSF12 strain on GenIII identification plate is shown in FIG. 2, the number of positive results is 63, the number of negative results is 17, and the number of reactions at the "boundary value" is 16; wherein 50 positive reactions and 9 negative reactions of the carbon source are tested; there were 12 reactions for chemosensitivity test and 7 negative reactions. Table 4 shows that the reaction strains showed 22 positive reactions and 2 negative reactions on the sugar alcohol substrate on the GenIII identification plate; table 5 shows that the strains had 1 in total negative and 5 positive reactions in the reaction of amino acid substrates in GenIII identification plates; as shown in Table 6, the strains showed 5 positive reactions and 6 negative reactions for the antibiotic substrates in the GenIII identification plate.
TABLE 4 results of sugar alcohol reactions of SSF12 strains in GenIII identification plates
TABLE 5 amino acid reaction results of SSF12 strains in GenIII identification plates
TABLE 6 antibiotic reaction results of SSF12 strains in GenIII identification plates
Molecular biological identification
Bacterial genomic DNA extraction method (bacterial genomic DNA extraction kit DP 302).
1. Taking 1-5mL of bacterial culture solution, centrifuging at 10,000rpm for 1min, and sucking the supernatant as much as possible.
2. 180uL of buffer was added to the bacterial pellet. The buffer system (1 mL) was: tris-HCl 20uL; EDTA ph=8.0 4ul; trition 12uL; sterile water 964uL; 0.02g of lysozyme.
3. After adding the buffer solution, the mixture is treated for more than 30 minutes at 37 ℃.
4. 20uL of protease K solution was added to the centrifuge tube and mixed well.
5. 220uL of buffer solution GB is added, the mixture is shaken for 15s, the mixture is placed for 10min (water bath kettle) at 70 ℃, the solution is clear in strain, and the mixture is centrifuged briefly to remove water drops on the inner wall of the tube cover.
6. Adding 220uL absolute ethyl alcohol, fully shaking and uniformly mixing for 15s, wherein flocculent precipitation possibly occurs at the moment, and performing short centrifugation to remove water drops on the inner wall of the tube cover.
7. Adding the solution obtained in the last step and flocculent precipitate into an adsorption column CB3 (the adsorption column is placed in a collecting pipe), standing for 2min, centrifuging at 12000rpm for 1min, pouring out waste liquid, and placing the adsorption column CB3 into the collecting pipe.
8. 500uL of buffer GD (please check whether absolute ethanol was added before use) was added to the adsorption column CB3, left for 2min, centrifuged at 12000rpm for 1min, the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube.
9. 600uL of rinse PW (checked before use if absolute ethanol is added) was added to the column CB3, left for 2min, centrifuged at 12000rpm for 1min, the waste liquid was poured off, and the column CB3 was placed in a collection tube.
10. And (3) repeating the step 811, placing the adsorption column CB3 back into a collecting pipe, centrifuging at 12000rpm for 2min, pouring out waste liquid, placing the adsorption column CB3 into a new centrifuge tube, and standing at room temperature for a plurality of minutes (5 min) to thoroughly dry residual rinse liquid in the adsorption material.
12. 50-200 uL (100 uL in the experiment) of elution buffer TE is suspended and dripped into the middle part of the adsorption film, the solution is placed for 2-5 min at room temperature and centrifuged for 2min at 12000rpm, and the solution is collected into a centrifuge tube.
The 16srDNA of the isolated strain was amplified by using universal primers,
F：5’-AGAGTTTGATCCTGGCTCA-3’；R：5’-GGTTACCTTGTTACGACTT-3’；
the reaction system (25. Mu.L) for PCR amplification comprises: premixTaqTM12.5. Mu.L, 1. Mu.L of each of the upstream and downstream primers (10 pmol/. Mu.L), 3. Mu.L of bacterial genomic DNA (1. Mu.g/. Mu.L), and ddH2O 7.5. Mu.L. The reaction conditions are as follows: 94 ℃ for 5min; denaturation at 94℃for 30s, 45s at 55℃for 2min at 72℃for 35 cycles; and at 72℃for 10min. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, the gel recovered product was ligated with pMD19-T vector and transferred into Trans-T1 competent cells, and positive clones were sent to Nanjing Jinsrui biological Co., ltd for sequencing by blue-white screening and colony PCR identification.
The results showed that the PCR band was single and clear, with a size of about 1500bp, which was consistent with the expected size (FIG. 3). A16 SrDNA sequence similarity alignment was performed using the BLAST program at NCBI, and a phylogenetic tree was constructed using MEGA7.0, which showed that the sequence similarity of strain SSF12 to strain Microbacterium arborescens strain ND was highest (99%). According to morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis results of the strain, the strain SSF12 is identified as Microbacterium arborescens (Microbacterium arborescens), the strain is named as Microbacterium arborescens SSF12, is classified and named as Microbacterium arborescens (Microbacterium arborescens), and is preserved in China general microbiological culture collection center of China Committee for culture Collection of microorganisms for 7 months and 14 days in 2022, with a preservation number of CGMCC No.25296 and a preservation address of North-West-Lou No. 1 and No. 3 in the Korean region of Beijing city.
Example 2
Measurement of cellulase Total Activity
Sucking 0-1.2 mL (0.0/0.2/0.4/0.6/0.8/1.0/1.2) of glucose standard solution of 1g/L respectively, adding deionized water to 2.00mL after an interval of 0.2mL, adding 3.00mL of DNS reagent, uniformly mixing, boiling in a boiling water bath for 5min, cooling with cold water, fixing the volume to 20.00mL with deionized water, and measuring the absorbance at 540 nm. And drawing a standard curve by taking absorbance as an X axis and the content of glucose as a Y axis. See fig. 5. The regression equation for the standard curve is: y=0.59550x+0.01517,R 2 = 0.9923, which indicates that the standard curve has a good linear relationship, and thus meets the requirements, and can be used as a standard curve for cellulase activity measurement. And (3) placing the reaction solution into an ultraviolet spectrophotometer to measure absorbance, and measuring the glucose content according to a standard curve to further calculate the enzyme activity of the cellulase.
Calculation of cellulase Activity
Definition of enzyme activity: the substrate is sodium carboxymethyl cellulose, the pH value is 6, the reaction condition is that the water bath is heated for 30min at the constant temperature of 50 ℃, the amount of cellulase required for catalyzing the sodium carboxymethyl cellulose to generate 1 mug of glucose every 30min is defined as an enzyme activity unit,
wherein: m is the glucose content calculated according to a standard curve equation, and mg;
n is the dilution factor of the enzyme solution; v is the volume of enzyme solution, mL;
t is the culture time, min;5.56 is the μmol number of 1mg glucose, 1000/180=5.56.
Measurement of filter paper enzyme activity (FPA) was used. A25 mL color comparison tube with a plug is added with a 1cm multiplied by 5cm filter paper strip (about 0.1 g), 1.00mL of 0.1mol/L citric acid buffer solution with pH of 4.5 is added to submerge the filter paper strip, 1.00mL of crude enzyme solution (blank group is 1.00mL of inactivated crude enzyme solution) is added, standing is carried out for 30min at 50 ℃, 3.00mL of DNS reagent is added, the mixture is uniformly mixed with boiling water bath for boiling for 5min, cooling is carried out with cold water, the volume is fixed to 20.00mL, and absorbance is measured at 540nm after uniform mixing. Each tube was repeated 3 times.
Determination of EG or Cx enzyme (endo beta-1, 4-glucosidase) Activity
The carboxymethyl cellulose (CMC) enzyme activity assay was used. To a 25mL cuvette with plug, 1.00mL crude enzyme solution (1.00 mL inactivated crude enzyme solution as blank) and 1.00mL1% CMC-Na solution (0.1 mol/L pH=4.5 citric acid buffer) were added, the mixture was allowed to stand at 50℃for 30min, 3.00mL DNS reagent was added, the mixture was boiled in a boiling water bath for 5min, cooled in cold water, the volume was set to 20.00mL with deionized water, and the absorbance was measured at 540nm after mixing. Each tube was repeated 3 times.
Determination of CBH or C1 enzyme (exo-beta-1, 4-glucosidase) Activity
Microcrystalline cellulose (MCC) enzyme activity assays were used. To a 25mL cuvette with plug, 1.00mL crude enzyme solution (1.00 mL inactivated crude enzyme solution as blank) and 1.00mL1% microcrystalline cellulose solution (0.1 mol/L pH=4.5 citric acid buffer) were added, and the mixture was allowed to stand at 50℃for 30min, 3.00mL DNS reagent was added, the mixture was boiled in a boiling water bath for 5min, cooled in cold water, the volume was set to 20.00mL, and absorbance was measured at 540nm after mixing. Each tube was repeated 3 times.
Determination of beta-1, 4-glucosidase Activity
1.00mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution as a blank group) and 1.00mL of 1% concentration salicin solution (0.1 mol/LpH =4.5 citric acid buffer solution) are added into a 25mL colorimetric tube with a plug, the temperature is 50 ℃ for 30min, 3.00mL of DNS reagent is added, the mixture is uniformly mixed, boiled in a boiling water bath for 5min, cooled in cold water, the volume is fixed to 20.00mL, and the absorbance of the mixture is measured at 540nm after uniform mixing. Each tube was repeated 3 times.
As a result, as shown in FIG. 6, the 4 enzyme activities of the strain were greatly different, the filter paper enzyme activity in the strain SSF12 was 33.45U/mL, the endoglucanase activity was 24.92U/mL, the exoglucanase activity was 31.88U/mL, and the beta-glucanase activity was 29.11U/mL.
Example 3 application of SSF12 Strain in straw degradation
The wheat straw crushed by the crusher is washed by distilled water, run out and then is placed into a constant temperature drying box to be dried for a few hours at 110 ℃ to constant weight. 200ml of fermentation medium (KH) was added to the flask 2 PO 4 1.00g、MgSO 4 ·7H 2 O 0.50g、(NH4) 2 SO 4 2.00g of NaCl 2.50g, yeast extract 2.50g, peptone 5.00g, constant volume 1L, pH value=7.0, 121 ℃ for 20 min), 10g of dried wheat straw and 10ml of bacterial liquid (1×10) are added 10 CFU/mL), four groups of 3 were made, each group was incubated in parallel at 37 ℃ with shaking at 200rpm, and the difference in weight change was the average degradation rate of the wheat straw, as shown in tables 7 and 8; .
TABLE 7 application of bacterial liquid in straw degradation
Table 8 degradation rates of various ratios of bacterial liquids to straw
Example 4 application of SSF12 Strain in straw composting
Straw 1 ton and 1 x 10 contained 8 The liquid fermentation liquid 50L of the CFU/mL arborescent microbacterium SSF12 is fully mixed, the mixed materials are added into a membrane type composting bin, the cover of the composting bin is of a membrane type structure, the water resistance can be realized, the heat dissipation can be prevented, the initial temperature can be kept at 32 ℃, the water content is 65%, the temperature can be kept at 55 ℃ for 15 days, and the composting is completed after the color of the composting becomes black brown and the texture is loose, for 50 days.
And detecting the C/N ratio by taking the straw compost subjected to natural fermentation as a control group. The C/N ratio can show the composting degree and represents the degradation condition of microorganisms on organic matters. The C/N ratio of SSF12 group was reduced from the original 49.93 to 18.16; the C/N ratio of the control group is reduced from the initial 54.12 to 22.13, which proves that the addition of SSF12 bacteria has obvious promotion effect on straw compost maturity.
EXAMPLE 5 saline-alkali tolerance analysis
The bacterial solution was inoculated at a concentration of 2% to four gradients of 4%, 6%, 8% and 10% of the salt concentration in a beef extract peptone medium prepared from NaCl at an OD600 of 0.5, and the culture was performed in parallel 3 times. Culturing at 37 deg.C 180r/min for 2-5 d, observing growth vigor and recording data, and measuring OD every 24 hr 600 Value, 5d was measured.
The bacterial solution was inoculated at a concentration of up to OD600 of 0.5 at 2% into four gradients of beef extract peptone medium pH 8, 9, 10 and 11, respectively, prepared with NaOH, in parallel 3 times. Culturing at 37 deg.C 180r/min for 2-5 d, observing growth vigor and recording data, and measuring OD every 24 hr 600 Value, 5d was measured. See table 9;
TABLE 9 salt and alkali resistance test of SSF12 strains
As the salt concentration and pH increased, the absorbance of the strain tended to increase, and by day 4, the absorbance of the strain tended to stabilize. The results of the salt tolerance test and the alkali tolerance test show that SSF12 can grow under the conditions that the concentration of sodium chloride is 10% and the pH=10, which indicates that SSF strains have stronger salt tolerance and alkali tolerance.
EXAMPLE 6 compost pot experiments
The pot experiment aims to analyze the influence of SSF12 compost on the growth condition of plants under high saline-alkali concentration from the sowing time, and analyze the growth promotion condition of SSF12 fermentation compost.
Each flowerpot is filled with equal amount of soil with high saline-alkali concentration, 10 wheat seeds are sown, and then the wheat seeds are divided into three groups of 3 parallel, and natural fermented compost (natural fermented compost group) is applied to the first group; a second group applied with equal amounts of SSF12 bacteria fermented compost (SSF 12 fermented compost group); the third group replaced compost with distilled water (non-composted group).
And observing and recording the emergence condition of the seeds every two days after sowing, and calculating the emergence rate. As a result, it was found that both of the composted groups had an improvement in the emergence rate of wheat seeds as compared to the non-composted group, wherein the emergence rate of SSF 12-fermented composted group was highest, reaching 78.93%, nearly 1.35 times higher than 58.46% of that of wheat composted by natural fermentation, and nearly 2.0 times higher than 39.36% of that of wheat in the non-composted group. The results show that the two groups of composting treatment can improve the germination rate of wheat, wherein the SSF12 fermentation composting treatment group can obviously improve the germination rate of wheat and can obviously reduce the toxic action of soil salt and alkali on wheat seeds.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A strain for degrading cellulose and resisting salt and alkalinity is characterized in that the strain is named as Microbacterium arborescens SSF12, is classified as Microbacterium arborescens (Microbacterium arborescens), and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 25296 and a preservation address of North Chen West Lu 1 No. 3 in the Korean region of Beijing city at the day of 7 months 14 of 2022.
2. Use of the microbacterium arborescens SSF12 of claim 1 in the fermentative production of cellulases.
3. Use of microbacterium arborescens SSF12 according to claim 1 in fermented straw compost.
4. Use of microbacterium arborescens SSF12 in fermented straw compost according to claim 3, characterized in that the straw is fermented by producing cellulases including filter paper enzymes, endoglucanases, exoglucanases and β -glucanases.
5. The use of the microbacterium arborescens SSF12 in the composting of fermented straw according to claim 3, wherein the cellulose degrading bacteria ferment to produce cellulase with the following enzyme activities: the filter paper has an enzyme activity of 33.45U/mL, the endoglucanase has an activity of 24.92U/mL, the exoglucanase has an activity of 31.88U/mL, and the beta-glucanase has an activity of 29.11U/mL.
6. Use of the microbacterium arborescens SSF12 of claim 1 in improving saline-alkali soil.
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