CN111057665B - Cellulose degrading bacterium n3 for producing IAA and application thereof - Google Patents
Cellulose degrading bacterium n3 for producing IAA and application thereof Download PDFInfo
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- CN111057665B CN111057665B CN201911267554.7A CN201911267554A CN111057665B CN 111057665 B CN111057665 B CN 111057665B CN 201911267554 A CN201911267554 A CN 201911267554A CN 111057665 B CN111057665 B CN 111057665B
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- cellulose
- iaa
- cellulose degrading
- straw
- promoting
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
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- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention provides an IAA-producing cellulose degrading bacterium n3 and application thereof, and relates to the technical field of agricultural microorganisms, wherein the cellulose degrading bacterium n3 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18613. The cellulose degrading bacteria n3 have strong capability of producing CMC enzyme, and can produce IAA, the CMC enzyme has the highest activity reaching 24.96U/mL, and the highest secretion of IAA can reach 19.07 mg/L. Therefore, the cellulose degrading bacteria n3 can be used for preparing a straw decomposition promoting microbial inoculum for producing IAA and/or CMC enzyme or having a growth promoting function, so that the cellulose degrading bacteria n3 can be applied to straw decomposition promoting and crop growth promoting, and straw returning efficiency and crop yield improvement are realized.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to an IAA-producing cellulose degrading bacterium n3 and application thereof.
Background
The straw returning field is a measure for increasing the yield of crops for fertilizing the soil fertility, which is generally regarded as important in the world at present, and plays an important role in improving the physical properties of soil, improving the organic matter level of the soil, increasing the biological activity of the soil, improving the nutrient supply level of the soil and the like while avoiding air pollution caused by the straw burning process. The straw has rich organic matters, nitrogen, phosphorus, potassium and other nutrient elements, can be converted into a nutrient form which is easy to absorb and utilize by crops, and the active effect of returning the straw to the field can be fully utilized, so that the resource utilization can be effectively improved, the yield of the crops can be increased, and the nutrient circulation can be promoted.
The straws mainly comprise substances which are difficult to degrade, such as lignin, cellulose, hemicellulose and the like, wherein the cellulose content is highest, and the straw decomposition speed is slow in a natural state. At present, the scholars at home and abroad generally consider that straw decomposing bacteria for producing cellulose degrading enzyme (such as carboxymethyl cellulose degrading enzyme, CMC) is applied when straw is returned to the field so as to accelerate the decomposition of waste such as straw and the like. However, whether functional microorganisms in the straw rotting agent can successfully colonize is influenced by the soil resource abundance, the competition strength of indigenous microorganisms and the like, so that the requirement on the area matching of the straw rotting agent is high, and the straw rotting agents applied to different soils in different areas are different, so that the strain is screened in the application area as much as possible to prepare the straw rotting agent so as to improve the straw rotting degree and the straw rate in the area.
The black sand ginger soil has the undesirable characteristics of dry shrinkage, wet swelling, easy drought and waterlogging, poor soil tilth and low fertility level, causes adverse effects on the growth of crops, and is typical low-yield soil in China. The black sand soil of the sand ginger is heavy in viscosity and poor in soil structure, the problems of drought, waterlogging, stiffness, thinness and the like are easy to occur in production, soil microorganisms and plough layer soil are low in fertility activity and not beneficial to colonization of exogenous strains, and therefore the application effect of the common straw rotting agent on the black sand of the sand ginger is poor. The sand ginger black soil is mostly a wheat-jade rotation area, the rotation time interval is short, and if straw decomposition is incomplete after the straw decomposition agent is applied, on one hand, previous crops cannot decompose in time and are accumulated and retained in soil for a long time, so that the seeding of the next crop is influenced; on the other hand, the straws which are not completely decomposed in the soil can cause that the wheat jade seeds can not be combined with the soil, so that the wheat jade seeds are difficult to germinate and compete for nutrients with crops, and the crop emergence rate is reduced and the yield is reduced.
Indoleacetic acid (IAA) is one of the plant hormones, is a signal substance that produces auxin-regulating substances, is ubiquitous in plants, and is an endogenous auxin ubiquitous in plants. The growth promoting effect of the auxin mainly promotes the growth of cells, particularly the elongation of the cells, and has positive significance on the aspects of crop growth and yield improvement. Therefore, the strain which has high-efficiency corrosion promotion and growth promotion functions is beneficial to the growth and development of the crops of the sand ginger black soil wheat jade rotation system and keeps the excellent properties of soil.
At present, most scholars at home and abroad only study the cellulose degradation capability or growth promotion capability of a certain strain, and are dedicated to study a certain function of a certain strain, but the strain with multiple functions is rarely seen.
Disclosure of Invention
In view of the above, the invention aims to provide a strain of efficient CMC-producing cellulose degrading bacteria n3 selected from sand ginger black soil, which can improve cellulose degradation rate and promote rotting of straws, thereby promoting field returning straw decomposition and improving field returning efficiency of straws; in addition, the strain can also produce IAA with high yield, promote the germination and growth of crop seeds and realize the yield increase of crops.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an IAA-producing cellulose degrading bacterium n3, which is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 18613.
The invention also provides application of the cellulose degrading bacterium n3 in preparation of IAA and/or CMC enzymes.
Preferably, when the cellulose degrading bacterium n3 is used for preparing IAA, the method comprises the following steps: adjusting the pH value of an LB culture medium containing 100 mg/LL-tryptophan to be 6.0-9.0, inoculating the bacterial suspension of the cellulose degrading bacteria n3, and performing shake culture; the volume of the bacterial suspension is 1% of the volume of the LB culture medium; OD of the bacterial suspension600The value is 0.8 to 1.2.
Preferably, the temperature of the shaking culture is 28-30 ℃, and the shaking speed is 160-180 rpm.
Preferably, when the cellulose-degrading bacterium n3 is used for preparing the CMC enzyme, the method comprises the following steps: adjusting the pH value of a liquid fermentation medium to 4.0-6.0, inoculating the cellulose degrading bacteria n3, and performing shake culture; the inoculation volume of the cellulose-degrading bacteria n3 is 1% of the volume of the liquid fermentation medium; the liquid fermentation medium comprises the following raw materials in concentration: 6g/L of sodium chloride, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of calcium chloride, 0.5g/L of monopotassium phosphate, 10g/L of yeast extract and 20g/L of straw.
Preferably, the LB culture medium or the liquid fermentation culture medium further comprises the following components in percentage by mass: 0.1% carbon source, 1% nitrogen source.
Preferably, the carbon source comprises one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose;
the nitrogen source comprises one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone.
The invention also provides application of the cellulose degrading bacterium n3 in preparation of a decay-promoting and growth-promoting bacterium agent.
Preferably, the type of the decay-promoting and growth-promoting microbial inoculum is a microbial aqua; when the microbial water agent is applied, the inoculation amount is 1-9 multiplied by 10 in terms of the amount of the cellulose degradation bacteria n36CFU/g straw or 1-9 multiplied by 107CFU/g soil.
The invention also provides application of the cellulose degrading bacteria n3 in promoting straw returning efficiency and crop yield increase.
The invention provides an IAA-producing cellulose degrading bacterium n3, which is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 18613. The cellulose degrading bacteria n3 have smooth surface, small bacterial colony, neat edge, opaqueness and slight yellow color; the cellulose degrading bacteria n3 are gram-negative bacteria, aerobic, chemoheterotrophic, catalase-positive, M.R-test-negative, VP-test-negative, starch hydrolysis-negative, gelatin hydrolysis-negative and citrate utilization-negative.
The cellulose degrading bacteria n3 can produce indoleacetic acid with high yield, has strong capability of producing CMC enzyme, has higher crop growth promoting and straw rotting promoting capabilities, the IAA secretion amount can reach 19.07mg/L to the maximum, and the CMC enzyme activity can reach 24.96U/mL to the maximum. Therefore, the cellulose degrading bacteria n3 can be used for preparing the straw-decomposing inoculant with the growth-promoting function, and can be applied to straw-decomposing promotion, straw returning efficiency promotion and crop yield increase.
The cellulose degrading bacterium n3 can be used for multiple purposes, and the maximum efficiency of the strain is exerted, so that the growth of crops can be promoted, and the yield of the crops can be increased; on the other hand, the microbial fertilizer can accelerate cellulose degradation, promote straw decay and strongly promote straw returning, has the potential of being used as a functional microbial fertilizer and contributes to the promotion of agricultural green development.
Biological preservation information
The cellulose degrading bacteria n3 are classified and named as Azospirillum zeae, are preserved in the China general microbiological culture collection management center in 2019, 09 and 23 days, and are located at the microbial research institute of China academy of sciences, No. 3, North Chen Xilu No.1, Beijing, the sunward area, and the preservation number is CGMCC No. 18613.
Drawings
FIG. 1 is a colony diagram of a cellulose-degrading bacterium n3 provided by the present invention;
FIG. 2 shows the CMC-producing enzyme capacity of different strains;
FIG. 3 shows IAA-producing ability of different strains;
FIG. 4 phylogenetic tree of n3 strain constructed from the 16S rDNA sequence;
FIG. 5 is a graph showing the effect of different pH values on the activity of CMC-producing enzyme by cellulose-degrading bacteria n 3;
FIG. 6 is a graph showing the effect of different liquid contents on the activity of CMC-producing enzyme by cellulose-degrading bacteria n 3;
FIG. 7 is a graph showing the effect of different nitrogen sources on the activity of CMC-producing enzyme by cellulose-degrading bacteria n 3;
FIG. 8 is a graph showing the effect of different culture times on IAA production by cellulose-degrading bacteria n 3;
FIG. 9 is a graph showing the effect of different culture times on the growth of cellulose-degrading bacteria n 3;
FIG. 10 is a graph showing the effect of different liquid contents on IAA production by cellulose-degrading bacteria n 3;
FIG. 11 is a graph showing the effect of different liquid contents on the growth of cellulose-degrading bacteria n 3;
FIG. 12 is a graph showing the effect of different initial pH on IAA production by cellulose-degrading bacteria n 3;
FIG. 13 is a graph showing the effect of different initial pH values on the growth of cellulose-degrading bacteria n 3;
FIG. 14 is a graph showing the effect of different carbon sources on IAA production by cellulose-degrading bacteria n 3;
FIG. 15 is a graph showing the effect of different carbon sources on the growth of cellulose-degrading bacteria n 3;
FIG. 16 is a graph showing the effect of different nitrogen sources on IAA production by cellulose-degrading bacteria n 3;
FIG. 17 is a graph showing the effect of different nitrogen sources on the growth of cellulose-degrading bacteria n 3;
FIG. 18 is a graph showing the influence of straw decomposition promoting ability of different strains.
Detailed Description
The invention provides an IAA-producing cellulose degrading bacterium n3, which is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 18613.
The cellulose degrading bacteria n3 are obtained by screening sand ginger black soil collected from agricultural demonstration scientific and technological park in Mongolian county of Anhui province, and after the verification, the cellulose degrading bacteria n3 are gram-negative bacteria, aerobic bacteria, chemoheterotrophic bacteria, catalase-positive bacteria, M.R test-negative bacteria, VP test-negative bacteria, starch hydrolysis-negative bacteria, gelatin hydrolysis-negative bacteria and citrate utilization-negative bacteria; the colony structure is shown in FIG. 1, and has smooth surface, small colony, neat edge, non-transparency and yellowish color.
The invention also provides application of the cellulose degrading bacterium n3 in preparation of IAA and/or CMC enzymes.
In the present invention, when IAA is produced using the cellulose-degrading bacterium n3, it preferably includes the steps of: adjusting the pH value of an LB culture medium containing 100mg/L L-tryptophan to be 6.0-9.0, inoculating the bacterial suspension of the cellulose degrading bacteria n3, and performing shake culture; the bacterial suspension is obtained by picking a ring of bacteria from a solid culture medium by using an inoculating ring, inoculating the ring of bacteria into a 25ml test tube containing 6ml of LB culture medium, plugging the test tube, and culturing the test tube in a shaking table at 30 ℃ and 180rpm overnight; OD of the bacterial suspension600The value is 0.8-1.2, and the inoculation volume is the volume of the LB culture medium1 percent. In the present invention, when the shake culture is performed, the liquid loading of the culture medium is preferably 25mL/250mL, and the culture time is preferably 15 hours, at which time the highest amount of IAA-producing cellulose-degrading bacterium n3 and the best growth ability of the strain are cultured. The temperature of the shaking culture is preferably 28-30 ℃, and the shaking speed is preferably 160-180 rpm.
In the present invention, when preparing the CMC enzyme using the cellulose-degrading bacterium n3, the method preferably comprises the steps of: adjusting the pH value of a liquid fermentation medium to 4.0-6.0, inoculating the cellulose degrading bacteria n3, and performing shake culture; the inoculation volume of the cellulose-degrading bacteria n3 is 1% of the volume of the LB culture medium; the liquid fermentation comprises the following raw materials in concentration: 6g/L of sodium chloride, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of calcium chloride, 0.5g/L of monopotassium phosphate, 10g/L of yeast extract and 20g/L of straw.
In the invention, the LB medium or the liquid fermentation medium preferably further comprises the following components in percentage by mass: 0.1% carbon source, 1% nitrogen source. The carbon source in the LB culture medium preferably comprises one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose, and is more preferably mannitol; the nitrogen source preferably includes one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, and more preferably yeast powder. The nitrogen source in the liquid medium of the present invention preferably includes one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, and more preferably yeast powder or peptone.
The invention also provides application of the cellulose degrading bacterium n3 in preparation of a decay-promoting and growth-promoting bacterium agent.
The type of the decay-promoting and growth-promoting microbial inoculum is preferably a microbial aqua; when the aqueous bacteria preparation is applied, the inoculation amount is preferably 1-9 x 10 based on the amount of the cellulose degrading bacteria n36CFU/g straw or 1-9 multiplied by 107CFU/g soil, more preferably 5X 106CFU/g straw or 5 x 107CFU/g soil.
The invention also provides application of the cellulose degrading bacteria n3 in promoting straw returning efficiency and crop yield increase. The application method and application amount of the cellulose-degrading bacteria n3 in the application of the invention are the same as those described above, and are not described in detail herein.
The IAA-producing cellulose-degrading bacterium n3 and its application provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of reagents:
LB culture medium: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, adjusting pH to 7.0-7.2, and sterilizing at 121 ℃ for 20 minutes (20 g of agar is added to the solid medium).
Inorganic salt culture medium: 2.0g of ammonium sulfate, 0.5g of sodium dihydrogen phosphate, 0.5g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.1g of calcium dichloride and 1000mL of distilled water, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 20 minutes.
Liquid fermentation medium: 6g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.1g of calcium chloride, 0.5g of potassium dihydrogen phosphate, 10g of yeast extract, 20g of straw and 1000mL of distilled water, and sterilizing for 20 minutes at 121 ℃.
Enrichment culture medium: 20g of sodium carboxymethylcellulose, 5g of microcrystalline cellulose, 5g of cellulose powder, 1g of dipotassium hydrogen phosphate, 1g of nitric acid, 0.2g of magnesium sulfate heptahydrate, 0.1g of copper chloride dihydrate, 0.02g of ferric trichloride and 1000mL of distilled water. Sterilizing at 121 deg.C for 20 min.
Carboxymethyl cellulose medium: 15g of sodium carboxymethylcellulose, 1g of ammonium nitrate, 1g of yeast extract, 0.5g of magnesium sulfate heptahydrate, 1g of monopotassium phosphate, 15g of agar and 1000mL of distilled water. Sterilizing at 121 deg.C for 20 min.
2. Strain screening
Weighing 10g of soil sample from Kaempferia galanga Hemsl collected from agricultural demonstration scientific and technological park in Mongolian county of Anhui province, inoculating the soil sample into 90mL of sterile water, shaking the soil sample in a shaking table at 28 ℃ and 150rpm for 30min, taking 1mL of soil suspension in a sterile operation table, adding 9mL of sterile water into the soil suspension to prepare the soil suspension with the concentration of 10mL-1The stock solution of (1). Separating and purifying the strains by a dilution plate method, and storing the pure cultured strains in a refrigerator at 4 ℃.
The basic properties of the soil tested are shown in table 1:
TABLE 1 basic Properties of the soil tested
And respectively inoculating the separated and purified strains on a sodium carboxymethylcellulose selective culture medium, standing for 20min by a Congo red staining method, measuring the diameter (D) of a bacterial colony and the diameter (H) of a transparent ring, and preliminarily judging the strength of the strains in the capacity of degrading cellulose according to the H/D value.
All strains with cellulose degradation capacity are screened out by a Congo red dyeing method, and the n3 strain has the highest capacity of degrading cellulose according to H/D results.
Preparing a crude enzyme solution: inoculating the strain into liquid culture medium containing wheat straw powder as sole carbon source, performing liquid shake culture at 37 deg.C for 60 hr, and culturing the fermentation liquid at 4 deg.C and 5000r min-1Centrifuging for 10min to obtain supernatant as crude enzyme solution. Taking 0.2mL of supernatant, putting the supernatant into a 25mL dry scale test tube, adding 1.8mL of 1% CMC-Na solution (prepared by 0.1mol/L citric acid-sodium citrate buffer solution with the pH value of 4.8) into a water bath at 50 ℃ for 30min, adding 3.0mL of DNS reagent into the water bath, boiling the water bath for 5min, stopping the reaction and developing color. The solution was cooled by a cold water shower, the volume was adjusted to 25mL, and the solution was shaken up and the absorbance was measured at a wavelength of 520 nm. The blank control is that the enzyme solution is inactivated in boiling water bath for 15min, and other conditions are not changed.
The enzyme activity X of the test is 1000 XGX25/0.2X 30X 180.
In the formula: x is enzyme activity (U cangue) of the sample; 1000, converting multiple; g is the glucose milligram corresponding to the light absorption value on the standard curve; 25, constant volume (mL); 0.2 enzyme addition (mL); 30 is action time (min); molecular weight of glucose (g/moL) 180. Under the above conditions, the enzyme activity was defined in terms of the international unit specification as the amount of enzyme catalyzing hydrolysis of the substrate (sodium carboxymethylcellulose) to 1pmol glucose per minute being 1 enzyme activity unit U.
The n3 in the 8 strains screened out has the strongest capacity of degrading cellulose, is obviously higher than other strains, and the CMC enzyme activity can reach 20.60 U.mL-1。
Qualitative determination comprises inoculating bacteria with cellulose degradation function to bacteria containing L-tryptophan (100 mg. L)-1) The strain was cultured in LB liquid medium (1 d) at 30 ℃ and 180rpm, 100. mu.L of the bacterial suspension was dropped on a white ceramic plate, and 100. mu.L of Salkowski colorimetric solution (50mL of 35% HClO) was added thereto4，1 mL 0.5mol·L-1FeCl3And is stored in a dark place), the white ceramic plate is kept in a dark place at room temperature for 30 minutes and then is observed, the reddish color shows that the white ceramic plate can secrete indoleacetic acid, the shade of the color shows that the white ceramic plate can produce the indoleacetic acid, the non-discoloring shows that the white ceramic plate can not produce the indoleacetic acid, and the positive control is 100 mu L of LB culture medium of non-inoculated bacteria liquid, and 100 mu L of Salkowski colorimetric liquid is added.
Quantitative determination, namely performing quantitative determination on the strains with the capability of secreting IAA screened by qualitative analysis, performing qualitative determination on the culture conditions, and determining the OD of the bacterial suspension by using a spectrophotometry method600Then sucking 5mL of bacterial suspension, centrifuging at 10000rpm for 10 minutes, taking 2mL of supernatant, adding an equal volume of Salkowski colorimetric solution, standing for 30 minutes in a dark room temperature, measuring the OD value of the solution when the wavelength is 530nm, and calculating the IAA content in the bacterial suspension by an IAA standard curve.
Through IAA qualitative analysis, 5 strains of bacteria have IAA production capacity which is n1, n3, n4, n5 and n8 respectively, and through quantitative determination, the result shows that the n3 strain has the strongest IAA production capacity, and the concentration can reach 19.07 mg.L-1Significantly higher than other strains.
The cellulose degrading bacteria n3 with the strongest capability of producing CMC enzyme and IAA and higher wheat straw rotting promoting capability can be screened out through the determination.
The strains screened and separated by the method are sequenced by Nanjing company, compared in a GenBank database according to the obtained 16S rDNA (SEQ ID NO.1) sequence result, homologous sequences are searched by Blast, and a phylogenetic tree is constructed by using MEGA5.0 software and a Neighbour-Joining method. Combining morphological analysis and the physiological and biochemical result characteristics of the strain, and identifying the strain as azospirillumzeae. The phylogenetic tree of the n3 strain constructed from the 16S rDNA sequence is shown in FIG. 4.
The physiological and biochemical properties of the strain were counted, as shown in table 2:
TABLE 2 physiological and biochemical characteristics of cellulose-degrading bacteria n3
Note: + indicates a positive reaction, and-indicates a negative reaction
Example 2
Aiming at different pH values, ventilation volumes and different nitrogen sources, the influence of the test on the CMC enzyme production capability of the strain is tested
1. Effect of initial pH of Medium on the ability to produce CMC enzyme
Inoculating the strain in liquid fermentation culture medium with wheat straw powder as sole carbon source, performing liquid shake-flask culture at 37 deg.C for 60h, and measuring OD with spectrophotometer520The value is obtained. Setting the initial pH values to be 4, 5, 6, 7, 8, 9 and 10 respectively, and measuring the content of the CMC-producing enzyme by using a spectrophotometer after culturing for 60 hours.
The results are shown in FIG. 2: when the pH value is 5.0, the CMC enzyme activity is highest and reaches 24.96 U.mL-1The enzyme activity is 24.85 U.mL when the pH value is 4.0-1The results show that the n3 strain has strong acid resistance, and the CMC enzyme activity is obviously higher than that under other pH conditions when the pH is 4.0 and 5.0.
2. Effect of ventilation on the ability to produce CMC enzyme
Inoculating the strain in liquid fermentation culture medium with wheat straw powder as sole carbon source, performing liquid shake-flask culture at 37 deg.C for 60h, and measuring OD with spectrophotometer520The value is obtained. Setting 25mL, 50mL, 75mL, 100mL and 150mL of culture solution, filling the culture solution into a 250mL triangular flask, culturing for 60h, and measuring the content of the CMC-producing enzyme by using a spectrophotometer.
The results are shown in FIG. 3: when the liquid loading volume of a 250mL triangular flask is 25mL and the ventilation volume is the maximum, the activity of the N3 strain for producing the CMC enzyme is the highest and is 15.33 U.mL-1。
3. Effect of Nitrogen Source on CMC-producing enzyme ability
Respectively adding 0.1% (m/V) nitrogen source into a liquid fermentation culture medium which takes wheat straw powder as a unique carbon source, wherein the nitrogen source comprises potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, carrying out shake-flask culture for 60h in a liquid at 37 ℃, and measuring the content of the strain CMC enzyme by using a spectro-gradiometer.
The results are shown in FIG. 4: different nitrogen sources have different influences on the CMC enzyme producing capability of the n3 strain, wherein the CMC enzyme producing capability is most obvious when yeast powder and peptone are used as nitrogen sources and respectively reaches 18.30 and 16.02 U.mL-1。
Example 3
Testing the influence of different pH values, ventilation volumes, different time, different carbon sources and different nitrogen sources on the yield and the growth volume of the strain IAA
1. Influence of fermentation time on yield and growth of strain IAA
Will contain 100 mg.L-150mL LB liquid medium (IAA detection medium) of L-tryptophan is filled into a 250mL triangular flask, a bacterial suspension with OD value of about 1 is inoculated according to the inoculation amount of 1% (V/V), shaking culture is carried out at 30 ℃, 180rpm, dynamic sampling is carried out for 10, 15, 20, 32, 44 and 56 hours respectively, and the growth condition (OD) of the bacterial strain is determined600) And IAA producing ability (OD)530) Three replicates per treatment were set.
The results are shown in FIG. 5: OD at 20h600The maximum value is reached, the strain grows for 20 hours and has a fading trend, the IAA content of the strain is basically consistent with the growth condition of the strain, the IAA content of the strain increases logarithmically in 10-15 hours, and the IAA content reaches the maximum value at 15 hours and is 18.66 mg.L-1After 15h, the IAA-producing ability of the strain gradually decreased.
2. Influence of pH value on IAA yield and growth of strain
Will contain 100 mg.L-1Adjusting the LB culture medium of L-tryptophan to different pH values (4, 5, 6, 7, 8, 9 and 10), respectively, placing 50mL LB liquid culture medium into a 250mL triangular flask, inoculating bacterial suspension with OD value of about 1 according to the inoculation amount of 1% (V/V), shake culturing at 30 ℃, 180rpm for 24h, dynamically sampling at 10, 15, 20, 32, 44 and 56h, respectively, and determining bacteriaGrowth status of the plants (OD)600) And IAA producing ability (OD)530) Three replicates per treatment were set.
The results are shown in FIG. 6: at pH 6.0, the OD of the strain600Value and OD530All values reach the maximum, OD6000.70, the IAA production concentration is 19.03 mg.L-1. The pH of the sand ginger black soil is acidic, which shows that the life habit of the bacterial strain is identical with that of the sand ginger black soil. The pH value is 7.0-9.0, the growth condition of the strain and the IAA content are relatively stable.
3. Effect of aeration on Strain IAA production and Strain growth
Will contain 100 mg.L-1The L-tryptophan LB liquid culture medium is filled into a 250mL triangular flask according to the proportion of 25mL, 50mL, 75mL, 100mL and 150mL, a bacterial suspension with the OD value of about 1 is inoculated according to the inoculation amount of 1% (V/V), shaking culture is carried out at 30 ℃ and 180rpm, dynamic sampling is carried out for 10, 15, 20, 32, 44 and 56 hours respectively, and the growth condition (OD) of the bacterial strain is determined600) And IAA producing ability (OD)530) Three replicates per treatment were set.
The results are shown in FIG. 7: since the strain n3 is aerobic bacteria, when the liquid loading volume of a 250mL triangular flask is 25mL, the growth condition and the IAA production condition are optimal, and the growth trend and the IAA production content of the n3 strain are generally reduced along with the increase of the liquid loading volume.
4. Influence of nitrogen source on yield and growth of strain IAA
In an inorganic salt medium (containing 100 mg. L) excluding ammonium sulfate-1L-tryptophan) is respectively added with 0.1 percent (W/V) nitrogen source, the nitrogen source comprises potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, 50mL of the mixture is taken and filled in a 250mL triangular flask, bacterial suspension with OD value about 1 is inoculated according to the inoculation amount of 1 percent (V/V), shaking culture is carried out for 24h at 30 ℃ and 180rpm, dynamic sampling is respectively carried out for 10, 15, 20, 32, 44 and 56h, and the growth condition (OD) of the bacterial strain is determined600) And IAA producing ability (OD)530)。
The results are shown in FIG. 8: growth amount (OD) of n3 strain using yeast powder as nitrogen source600) The maximum value is 0.64, and when yeast powder is used as a nitrogen source, the IAA content (OD) of the strain is generated530) The highest of the number of the channels is,up to 36.18 mg.L-1。
5. Influence of carbon Source on the yield of IAA Strain and the growth of Strain
In an inorganic salt medium (containing 100 mg. L)-1L-tryptophan) is respectively added with 1% (W/V) of carbon source, the carbon source comprises glucose, mannitol, sucrose, maltose, xylose, lactose and fructose, 50mL of the carbon source is taken and filled in a 250mL triangular flask, bacterial suspension with OD value about 1 is inoculated according to the inoculation amount of 1% (V/V), shaking culture is carried out for 24h at 30 ℃ and 180rpm, dynamic sampling is carried out for 10, 15, 20, 32, 44 and 56h respectively, and the growth condition (OD) of the bacterial strain is measured600) And IAA producing ability (OD)530)。
The results are shown in FIG. 9: when mannitol is used as a carbon source, the highest IAA production capacity can reach 7.36 mg.L-1The growth amount of the strain also reaches the maximum.
Example 4:
straw corrosion promotion test of microbial inoculum
The test process comprises the following steps: weighing 5g of crushed wheat straw powder which is sieved by a 20-mesh sieve in a 250mL triangular flask, adding 30mL of water, 2g of sodium nitrate and 10mL of bacterial liquid centrifugally resuspended in sterile water to enable the concentration of the bacterial liquid to reach 108cfu·mL-1And (4) taking out the straws after 15 days of culture, repeatedly cleaning the side walls by using distilled water, drying at 80 ℃ to constant weight, additionally arranging sterile water instead of bacterial liquid, performing other steps in a consistent manner, and repeating the steps for each treatment.
The degradation test is shown in fig. 15: after 15 days of degradation, the degradation rate of the wheat straw treated by adding the microbial inoculum reaches 15.1 percent, which is higher than that of a control 9.8 percent.
Example 5:
corn growth promotion test of microbial inoculum
Test soil: agricultural demonstration garden from Mongolian county of Anhui province
The test method comprises the following steps: uniformly spreading corn seeds in a culture dish attached with clean filter paper, soaking for 2 days at 28 ℃ by using distilled water, and selecting the seeds with good and regular buds for sowing. Planting 2 corn plants in a flowerpot (with upper opening diameter of 5cm, lower bottom diameter of 3cm and height of 5cm) as culture container with soil of 5.0kg per pot, culturing test strain n3, and making into bacteria liquidAgent according to 5X 108The inoculation amount of CFU/g was inoculated into the soil, and 5 replicates per group were used without inoculum as a control. Before sowing, the maize seeds after germination acceleration are respectively soaked in the bacterial liquid. And (3) placing the soaked seeds in each pot at a distance of 1cm from the upper edge of the soil, sowing the seeds in the pots, covering a layer of floating soil, sowing one seed in each pot of corn, and planting 5 pots in each treatment. After sowing, the pots were placed in a climatic chamber (30 ℃ in the day, 25 ℃ at night, 16 hours for light). All treatments were randomized under the same conditions and pot watered at the same time. The dosages of the N, K fertilizers are 2.0g of urea and 1.4g of potassium chloride in each pot respectively.
And (3) harvesting a sample: and sampling the corn after the corn grows for 49 days, and measuring the plant height, the dry weight and the fresh weight of the corn.
TABLE 3 Effect of inoculum strain n3 on maize plants
Note: indicates significant difference between two treatments (P <0.05), and indicates very significant difference between two treatments (P <0.01)
The results are shown in Table 3: the strain n3 has the function of promoting the growth of plants, and in the test, after the inoculation treatment, the plant height and the SPAD value of the corn plants are increased and have obvious difference (P is less than 0.5) compared with the control treatment; the total potassium differed very significantly from the control treatment (P < 0.01). Wherein, compared with the control treatment, the plant height and the SPAD value of the corn are respectively increased by 18.3 percent and 5.24 percent, and the total potassium is increased by 15.6 percent. To sum up: the inoculated plants are obviously superior to the control treatment in the aspects of shape and nutrient absorption.
The cellulose degrading bacterium n3 for producing IAA and the application thereof provided by the invention have the capability of producing IAA and CMC enzyme, and can promote the decay of straws and improve the cellulose degradation rate, thereby promoting the straw returning efficiency, reducing the environmental pollution and realizing the yield increase of crops.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> agriculture university of Anhui
<120> IAA-producing cellulose degrading bacterium n3 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 560
<212> DNA
<213> Azospirillum zeae
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cctacgggag gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagcaatgc 60
cgcgtgagtg atgaaggcct tagggttgta aagctctttc gcacgcgacg atgatgacgg 120
cagcgtgaga agaagccccg gctaacttcg tgccagcagc cgcggtaata cgaagggggc 180
tagcgttgtt cggaattact gggcgtaaag ggcgcgtagg cggcctgttt agtcagaagt 240
gaaagctccg ggctcaacct gggaatagct tttgatactg gcaggcttga gttccggaga 300
ggatggtgga attcccagtg tagaggtgaa attcgtagat attgggaaga acaccggtgg 360
cgaaggcggc catctggacg gacactgacg ctgaggcgcg aaagcgtggg gagcaaacag 420
gattagatac cctggtagtc cacgccgtaa acgatgaatg ctagacgtcg gggtgcatgc 480
acttcggtgt cgccgctaac gcattaagca ttccgcctgg ggagtacggc cgcaaggtta 540
aaactcaaag gaattgacgg 560
Claims (10)
1. IAA-producing cellulose-degrading bacterium, Azospirillum maydis (A)Azospirillum zeae) n3, which is characterized in that the preservation number is CGMCC No. 18613.
2. Use of the cellulose degrading bacterium azospirillum zeae n3 as claimed in claim 1 for the preparation of IAA and/or CMC enzymes.
3. The use of claim 2, wherein the cellulose degrading bacterium azospirillum zeae n3 is used for preparing IAA, and the method comprises the following steps: adjusting the pH value of an LB culture medium containing 100mg/L L-tryptophan to be 6.0-9.0, inoculating the bacterial suspension of the cellulose degrading bacteria, namely the azospirillum zeae n3, and performing shake culture; the volume of the bacterial suspension is 1% of the volume of the LB culture medium; OD of the bacterial suspension600The value is 0.8 to 1.2.
4. The use according to claim 3, wherein the temperature of the shaking culture is 28-30 ℃ and the shaking speed is 160-180 rpm.
5. The use of claim 2, wherein the cellulose-degrading bacterium azospirillum zeae n3 is used for preparing CMC enzyme, and the method comprises the following steps: adjusting the pH value of a liquid fermentation medium to 4.0-6.0, inoculating the cellulose degrading bacteria, namely the azospirillum zeae n3, and performing shake culture; the inoculation volume of the cellulose degrading bacteria, namely the azospirillum zeae n3 is 1% of the volume of the liquid fermentation medium; the liquid fermentation medium comprises the following raw materials in concentration: 6g/L of sodium chloride, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of calcium chloride, 0.5g/L of monopotassium phosphate, 10g/L of yeast extract and 20g/L of straw.
6. The application of claim 5, wherein the LB culture medium or the liquid fermentation culture medium further comprises the following components in percentage by mass: 0.1% carbon source, 1% nitrogen source.
7. The use of claim 6, wherein the carbon source comprises one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose;
the nitrogen source comprises one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone.
8. The use of the cellulose degrading bacterium azospirillum zeae n3 in the preparation of a decay promoting growth-promoting microbial inoculum according to claim 1.
9. The use according to claim 8, wherein the type of the corrosion-promoting and growth-promoting microbial inoculum is a microbial aqua; when the microbial water agent is applied, the inoculation amount is 1-9 multiplied by 10 according to the amount of the cellulose degrading bacteria azospirillum zeae n36CFU/g straw or 1-9 multiplied by 107CFU/g soil.
10. The use of the cellulose degrading bacterium, azospirillum zeae n3, according to claim 1, for promoting straw return efficiency and crop yield increase.
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