CN111172051A - Method for expanding culture of rice straw degradation bacterial line - Google Patents

Abstract

The invention relates to the technical field of agricultural biology, in particular to an expanded culture method of a rice straw degradation bacterial line. Inoculating the stable straw degradation bacterial system into 4-10L of fresh enrichment medium according to the inoculation amount of 8-10 wt%, and standing and culturing at room temperature until filter paper strips in the medium disintegrate; inoculating the 4-10L of culture into 40-100L of enrichment medium, and standing for culture until the filter paper strips in the medium disintegrate to complete the expanding culture; the invention takes the enrichment culture medium as the strain expanding culture medium, and a large amount of rice straw degrading strains with stable degrading flora can be obtained by amplifying culture step by step and can be used for decomposing rice straws in returning field.

Description

Method for expanding culture of rice straw degradation bacterial line

Technical Field

The invention relates to the technical field of agricultural biology, in particular to an expanded culture method of a rice straw degradation bacterial line.

Background

China is a big agricultural country, and a large amount of crop straws are idle or burnt every year, so that resources are wasted, and serious air pollution is caused. The rice is planted in one of three grain crops in China in many areas, the practical problems of low temperature, short stubble period and the like in winter after autumn harvest seriously affect the implementation of returning the rice straws to the field in the planting area, the straw returning not only can effectively increase the organic matter content of soil, improve the soil fertility and improve the soil structure, but also solves the problems of reutilization of agricultural waste straws and environmental pollution to a certain extent. However, the straw containing rich cellulose has a complex structure, is slowly decomposed in the returned soil and affects the planting of the next crop, and the straw returning method is not easily accepted by wide farmers and is difficult to popularize.

The rice straw degrading bacteria system can accelerate the decomposition of the returned rice straw, and brings hope for the popularization and implementation of the rice straw returning mode. At present, most of straw decomposition agents in the market are composite bacterial systems compounded by a plurality of pure bacteria, the original bacterial sources are different, the synergistic effect among the bacterial is poor, the straw decomposition agents are easy to generate antagonistic action with indigenous bacteria after being scattered into returned straws, and the straw decomposition effect is not ideal. In recent years, a plurality of researches take various samples such as rotten straws, rotten fallen leaves, rotten wood, rotten grass, animal manure, compost, sawdust, edible fungi cultivation leftovers, wheat field soil, forest soil, perennial straw returning soil, garden soil, grassland soil and the like as bacteria sources, and take the straws as target degradants, and a plurality of groups of straw degradation bacteria systems are obtained through domestication and enrichment culture. However, the complexity of the composition of the degrading strain determines the difficulty of the expanding culture, the strain contains dozens of even hundreds of microorganisms and strains which can be cultured and can not be cultured, the strains can not be completely separated out and compounded after the expanding culture, all the strains can only be cultured together, and all the functional strains are ensured not to be lost in the culture process. Therefore, it is difficult to expand the degrading strains according to the conventional expansion culture method.

Disclosure of Invention

The invention aims to provide a rice straw degradation bacterial line expanding culture method.

In order to achieve the purpose, the invention adopts the technical scheme that:

an expanded culture method of a rice straw degradation bacterial line,

1) inoculating the stable straw degradation bacterial system into 4-10L of fresh enrichment medium according to the inoculation amount of 8-10 wt%, and standing and culturing at room temperature until filter paper strips in the medium disintegrate;

2) inoculating the 4-10L of culture into 40-100L of enrichment medium, and standing for culture until the filter paper strips in the culture medium disintegrate to complete the expanding culture;

the formula of the enrichment culture medium is that each liter of culture medium contains the following components: K2HPO41.0-3.0 g, (NH4)2SO41.0-3.0g, NaNO31.0-3.0 g, MgSO4 & 7H2O 0.5.5-1.5 g, FeSO4 & 7H2O 0.01.01-0.05 g, KCl0.5-1.5g, sodium pyruvate 0.3-1.2g, yeast extract powder 0.5-1.5g, peptone 0.5-1.5g, soluble starch 0.5-1.5g, straw powder 5-10g, D-sorbitol 0.5-1.5g, trace element mixed solution 1-3ml, calcium solution 1-3ml, filter paper strip 2 pieces, and pH value is adjusted to 7.0-7.5.

The formula of the mixed solution of the trace elements in the culture enrichment medium is as follows (g/L): ZnSO4 & 7H2O 1.5.5-3.5 g, CoCl2 & 6H2O 0.15.15-0.50 g, Na2MoO4 & 2H2O 0.10.10-0.50 g, MnSO4 & H2O 1.5.5-3.0 g, CuSO4 & 5H2O0.15-0.35g, H3BO30.3-0.12 g, NiCl2 & 6H2O 0.06.06-0.15 g and KI 0.06-0.15 g;

the calcium solution is CaCl₂The aqueous solution of (1) has a concentration of 3%.

The straw powder is prepared by naturally drying and crushing rice straws and then sieving the rice straws with a 40-mesh sieve for later use.

The stable straw degradation bacterial system is characterized in that rice straws are used as a bacterial source sample, an enrichment culture medium is added, a filter paper strip is used as a cellulose degradation indicator in the enrichment process, and a flora is formed after the indicator in the culture medium is disintegrated, namely the stable straw degradation bacterial system.

The rice straw degrading bacterial line comprises bacteria of Arcobacter, Bacteroides, Pseudomonas, Macellibacter, Comamonas, Anaerocella, dechlorobacter, desulfomicbium, enterococcus, lysinibacillus and treponema.

The filter paper strips are made of Hangzhou paper (60cm x 60cm) and cut into filter paper strips.

The invention has the beneficial effects that:

the rice straw degrading bacterial line is a bacterial line with stable flora and obtained by taking rice straws as target degradation products, and the obtained bacterial line can be gradually expanded and cultured by the expanding culture method, so that the using amount of the straw degrading bacterial line required by field straw returning work can be further realized. The strain contained in the rice straw degrading bacterial system after propagation is derived from the rice straw body, and the bacterial system is not antagonized by indigenous bacteria carried by the straw when being applied to the straw after being prepared into a microbial inoculum, so that the bacterial system is beneficial to decomposition of the straw. The expanding culture method of the invention creates feasibility for subculture and expanding culture of the strain system on the principle of comprehensive nutrition and cheap and easily obtained raw materials when designing the formula of the enrichment culture medium.

Drawings

FIG. 1 is a drawing of a rice straw-degrading strain-screened ligninase-producing strain plate.

FIG. 2 is an electropherogram of an amplification product of 16SrDNA of a ligninase-producing strain selected from a rice straw-degrading strain line according to an embodiment of the present invention.

Detailed Description

The present invention will be further explained with reference to examples.

Example 1

Rice straw degrading bacterial system expanding culture

1) And (3) sucking 40ml of the obtained stable straw degradation bacterial system, inoculating the stable straw degradation bacterial system into 4L of fresh enrichment medium according to the inoculation amount of 10%, and performing standing culture at room temperature until filter paper strips in the medium are disintegrated.

2) And (3) inoculating 4L of the culture into 40L of enrichment medium, and standing for culture until the filter paper strips in the culture medium are disintegrated, thus completing the propagation.

The culture medium formula for expanding culture contains the following components in each liter of culture medium: K2HPO41.0-3.0 g, (NH4)2SO41.0-3.0g, NaNO31.0-3.0 g, MgSO4 & 7H2O 0.5.5-1.5 g, FeSO4 & 7H2O 0.01.01-0.05 g, KCl0.5-1.5g, sodium pyruvate 0.3-1.2g, yeast extract powder 0.5-1.5g, peptone 0.5-1.5g, soluble starch 0.5-1.5g, straw powder 5-10g, D-sorbitol 0.5-1.5g, trace element mixed solution 1-3ml, calcium solution 1-3ml, filter paper strip 2 pieces, and pH value is adjusted to 7.0-7.5. Wherein the formula of the mixed solution of the trace elements in the culture medium is as follows (g/L): ZnSO4 & 7H2O 1.5.5-3.5 g, CoCl2 & 6H2O 0.15.15-0.50 g, Na2MoO4 & 2H2O 0.10.10-0.50 g, MnSO4 & H2O 1.5.5-3.0 g, CuSO4 & 5H2O0.15-0.35g, H3BO30.3-0.12 g, NiCl2 & 6H2O 0.06.06-0.15 g, KI 0.06-0.15 g; the calcium solution is a CaCl2 water solution with the concentration of 3%.

The strain includes Arcobacter, Bacteroides, Pseudomonas, Macellibacter, Comamonas, Anaerocella, dechlorobacter, desulformobium, enterococcus, lysinubacter and treponema.

Example 2

Screening and identification of lignin enzyme producing strain of rice straw degrading strain system after propagation

1. Experimental Material

1.1 bacterial sources

The test strain F is a rice straw degradation strain.

1.2 culture Medium

screening of lignin-degrading bacteria (screening of aniline blue plate) comprises 10g of yeast extract powder, 10g of glucose, 1.0g of aniline blue, 20g of agar, natural pH, 1000mL of distilled water (prepared with 1.0% aniline blue (Azure-B) mother liquor, and 1.0mL of mother liquor is added to each 100mL of culture medium before sterilization), ② manganese chloride screening plate comprises MnC 12.4 H200.15g of tetrahydrate, 20g of agar and 1000mL of distilled water, and 5.0g of peptone, ② sodium carboxymethylcellulose (CMC) -Na 5.0g of sodium chloride (NaCl), ② calcium carbonate (CaCo32.0g of mother powder), 1.0g of yeast, pH 7.0 g of PCS (PCS) is added to the final concentration of 0.005% after the culture medium is sterilized and cooled to about 50 ℃).

screening of cellulose-degrading bacteria ① g CMC-Na 20.0 g), ammonium sulfate ① g NH4)2SO42.0g, magnesium sulfate heptahydrate ① g MgSO 4.7H 2O 0.5.5 g, potassium dihydrogen phosphate ① g KH2PO41.0 g), sodium chloride ① g NaCl) 0.5g, Congo red 0.2g, agar 20.0g, distilled water 1000mL, pH 7.2, beef extract peptone inclined plane ① g beef extract 3 g), peptone 10g, sodium chloride ① g NaCl5 g), agar 20g, distilled water 1000mL, pH 7.4-7.6

1.3 drugs and reagents

Agar Bio-engineering (Shanghai) Inc.; sodium carboxymethylcellulose (CMC-Na) tokyo chemical industries co; ammonium sulfate ((NH4)2SO4) Citra Daloc Chemicals, Tianjin; magnesium sulfate heptahydrate (MgSO4 & 7H2O) Tianjinbo chemical Co., Ltd.; potassium dihydrogen phosphate (KH2PO4), calcium carbonate (CaCO3), and Yongda chemical reagent of Tianjin, glucose; congo red Shanghai leaf Biotech limited; chemical reagent of sodium chloride (NaCl) national drug group ltd; yeast extract powder Angel Yeast Co., Ltd; peptone, beef extract, beijing obo biotechnology, llc; aniline blue Shanghai leaf Biotech Co., Ltd; manganese chloride tetrahydrate (MnCl2 & 4H2O) Shanghai-sourced leaf Biotech Co., Ltd

alpha-naphthol Beijing Promega science and technology Limited, Hengxing chemical reagent manufacturing Limited liability company of absolute ethyl alcohol (CH2CHOH) Tianjin, and genome extraction kit Beijing kang, century Biotechnology Limited company

Shenyang applied ecology research institute of Chinese academy of sciences with 0.5 × TAE buffer solution; agarose Beijing Kulaibo science and technology, Inc.; goldview dye liquor, Beijing Boda Take BioGene technology, Inc.; 10 × buffer TaKaRa; loading buffer TaKaRa company; coli T14Marker TaKaRa; DL2000marker TaKaRa; taq DNA polymerase TaKaRa Co

Primers 27F 5 '-AGAGATTTGATCMTGGCTCAG-3' limited company of Shanghai Biotechnology engineering

Primers 1492R 5 '-TACGGHTACCTTGTTACG-3' Shanghai BioEngineers Limited

1.4 Experimental Equipment and Instrument

1.4.1 Experimental Equipment

Test tubes, marker pens, graduated cylinders, glass rods, pH paper, 500mL Erlenmeyer flasks, 250mL Erlenmeyer flasks, pipette tips, EP tubes, 0.22 μm filters, plastic plates, analytical balances, pipettes (specification: 10 μ L, 200 μ L, 1000 μ L).

1.4.2 Experimental instruments

Desk-top low temperature high speed centrifuge: germany Albender GmbH; constant temperature culture shaker: shanghai Zhicheng analytical instruments manufacturing, Inc.; top loading balance: Mettler-Tollido instruments (Shanghai) Co., Ltd

Analytical balance: shanghai Minqiao precision scientific instruments, Inc.; vertical pressure steam sterilizer:

LDZX-50KBS, Shanghai Shenan medical instruments factory; insulation can: tianjin City south suburb east mud iron works; electric heating constant temperature air blast drying cabinet: shenyang medical facilities; superclean bench: suzhou City, Antai air technologies, Inc.; gel imager: model number of ProteinSimple, USA:

FluorChem HD 2; numerical control super constant temperature bath: ningbo City Haimeitian Instrument works; electrophoresis apparatus: united states thermoelectric corporation; a refrigerator: the model BCD-215TD GA of Haier GmbH of Qingdao, China; PCR Instrument Biometra, Germany; a micro high-speed centrifuge: WTL-6K; hunan instruments centrifuge Limited Inc.; a pH meter: Mettler-Tollido instruments (Shanghai) Inc.; a vortex mixer:

scientific Industries Vortex-Genie 2; scientific Industries, USA.

2. Experimental methods

2.1 Primary reagent preparation

Agarose gel 0.8% agarose gel, weighing electrophoresis-grade agarose 0.24g, placing in a triangular flask, adding 30ml of AE electrophoresis buffer solution, and wrapping a perforated preservative film on the bottle mouth. The flask was placed in a microwave oven and added until the agarose had completely melted. Add 2. mu.L of gold view dye.

TAE Buffer ① 50 × TAE Buffer) weighing 242g Tris, Na2EDTA.2H2O 18.612g in 1L beaker, adding about 800ml deionized water into the beaker, stirring well, adding 57.1ml deionized waterGlacial acetic aciddissolving, adjusting pH to 8.3 with NaOH, adding deionized water to 1L, and storing at room temperature, 0.5 × TAE using the solution is diluted 100 times, i.e. 0.5 × TAE PCR system 50 μ L is divided into 12 parts, including double distilled water 451.2 μ L, 10 × buffer 60 μ L, dNTP 48 μ L, upstream primer (27F)12 μ L, downstream primer (1492R)12 μ L, Taq DNA polymerase 4.8 μ L, template (total genome DNA)1 μ L

2.2 Medium plate preparation

Accurately calculating and weighing the actual dosage of each medicine according to the formula, weighing distilled water into a beaker by using a dosing cylinder, adding each medicine (except agar), and stirring until the medicines are dissolved. And adjusting the pH value of the solution. And (3) subpackaging the prepared culture medium into triangular flasks, directly putting the weighed agar into the triangular flasks, and sleeving sealing films on the triangular flasks. Placing the triangular flask in a sterilizing pot, and sterilizing at 121 deg.C for 15 min. And after the sterilization is finished, cooling to 50-60 ℃, and pouring into a culture dish in a sterile room.

2.3 bevel preparation

Accurately calculating and weighing the actual dosage of each medicine according to the formula, weighing distilled water into a beaker by using a dosing cylinder, boiling the liquid culture medium, adding agar, and continuously heating until the agar is completely melted. During the heating process, the stirring is kept to prevent the agar from precipitating at the bottom of the bottle, and the fire power is controlled to prevent the culture gene from boiling and overflowing the container. After the agar had completely melted, the water lost by evaporation was made up with hot water. Before solidification, the mixture is subpackaged into test tubes, the test tubes are preferably 1/5-1/4 of the height, and test tube plugs are covered. Wrapping with newspaper, and sterilizing at 121 deg.C for 15 min. After the bacteria are finished, placing the sterilized test tube culture medium in an inclined manner before solidification, wherein the length of the placed inclined plane is preferably that the culture medium does not exceed half of the total lineation length of the test tube.

2.4 Experimental procedures

2.4.1 Selective plate Primary screening

Taking 36 dishes of aniline blue plate and Congo red plate, numbering A, B, C, D, E, F, G, H, I respectively, and 45 pieces of sterile water. Firstly, taking 5 pieces of 9mL sterile water, and numbering 10-1, 10-2, 10-3, 10-4 and 10-5 in sequence. Sucking 1mL of original bacterial liquid into a test tube of 10-1 by using a 100-1000-microliter pipette, and uniformly mixing. And (4) replacing a pipette tip, repeatedly blowing and sucking in a test tube with 10-1 dilution for several times in the same way, accurately transferring 1mL of bacteria liquid to be tested into a test tube with 10-2, and repeating the operation until the test tube with 10-5 is obtained. And respectively sucking 100 mu L of the bacterial fluid corresponding to the dilution gradient to the surface of the sterile plate by using a pipette of 20 mu L-200 mu L (wherein, according to the result of the pre-experiment, the dilution gradient of the sterile plate numbered A, B is 10-2 and 10-3, and the dilution gradient of the sterile plate numbered C, D, E, F, G, H, I is 10-4 and 10-5). And (3) lightly coating the surface of the plate by using a coating rod which is sterilized and dried in advance, so that the bacteria liquid is uniformly spread on the whole surface of the plate. After coating, numbering the culture medium, placing the culture dish upside down in a constant temperature incubator at 28 ℃ for culture, and observing the growth condition every day.

2.4.2 streaking Single colonies

From the above culture plate, single colonies (total 10, numbered 1-10) which produced a transparent circle were picked up, streaked by a divisional streaking method, and the streaked plate was placed in a constant temperature incubator at 28 ℃ and subjected to inverted culture for 48 hours, and the results of separation were observed.

2.4.3 test tube pure culture

A single colony on the plate-streaked medium was inoculated into a beef extract peptone liquid test tube medium and cultured in a shaker at 30 ℃ for 24 hours. The culture results were observed.

2.4.4 Collection of cells

1.5mL of the overnight cultured bacterial cells were transferred into a 2.0mL centrifuge tube, and after centrifugation at 12000rpm for 30 seconds, the supernatant was completely discarded.

2.4.5 genomic DNA extraction

② extracting ② 16 ② SrDNA ② according ② to ② the ② specification ② of ② a ② bacterial ② genome ② extraction ② kit ② of ② Beijing ② kang ②, ② the ② century ② Biotechnology ② Limited ② company ②, ② carrying ② out ② agarose ② gel ② electrophoresis ② (② Marker ②: ② lambda ② - ② E ②. ② coli ② T ② 14 ② Marker ②) ② after ② extraction ② is ② finished ②, ② checking ② the ② extraction ② result ②, ② storing ② the ② sample ② in ② a ② refrigerator ② at ② - ② 4 ② ℃ ② (② preparing ② 0.8 ②% ② agarose ② gel ②, ② weighing ② 0.24 ② g ② agarose ②, ② placing ② the ② agarose ② in ② a ② triangular ② flask ②, ② adding ② 30 ② mL ② TAE ② electrophoresis ② buffer ② solution ②, ② wrapping ② a ② perforated ② preservative ② film ② at ② a ② bottle ② opening ②, ② heating ② the ② triangular ② flask ② in ② a ② microwave ② oven ② until ② agarose ② is ② completely ② melted ②, ② cleaning ② and ② drying ② the ② inner ② groove ② of ② the ② organic ② glass ②, ② placing ② the ② organic ② glass ② in ② a ② gel ② making ② mold ②, ② selecting ② a ② gel ② making ② comb ② with ② a ② proper ② pore ② size ②, ② vertically ② erecting ② the ② gel ② making ② comb ② at ② one ② end ② of ② the ② electrophoresis ② gel ② making ② mold ②, ② enabling ② the ② bottom ② of ② the ② gel ② making ② comb ② to ② be ② 1 ② mm ② away ② from ② the ② bottom ② of ② the ② electrophoresis ② gel ② making ② mold ②, ② adding ② 2 ② mu ② L ② Goldview ② dye ② solution ② when ② the ② agarose ② gel ② is ② cooled ② to ② about ② 60 ② ℃ ②, ② carefully ② pouring ② the ② agarose ② gel ② on ② the ② agarose ② gel ② making ② comb ②, ② slowly ② pouring ② the ② agarose ② gel ② on ② the ② surface ② of ② the ② agarose ② gel ②, ② slowly ② forming ② agarose ② gel ②, ② enabling ② the ② agarose ② gel ② to ② be ② about ② 3 ② mm ②, ② after ② the ② agarose ② gel ② is ② unfolded ②, ② after ② the ② agarose ② gel ② is ② formed ②, ② placing ② the ② agarose ② gel ② in ② the ② agarose ② gel ② making ② agarose ② gel ②, ② keeping ② a ② power ② supply ②, ② placing ② the ② agarose ② gel ②, ② keeping ② a ② horizontal ② electrophoresis ② buffer ②, ② placing ② a ② horizontal ② electrophoresis ② groove ②, ② lifting ②, ② keeping ② a ② horizontal ② electrophoresis ②, ② lifting ② the ② agarose ② gel ②, ② placing ② a ② horizontal ② electrophoresis ② sample ②, ② placing ② a ② power ② supply ②, ② lifting ② - ② 20 ② mu ② L ② - ② 20 ② mu ② - ② L ② electrophoresis ② sample ②, ② lifting ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ② electrophoresis ② sample ②, ② placing ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ② electrophoresis ② sample ②, ② lifting ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ② electrophoresis ② sample ②, ② placing ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ② electrophoresis ② sample ②, ② lifting ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ② rack ②, ② lifting ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ② rack ②, ② lifting ② glass ②, ② lifting ② a ② horizontal ② electrophoresis ② sample ②, ② lifting ② glass ②, ② lifting ② a ② horizontal ② electrophoresis ② buffer ②, ② lifting ② a ② horizontal ②

2.4.6 amplification of 16SrDNA and purification of PCR products

④ taking ④ out ④ the ④ reagents ④ related ④ to ④ PCR ④ amplification ④ from ④ a ④ refrigerator ④, ④ preparing ④ a ④ PCR ④ reactant ④ solution ④, ④ ② ④ adding ④ the ④ reagents ④ into ④ a ④ sterilized ④ 0.2 ④ ml ④ PCR ④ tube ④ according ④ to ④ the ④ sequence ④ of ④ double ④ distilled ④ water ④, ④ 10 ④ x ④ buffer ④, ④ dNTP ④, ④ an ④ upstream ④ primer ④ (④ 27 ④ F ④) ④, ④ a ④ downstream ④ primer ④ (④ 1492 ④ R ④) ④, ④ Taq ④ DNA ④ polymerase ④ and ④ a ④ template ④ (④ total ④ genomic ④ DNA ④) ④, ④ uniformly ④ shaking ④ and ④ centrifuging ④ for ④ a ④ short ④ time ④, ④ inserting ④ the ④ PCR ④ thin ④ - ④ wall ④ tube ④ with ④ the ④ sample ④ into ④ a ④ PCR ④ instrument ④, ④ and ④ running ④ according ④ to ④ the ④ following ④ program ④ that ④ the ④ PCR ④ thin ④ - ④ wall ④ tube ④ with ④ the ④ sample ④ is ④ pre ④ - ④ denatured ④ at ④ 94 ④ ℃ ④ for ④ 5 ④ min ④, ④ the ④ PCR ④ thin ④ - ④ wall ④ tube ④ with ④ the ④ sample ④ is ④ denatured ④ at ④ 94 ④ ℃ ④ for ④ 1 ④ min ④, ④ annealed ④ at ④ 55 ④ ℃ ④ for ④ 1 ④ min ④, ④ and ④ extended ④ at ④ 72 ④ ℃ ④ for ④ 2 ④ min ④, ④ fully ④ extended ④ at ④ 72 ④ ℃ ④ for ④ 15 ④ min ④, ④ and ④ stored ④ at ④ 10 ④ ℃ ④ until ④ the ④ sample ④ is ④ taken ④ out ④. ④

2.4.7 agarose gel electrophoresis

The preparation method of the agarose electrophoresis gel is as above, and the marker is DL2000marker (TaKaRa company)

2.4.8 Provisioning Co Ltd

Sending to Shanghai biological engineering Co., Ltd for sequencing.

2.4.9 database comparison on the web

Based on the sequencing results, the most similar sequences were searched in the NCBI database using a BLAST search tool to determine the species of the selected strains.

2.4.10 slant preservation of Strain

And respectively inoculating the strains with the numbers of 1-10 to a beef extract peptone inclined plane, culturing at 28 ℃, transferring to a refrigerator at 4 ℃ after 96 hours of full growth of thalli, and preserving. And the screening plate was sealed with a sealing film and stored in a refrigerator also at 4 ℃.

Results of the experiment

3.1 Selective plate screening results

through a selective plate partition scribing method, 10 strains which can enable the aniline blue plate to fade and produce Lip (lignin peroxidase) enzyme are screened out in total, the number of the strains is 1-10 (shown in figure 1), and the manganese chloride screening plate, the α -naphthol screening plate and the congo red culture medium do not have a bacteriostatic zone and need to be further screened repeatedly.

3.2 gel electrophoresis of PCR-purified products

The PCR purified product adopts a marker DL2000, and the agarose gel electrophoresis pattern is shown in FIG. 2:

② 3.3 ② identification ② of ② the ② Strain ② genus ② the ② strain ② sequencing ② results ② are ② compared ② with ② Genbank ②, ② the ② results ② show ② that ② 10 ② strains ② belong ② to ② Pseudomonas ② kuuykendalii ② strain ② (② Pseudomonas ② genus ②) ②, ② Pseudomonas ② Alcaligenes ② (② Pseudomonas ② Alcaligenes ②) ②, ② Chryseobacter ② molle ② strain ② (② Chryseobacterium ② genus ②) ②, ② Alcaligenes ② sp ② (② Alcaligenes ② genus ②) ②, ② Achromobacter ② sp ② (② Achromobacter ② genus ②) ②, ② Pseudosciaenopsis ② sp ②, ② (② Pseudoacidobacter ② genus ②) ②, ② Pseudorhizobium ② aureus ②, ② Pseudomonas ② inodorsum ②, ② Pseudomonas ② intermedium ② (② Pseudomonas ② Alcaligenes ② genus ②) ②, ② Pseudomonas ② aureodorsum ② endonidus ② (② Pseudomonas ② Alcaligenes ② genus ②) ②, ② Pseudomonas ② kukendalensis ② strain ② (② Comastomonas ② comonella ② strain ②) ②, ② and ② Pseudomonas ② testosteroni ② strain ② (② Comastomonas ② genus ②) ②. ②

Analysis of the affiliation of the screened ligninase-producing bacteria

Claims (4)

1. An expanded culture method of a rice straw degradation bacterial line, which is characterized in that,

1) inoculating the stable straw degradation bacterial system into 4-10L of fresh enrichment medium according to the inoculation amount of 8-10 wt%, and standing and culturing at room temperature until filter paper strips in the medium disintegrate;

2) inoculating the 4-10L of culture into 40-100L of enrichment medium, and standing for culture until the filter paper strips in the medium disintegrate to complete the expanding culture;

the formula of the enrichment culture medium is that each liter of culture medium contains the following components: K2HPO41.0-3.0 g, (NH4)2SO41.0-3.0g, NaNO31.0-3.0 g, MgSO4 & 7H2O0.5-1.5g, FeSO4 & 7H2O 0.01.01-0.05 g, KCl0.5-1.5g, sodium pyruvate 0.3-1.2g, yeast extract powder 0.5-1.5g, peptone 0.5-1.5g, soluble starch 0.5-1.5g, straw powder 5-10g, D-sorbitol 0.5-1.5g, trace element mixed solution 1-3ml, calcium solution 1-3ml, filter paper strip 2 pieces, and pH value 7.0-7.5.

2. The method for the expanded culture of the rice straw-degrading bacterial line according to claim 1, wherein the formulation of the mixed solution of the trace elements in the culture enrichment medium is as follows (g/L): ZnSO 4.7. 7H2O1.5-3.5g, CoCl 2.6H 2O 0.15.15-0.50 g, Na2MoO 4.2H 2O 0.10.10-0.50 g, MnSO 4.H 2O 1.5.5-3.0 g, CuSO 4.5H 2O 0.15.15-0.35 g, H3BO30.3-0.12 g, NiCl 2.6H 2O 0.06.06-0.15 g and KI 0.06-0.15 g;

the calcium solution is CaCl₂The aqueous solution of (a) to (b),the concentration was 3%.

3. The method for the expanded culture of the rice straw-degrading bacterial line of claim 1, wherein the straw powder is rice straw which is naturally dried and crushed and then screened by a 40-mesh sieve for later use.

4. The method for the expanded culture of the rice straw degradation bacterial system according to claim 1, wherein the stable straw degradation bacterial system is obtained by taking rice straws as a bacterial source sample, adding an enrichment medium, taking a filter paper strip as a cellulose degradation indicator in the enrichment process, and disintegrating the indicator in the medium to form a flora.

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