CN110643528B - Kluyveromyces intermedia with good degradation effect on cellulose - Google Patents

Abstract

A Kluyveromyces intermedia strain with a good degradation effect on cellulose belongs to the technical field of microbiology, and is characterized in that: by utilizing an enrichment culture method, a strain B1-2 is separated from frozen soil collected in the desert river county of the great Khingan region of Heilongjiang province, and the strain B1-2 can fully degrade straws with the thickness of 10 centimeters within 72 days; directly separating out a strain B1-2 through a sodium carboxymethyl cellulose flat plate; through physiological and biochemical and 16S rDNA identification, the strain B1-2 is identified as Kluyvera intermedia of Kluyvera intermediate, and the preservation date of the strain is as follows: 11 and 30 months in 2018; the preservation number is: CGMCC No. 16849; degradation experiments show that the strain B1-2 has good cellulose degradation effect.

Description

Kluyveromyces intermedia with good degradation effect on cellulose

Technical Field

The invention relates to a Kluyveromyces intermedia strain with a good cellulose degradation effect, and belongs to the technical field of microbiology.

Background

Cellulose is a linear glucan in which glucose residues are linked together by β -1, 4-glucosidic bonds, and is widely present in plants. Corn stover is a huge biomass agricultural waste in the world, is rich in cellulose components, and can play an important role in many fields. But the existing utilization condition causes a considerable part of corn straws to be used with low value and directly discarded, and the subsequent result is that resources are wasted and the environment is polluted. Cellulose is the most widespread carbohydrate group in nature and is also the largest renewable resource on earth. At present, only a small part of cellulose in nature is utilized, and most of cellulose is wasted and causes environmental pollution. The cellulose substances can be converted into feed, culture medium, organic fertilizer, chemical raw material, etc. by decomposing with microorganism. Cellulases are important enzyme substances in the cellulose degradation process, including class 3 soluble extracellular enzymes: 1, 4-beta-endoglucanases, 1, 4-beta-exoglucanases and beta-glucosidases. Many microorganisms, including many bacteria, fungi and actinomycetes, have the ability to produce cellulases. However, the cellulase activity of the currently obtained strains is generally low, and even the strains with high enzyme activity have instability phenomenon in subsequent culture, which are factors influencing the mass production of cellulase. Screening for strains with high cellulase activity remains an object of much effort. The cellulose degradation capability and biological characteristics of the strain are measured, and the excellent strain capable of degrading cellulose efficiently and quickly is obtained. The research separates and screens the Kluyveromyces intermedia capable of efficiently degrading cellulose from frozen soil collected in the desert and river counties of the great Khingan region of the Heilongjiang province, measures the cellulose degradation effect of the Kluyveromyces intermedia, and provides a theoretical basis for future degradation of straws by biological methods such as straw degradation and straw returning.

At present, various strains capable of degrading cellulose are screened from different soils by researchers at home and abroad, such as: acetobacter xylinus (Feth el Zahar Haichar, et al 2007.) and the like, although most strains are capable of degrading cellulose, the degradation efficiency of these degrading bacteria is not so high in general.

How to find a bacterium with obvious degradation effect on cellulose becomes a big problem to be solved urgently? Therefore, the invention provides the Kluyveromyces intermedia with good degradation effect on the cellulose, and is necessary to find the Kluyveromyces intermedia capable of efficiently degrading the cellulose.

Disclosure of Invention

In order to overcome the problem that the degrading bacteria found at present are not very high in cellulose degrading efficiency, the invention provides a Kluyveromyces intermedia (preservation number: CGMCC No.16849) with good cellulose degrading effect.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the Kluyveromyces intermedia (preservation number: CGMCC No.16849) with good degradation effect on cellulose is specifically constructed and verified as follows:

the soil sample used in the experiment was collected from frozen soil (0-15 cm) collected in the region of Daxing AnLing, Heilongjiang province, desert and county. Soil samples were screened through a20 mesh screen to remove rocks and plant debris and then packed into sealed bags for storage at 4 ℃.

Reagent: the 3, 5-dinitrosalicylic acid is purchased from Beijing Baiolai science and technology Limited, Congo red is purchased from Hongyu reagent factory in the east river of Tianjin, and other conventional reagents are analytically pure or higher purity.

Culture medium: LB culture medium: qingdao GaoKeyuan Haibo Biotech Co., Ltd. Sodium Carboxymethylcellulose Medium (CMC): 15.0g of CMC-Na, 6.0g of NaCl, 1.0g of yeast powder, (NH)₄)₂SO₄ 2.0g，MgSO₄ 0.1g，KH₂PO₄ 0.1g，CaCl₂ 0.1g，K₂HPO₄0.5g, agar 18g, distilled water 1000mL, pH 7.0. And (3) sterilization conditions: autoclaving at 120 ℃ for 20 min. Fermentation medium: 1.0% of yeast powder, 0.5% of NaCl, 1.0% of peptone, 1.0% of sodium carboxymethylcellulose and K₂HPO₄0.1％。

Enrichment, domestication and separation of degrading bacteria: weighing 5g of soil sample, adding the soil sample into 100mL of CMC liquid culture medium which takes sodium carboxymethylcellulose as a unique carbon source, placing the CMC liquid culture medium in a shaking table, and carrying out shaking culture at 30 ℃ and 150r/min for 7 days. After 7 days, the culture broth was inoculated at 10% into the same liquid medium, and the culture was continued. After 3 times of enrichment culture, respectively diluting the bacterial liquid to 10-3, 10-4 and 10^-5Streaking in LB solid culture medium, picking out color and shape after 5 daysThe same colonies were purified and analyzed by a conventional microscopic method to obtain the B1-2 strain. Identification of degrading strains: (1) physiological and biochemical identification: the strains to be tested were inoculated on LB solid plates and identified with reference to "microbiology experiments" (Zhao and, Hao Shao, Hao Jiang. 2002.) and "Bergey's Manual of Systematic Bacteriology (second evaluation)" (George MG, Julia AB, Timothy GL.2004.). (2)16S rDNA identification: since the 16S rDNA sequence of the same species, intergeneric bacteria is directly highly conserved, homology analysis of 165rDNA sequences is commonly used as a systematic classification between bacteria. Taking the total DNA of each strain as a template, carrying out 16S rDNA sequence amplification on the pure culture strains in the degraded community by adopting a PCR technology, wherein the amplification primers adopt general primers: 27-F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492-R (5'-TACCTTGTTACGACTT-3') (Liuchuangguang, Yangposhan, Luxinzhong, etc.. 2010.). The PCR reaction conditions are as follows: 5 minutes at 94 ℃; 1 minute at 94 ℃, 1 minute at 52 ℃, 2 minutes at 72 ℃ and 30 cycles; the reaction system was 25. mu.L at 72 ℃ for 10 minutes. Sequencing of the PCR-purified products was done by Jinzhi, Suzhou, and the sequencing results were compared with BLAST homology in Genbank databases and identified as Kluyvera intermedia by 165rDNA analysis, B1-2.

And (3) determining the degradation effect of the strain B1-2: (1) determination of the ratio of the diameter of the hydrolysis ring to the diameter of the colony: inoculating the grown colony points on a CMC plate, culturing for 48 hours at 37 ℃, staining for 1 hour with 1% Congo red, decoloring for 1 hour with 5% NaCl, observing and recording the diameter of a hydrolysis ring and the diameter of a colony. (2) Determination of cellulase activity: picking single colony in seed culture solution at 37 deg.c and oscillating at 170r/min for 24 hr, inoculating bacterial liquid of cellulose degrading bacteria in the amount of 1% into prepared fermenting culture solution at 37 deg.c and 200r/min for oscillating culture for 36 hr. After shaking culture, 2mL of the bacterial liquid is put into a clean centrifugal tube and centrifuged for 10 minutes at 5000r/min, the supernatant is gently sucked by a gun head and transferred into a 10mL centrifugal tube to be diluted by 10 times to be used as crude enzyme liquid of the cellulase, and the extinction degree of the crude enzyme liquid is measured at 520nm by a 3, 5-dinitrosalicylic acid method (DNS). (3) Determination of degradation efficiency of cellulose-degrading bacteria in indoor simulated farmlands: selecting a transparent jar, adding straw and bacterial manure at the bottom of the jar, covering a soil sample with the thickness of 10 cm above the transparent jar, pouring out the upper soil layer after 72 days, exposing the straw layer, taking 15g of straw which is not dipped with soil, and marking the straw as wet weight (S). The heating table was opened and a piece of white paper was placed on the table to avoid staining the table top, and 15g of straw was laid on the paper. The temperature of the heating table is set to 200 ℃, 15G of straws can be dried after observation for about 30 minutes, and the dry weight (G) is obtained by subtracting the weight of paper from the total weight. And (3) burning the dried straws in the rice vat, weighing the total weight of the straws, pouring the ash after burning, measuring the weight of the rice vat, and subtracting the weight of the rice vat from the total weight to obtain the weight (H) of the ash. Subtracting the ash weight from the dry weight to obtain the combustible organic matter weight (K), calculating the combustible organic matter content of the normal 15g of straws according to the operation to obtain a standard value (A), and finally calculating the straw decomposition rate (A-K)/A. The final average value of the efficiency of the bacteria to degrade cellulose was 87.17%.

The invention has the beneficial effects that: the Kluyveromyces intermedia with good degradation effect on cellulose is obtained by separating a strain B1-2 from frozen soil collected from the desert river county in the great Khingan region of Heilongjiang by an enrichment culture method, wherein the strain B1-2 can fully degrade straws with the thickness of 10 cm within 72 days. Strain B1-2 was isolated directly from the sodium carboxymethyl cellulose plates. Through physiological and biochemical and 16S rDNA identification, the strain B1-2 is identified as Kluyvera intermedia, and degradation experiments show that the strain B1-2 has good cellulose degradation effect.

Strain preservation information:

classifying and naming strains: kluyveromyces intermedia (Kluyvera intermedia);

the preservation organization: china general microbiological culture Collection center;

and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;

the preservation date is as follows: 11 and 30 months in 2018;

the preservation number is: CGMCC No. 16849;

the name, strain number or symbol assigned to its culture by the depositor is requested: b1-2;

culture conditions of the culture, conditions for detection of survival (medium composition, pH, temperature, etc.): LB culture medium; pH: 7-9; temperature: 30 ℃;

the culture was isolated from where and what substrate: separating and separating frozen soil collected from the desert river county in the great Xingan AnLing region of Heilongjiang province.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 is a diagram showing the sequencing result of 16S rDNA of a Kluyveromyces intermedia with a good cellulose degradation effect.

FIG. 2 is a graph showing the staining result of Congo red which is a strain B1-2 of Kluyveromyces intermedia having a good degradation effect on cellulose.

FIG. 3 is a bacterial strain B1-2 of Kluyveromyces intermedia with good degradation effect on cellulose, and the bacterial strain B1-2 is a bacterial orthophase connection phylogenetic tree constructed based on a 16S rDNA sequence.

Detailed Description

In the context of this specification, unless otherwise indicated, any terms used herein have the meanings commonly understood in the art by those skilled in the art, and experimental procedures without specification to details are carried out according to conventional experimental procedures or according to the instructions recommended by the supplier.

Example one

1 materials and methods

1.1 Experimental instruments

TGL-16C high speed centrifuge (shanghai ann pavilion scientific instruments factory), FA2004 electronic balance (shanghai hei instruments ltd.), P330-31 ultra-low ultraviolet spectrophotometer (Implen GmbH, Munich, Germany), BOXUN vertical pressure steam sterilization pot (shanghai bosch industries ltd.), SW-CJ-IFD ultra clean bench (sujing group suzhou antai air technology ltd.), micropipette (Biotech).

1.2 materials

1.2.1 soil sample to be tested

The soil sample used in the experiment was collected from frozen soil (0-15 cm) collected in the region of Daxing AnLing, Heilongjiang province, desert and county. Soil samples were screened through a20 mesh screen to remove rocks and plant debris and then packed into sealed bags for storage at 4 ℃.

1.2.2 reagents

The 3, 5-dinitrosalicylic acid is purchased from Beijing Baiolai science and technology Limited, Congo red is purchased from Hongyu reagent factory in the east river of Tianjin, and other conventional reagents are analytically pure or higher purity.

1.2.3 Medium

LB culture medium: qingdao GaoKeyuan Haibo Biotech Co., Ltd.

Sodium Carboxymethylcellulose Medium (CMC): 15.0g of CMC-Na, 6.0g of NaCl, 1.0g of yeast powder, (NH)₄)₂SO₄2.0g，MgSO₄ 0.1g，KH₂PO₄ 0.1g，CaCl₂ 0.1g，K₂HPO₄0.5g, agar 18g, distilled water 1000mL, pH 7.0.

Fermentation medium: 1.0% of yeast powder, 0.5% of NaCl, 1.0% of peptone, 1.0% of CMC-Na and K₂HPO₄0.1％。

And (3) sterilization conditions: autoclaving at 120 ℃ for 20 min.

1.3 enrichment, acclimatization and separation of degrading bacteria

Weighing 5g of soil sample, adding the soil sample into 100mL of CMC liquid culture medium which takes sodium carboxymethylcellulose as a unique carbon source, placing the CMC liquid culture medium in a shaking table, and carrying out shaking culture at 30 ℃ and 150r/min for 7 days. After 7 days, the culture broth was inoculated at 10% into the same liquid medium, and the culture was continued. After 3 times of enrichment culture, the bacterial liquid is respectively diluted to 10^-3、10^-4、10^-5Streaking and culturing in LB solid culture medium, and 5 days later, picking out colony with different color and shape for purification.

1.4 identification of degrading strains

1.4.1 physiological and biochemical assays

The strains to be tested were inoculated on LB solid plates and identified with reference to "microbiology experiments" (Zhao and, Hao Shao, Hao Jiang. 2002.) and "Bergey's Manual of Systematic Bacteriology (second evaluation)" (George MG, Julia AB, Timothy GL.2004.).

1.4.216S rDNA identification

Taking the total DNA of each strain as a template, carrying out 16S rDNA sequence amplification on the pure culture strains in the degraded community by adopting a PCR technology, wherein the amplification primers adopt general primers: 27-F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492-R (5'-TACCTTGTTACGACTT-3') (Liuchuangguang, Yangposhan, Luxinzhong, etc.. 2010.). The PCR reaction conditions are as follows: 94 ℃ for 5 min: 1min at 94 ℃, 1min at 52 ℃, 2min at 72 ℃ and 30 cycles; the reaction system is 25 mu L at 72 ℃ for 10 min. The amplified product was recovered by using a kit (Axygen, USA), and then ligated with pMD19-T vector (TaKaRa, Japan), transformed into E.coli DH 5. alpha. and sent to Jinzhi, Suzhou for sequencing, and the results were subjected to homology alignment by Genbank to determine its classification.

1.5 determination of the degradation Effect of Strain B1-2

1.5.1 determination of the ratio of the diameter of the hydrolysis Ring to the diameter of the colony

The grown colonies were spotted on CMC plates and cultured at 37 ℃ for 48 hours, stained with 1% Congo red for 1 hour, destained with 5% NaCl for 1 hour, and the diameter of the hydrolytic loop and the diameter of the colony were observed and recorded.

1.5.2 determination of cellulase Activity

Picking single colony in seed culture solution at 37 deg.c and oscillating at 170r/min for 24 hr, inoculating bacterial liquid of cellulose degrading bacteria in the amount of 1% into prepared fermenting culture solution at 37 deg.c and 200r/min for oscillating culture for 36 hr. After shaking culture, 2mL of the bacterial liquid is put into a clean centrifugal tube and centrifuged for 10 minutes at 5000r/min, the supernatant is gently sucked by a gun head and transferred into a 10mL centrifugal tube to be diluted by 10 times to be used as crude enzyme liquid of the cellulase, and the extinction degree of the crude enzyme liquid is measured at 520nm by a 3, 5-dinitrosalicylic acid method (DNS).

1.5.3 determination of degradation efficiency of cellulose-degrading bacteria in indoor simulated farmlands

Selecting a transparent jar, adding straw and bacterial manure at the bottom of the jar, covering a soil sample with the thickness of 10 cm above the transparent jar, pouring out the upper soil layer after 72 days, exposing the straw layer, taking 15g of straw which is not dipped with soil, and marking the straw as wet weight (S). The heating table was opened and a piece of white paper was placed on the table to avoid staining the table top, and 15g of straw was laid on the paper. The temperature of the heating table is set to 200 ℃, 15G of straws can be dried after observation for about 30 minutes, and the dry weight (G) is obtained by subtracting the weight of paper from the total weight. And (3) burning the dried straws in the rice vat, weighing the total weight of the straws, pouring the ash after burning, measuring the weight of the rice vat, and subtracting the weight of the rice vat from the total weight to obtain the weight (H) of the ash. Subtracting the ash weight from the dry weight to obtain the combustible organic matter weight (K), calculating the combustible organic matter content of the normal 15g of straws according to the operation to obtain a standard value (A), and finally calculating the straw decomposition rate (A-K)/A. The final average value of the efficiency of the bacteria to degrade cellulose was 87.17%.

2 results and analysis

2.1 pure culture and isolation of Strain B1-2

After 2 months of purification, the bacteria can grow out on the CMC plate, colonies with different colors and forms are selected for purification, and the colonies are analyzed by combining a conventional microscopic examination method to finally obtain the B1-2 strain.

2.2 identification of physiological and biochemical experiments

The results of conventional physiological and biochemical identification of bacteria are shown in Table 1.

Table 1: physiological and biochemical identification of degrading strain B1-2

2.316S rDNA Gene sequence analysis

Since the 16S rDNA sequence of the same species, intergeneric bacteria is directly highly conserved, homology analysis of the 16S rDNA sequence is commonly used as a systematic classification between bacteria. B1-2 was identified as Kluyvera intermedia by 16S rDNA analysis. Blast results and GenBank accession numbers of the 16S rDNA sequence of this strain are shown in table 2.

Table 2: b1-2 strain 16S rDNA sequence classification identification

2.4 cellulase degradation Properties of B1-2 Strain

2.4.1 determination of the ratio of the diameter of the hydrolysis Ring to the diameter of the colony

The grown colonies were spotted on CMC plates and cultured at 37 ℃ for 48 hours, stained with 1% Congo red for 1 hour, destained with 5% NaCl for 1 hour, and the diameter of the hydrolytic loop and the diameter of the colony were observed and recorded.

Table 3: b1-2 strain hydrolysis ring diameter and colony diameter

2.4.2 determination of cellulase Activity

Picking single colony in seed culture solution at 37 deg.c and oscillating at 170r/min for 24 hr, inoculating bacterial liquid of cellulose degrading bacteria in the amount of 1% into prepared fermenting culture solution at 37 deg.c and 200r/min for oscillating culture for 36 hr. After shaking culture, 2mL of the bacterial liquid is put into a clean centrifugal tube and centrifuged for 10 minutes at 5000r/min, the supernatant is gently sucked by a gun head and transferred into a 10mL centrifugal tube to be diluted by 10 times to be used as crude enzyme liquid of the cellulase, and the extinction degree of the crude enzyme liquid is measured at 520nm by a 3, 5-dinitrosalicylic acid method (DNS).

Table 4: cellulase activity of B1-2 strain

2.4.3 determination of degradation efficiency of cellulose-degrading bacteria in indoor simulated farmlands

Selecting a transparent jar, adding straw and bacterial manure at the bottom of the jar, covering a soil sample with the thickness of 10 cm above the transparent jar, pouring out the upper soil layer after 72 days, exposing the straw layer, taking 15g of straw which is not dipped with soil, and marking the straw as wet weight (S). The heating table was opened and a piece of white paper was placed on the table to avoid staining the table top, and 15g of straw was laid on the paper. The temperature of the heating table is set to 200 ℃, 15G of straws can be dried after observation for about 30 minutes, and the dry weight (G) is obtained by subtracting the weight of paper from the total weight. And (3) burning the dried straws in the rice vat, weighing the total weight of the straws, pouring the ash after burning, measuring the weight of the rice vat, and subtracting the weight of the rice vat from the total weight to obtain the weight (H) of the ash. Subtracting the ash weight from the dry weight to obtain the combustible organic matter weight (K), calculating the combustible organic matter content of the normal 15g of straws according to the operation to obtain a standard value (A), and finally calculating the straw decomposition rate (A-K)/A. The final average value of the efficiency of the bacteria to degrade cellulose was 87.17%.

Table 5: degradation efficiency of B1-2 strain in indoor simulated farmland

At present, various cellulose degrading strains are screened from different soils by domestic and foreign researchers, and although most of the strains can degrade cellulose, the degrading efficiency of the degrading strains is not high under normal conditions. The cellulose degrading strain B1-2 screened by the experiment can fully degrade cellulose within 72 days, and has obvious degradation advantage and practical value compared with the strain with poor fiber degrading effect. Therefore, the cellulose-degrading strain B1-2 is an important cellulose-degrading strain. In the experiment, the strain B1-2 is separated and screened out by an enrichment culture method through a sodium carboxymethyl cellulose culture medium, and the strain B1-2 can fully degrade straws in 72 days. Strain B1-2 was identified as Kluyvera intermedia based on physiological and biochemical identification and 16S rDNA sequence analysis, accession no: CGMCC No. 16849.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined by the appended claims and their equivalents.

Claims (1)

1. The Kluyveromyces intermedia with good degradation effect on cellulose is characterized by comprising the following components in percentage by weight: by utilizing an enrichment culture method, a strain B1-2 is separated from frozen soil collected in the desert river county of the great Khingan region of Heilongjiang province, and the strain B1-2 can fully degrade straws with the thickness of 10 centimeters within 72 days; directly separating out a strain B1-2 through a sodium carboxymethyl cellulose flat plate; through physiological and biochemical and 16S rDNA identification, the strain B1-2 is identified as Kluyvera intermedia of Kluyvera intermediate, and degradation experiments show that the strain B1-2 has good cellulose degradation effect;

strain preservation information:

classifying and naming strains: kluyveromyces intermedia (Kluyvera intermedia);

the preservation organization: china general microbiological culture Collection center;

and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, Beicheng;

the preservation date is as follows: 11 and 30 months in 2018;

the preservation number is: CGMCC No. 16849.

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