A Serratia marcescens strain with high efficiency in degrading lignin and its application

Abstract

The present invention relates to a strain of Serratia marcescens GZAUS341020 with high efficiency in degrading lignin and its application. A strain of Serratia marcescens GZAUS341020 with high efficiency in degrading lignin was isolated from environmental samples. This strain is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 64617. The obtained Serratia marcescens strain has a good lignin degradation rate under different temperature conditions, and also has good cellulase, lipase, and amylase activities, and can be used for the biodegradation of agricultural straw.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a strain of Serratia marcescens with high efficiency in degrading lignin and its application. Background Art

[0002] Straw mainly refers to crop straw, which is the residual and inedible part after crop harvest. Straw contains most of the products stored by plants through photosynthesis, including organic substances, mineral elements, etc., and is considered a renewable biological resource with multiple uses. China is a large agricultural country with a huge output of crop straw, with an annual output exceeding 800 million tons, among which corn straw, rice straw, sorghum straw, etc. account for a large proportion. However, the lignocellulose content in various crop straws is as high as 10% - 15%. Lignin is a highly complex macromolecular biopolymer with an amorphous three-dimensional network structure, mainly composed of three aromatic monomers: coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These alcohol monomers can form different lignin subunit structural groups, constituting structures with different degrees of stability. Among them, the lignin configurations of corn straw and rice straw are the most complex. Therefore, corn straw and rice straw have strong resistance to enzymatic hydrolysis and hydrolysis, making lignin difficult to degrade. This not only reduces the application efficiency of various straws in feed and substrate, but also turns the valuable crop straw resources into agricultural waste.

[0003] Currently, in the field of biological degradation of lignin, only white rot fungi can completely degrade lignin into CO 2 and H 2 O under normal temperature and pressure environment. However, there are problems such as poor environmental adaptability, long pretreatment period, and easy spore contamination in the degradation of lignin by fungi. Bacteria have relatively strong adaptability to complex environments. However, according to existing reports, the degradation efficiency of bacteria for lignin is mostly about 20%, which is relatively low compared to fungi.

[0004] To solve the above problems, the present invention provides a strain of Serratia marcescens, whose degradation efficiency for lignin is above 40% under the condition of 20°C - 40°C, and it has good ability of other organic matter hydrolases. Summary of the Invention

[0005] The object of the present invention is to provide a strain of Serratia marcescens GZAUS341020.

[0006] Another object of the present invention is to provide the application of Serratia marcescens GZAUS341020 in the preparation of a biodegradant.

[0007] Another object of the present invention is to provide the application of Serratia marcescens GZAUS341020 in the preparation of an agricultural straw biodegradant

[0008] The method for isolating and screening the Serratia marcescens GZAUS341020 of the present invention comprises the following steps:

[0009] (1) Collect forest soil in southeastern Guizhou; sieve the soil, discard large pieces of soil, and keep the fine soil sample;

[0010] (2) Prepare LB liquid medium, sterilize it at 121 °C for 20 min for later use. The formula includes: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L;

[0011] (3) Take 10 g of sieved soil, add it to LB liquid medium, and enrich and culture it at 30 °C and 180 rpm for 48 h;

[0012] (4) Prepare LB solid medium, sterilize it at 121 °C for 20 min for later use. The formula includes: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, 15 g / L purified agar powder;

[0013] (5) Take 10 -2 μL of the bacterial dilution with a dilution concentration is spread on an LB solid plate, and each treatment has three replicates;

[0014] (6) After culturing at 30 °C for 24 - 72 h, pick single colonies with phenotypic differences and streak them on LB medium for purification culture;

[0015] (7) Identify the isolated Serratia marcescens.

[0016] Preferably, the culture time in step (6) of the present invention is 30 h.

[0017] The identification method of the Serratia marcescens of the present invention includes morphological identification and molecular biological identification.

[0018] The morphological identification method of the Serratia marcescens of the present invention is: after inoculating the Serratia marcescens strain into LB medium and culturing it for 48 h, bright red colonies can be seen on the LB medium. The colonies are round, moist, and have neat edges.

[0019] The molecular biological identification method of the Serratia marcescens described in the present invention is as follows: After culturing the Serratia marcescens strain in LB medium for 48 hours, it is sent to Guangzhou Aiji Biotechnology Company for 16S sequencing. Using 27F: (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R: (5-GGTTACCTTGTTACGACTT-3) as primers, in the GenBank database of NCBI, the 16S sequence of Serratia marcescens is retrieved by BLAST, identified, and a strain phylogenetic tree is constructed.

[0020] Beneficial effects:

[0021] 1. The obtained Serratia marcescens GZAUS341020 of the present invention has a high degradation rate of lignin at different temperatures. Among them, the degradation rate reaches 52% at 40 °C.

[0022] 2. The obtained Serratia marcescens GZAUS341020 of the present invention has a high amylase activity at different temperatures. Among them, the amylase activity is the highest at 40 °C, reaching 32 U / mL.

[0023] 3. The obtained Serratia marcescens GZAUS341020 of the present invention has a high lipase activity at different temperatures. Among them, the lipase activity is the highest at 20 °C, reaching 35 U / mL.

[0024] 4. The obtained Serratia marcescens GZAUS341020 of the present invention has a high cellulase activity at different temperatures. Among them, the cellulase activity is the highest at 20 °C, reaching 45 U / mL.

[0025] 5. Applying the obtained Serratia marcescens GZAUS341020 strain of the present invention to the biodegradation of agricultural straw solves the problems of poor environmental adaptability, long pretreatment period, easy spore contamination, and low lignin degradation efficiency of fungi in lignin degradation.

[0026] Description of preservation information:

[0027] Strain name: Serratia marcescens GZAUS341020;

[0028] Preservation unit: Guangdong Microbial Culture Collection Center;

[0029] Preservation address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou;

[0030] Preservation date: May 13, 2024;

[0031] The preservation number is GDMCC NO: 64617. Description of the Drawings

[0032] Figure 1 It is a schematic diagram of the purification culture of Serratia marcescens GZAUS341020;

[0033] Figure 2 It is the phylogenetic tree of Serratia marcescens GZAUS341020;

[0034] Figure 3 It is the lignin degradation rate of Serratia marcescens GZAUS341020 under different temperature conditions;

[0035] Figure 4 It is the amylase activity of Serratia marcescens GZAUS341020 under different temperature conditions;

[0036] Figure 5 It is the lipase activity of Serratia marcescens GZAUS341020 under different temperature conditions;

[0037] Figure 6 It is the cellulase activity of Serratia marcescens GZAUS341020 under different temperature conditions;

[0038] Figure 7 It is the growth curve of Serratia marcescens GZAUS341020 under different temperature conditions. Detailed Implementation Modes

[0039] Example 1

[0040] Application of Serratia marcescens GZAUS341020 in the preparation of a biodegradant.

[0041] Example 2

[0042] Application of Serratia marcescens GZAUS341020 in the preparation of an agricultural straw biodegradant.

[0043] Example 3 Isolation and Screening Method of Serratia marcescens GZAUS341020

[0044] (1) Collect forest soil in Southeast Guizhou; sieve the soil, discard large pieces of soil, and keep fine soil samples;

[0045] (2) Prepare LB liquid culture medium, sterilize at 121°C for 20 min and set aside. The formula includes: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride;

[0046] (3) Take 10 g of sieved soil, add LB liquid medium, and culture at 30°C and 180 rpm for 48 h;

[0047] (4) Prepare LB solid culture medium, sterilize at 121°C for 20 min and set aside. The formula includes: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L purified agar powder;

[0048] (5) Take 10 -2 100 μL of the diluted solution containing bacteria was spread on LB solid plates, with three replicates for each treatment;

[0049] (6) After culturing at 30°C for 30 h, single colonies with different phenotypes were picked and streaked onto LB medium for purification;

[0050] (7) Identify the isolated Serratia marcescens.

[0051] Example 4 Isolation and Screening Method for Serratia marcescens GZAUS341020

[0052] (1) Collect forest soil in Southeast Guizhou; sieve the soil, discard large pieces of soil, and keep fine soil samples;

[0053] (2) Prepare LB liquid culture medium, sterilize at 121°C for 20 min and set aside. The formula includes: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride;

[0054] (3) Take 10 g of sieved soil, add LB liquid medium, and culture at 30°C and 180 rpm for 48 h;

[0055] (4) Prepare LB solid culture medium, sterilize at 121°C for 20 min and set aside. The formula includes: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L purified agar powder;

[0056] (5) Take 10 -2100 μL of the diluted bacterial solution with the diluted concentration was spread on the LB solid plate, with three replicates for each treatment;

[0057] (6) After culturing at 30 °C for 70 h, single colonies with phenotypic differences were picked and streaked on the LB medium for purification culture;

[0058] (7) Identify the isolated Serratia marcescens.

[0059] Identification method of Serratia marcescens GZAUS341020 in Example 5

[0060] (1) Morphological identification

[0061] After inoculating the strain of Serratia marcescens GZAUS341020 into the LB medium and culturing for 48 h, bright red colonies were visible on the LB medium. The colonies were round, moist, and had neat edges.

[0062] (2) Molecular biology identification

[0063] After culturing the strain of Serratia marcescens GZAUS341020 in the LB medium for 48 h, it was sent to Guangzhou Aiji Biotechnology Company for 16S sequencing. Using 27F: (5-AGAGTTTGATCC TGGCTCAG-3) and 1492R: (5-GGTTACCTTGTTACGACTT-3) as primers, the 16S sequence of Serratia marcescens was retrieved and identified by BLAST in the GenBank database of NCBI, and the strain phylogenetic tree was constructed.

[0064] To verify the effectiveness of the present invention, the invention team conducted a series of tests, as follows:

[0065] 1. Reagents and instruments

[0066] 1.1 Main reagents and consumables

[0067] Table 1 Main instruments and equipment

[0068]

[0069] 1.2 Main instruments and equipment

[0070] Table 2 Main instruments and equipment

[0071]

[0072]

[0073] 2. Isolation and Screening Method of Serratia marcescens GZAUS341020

[0074] (1) Collect forest soil in southeastern Guizhou area, sieve the soil, discard large pieces of soil, and keep the fine soil samples.

[0075] (2) Prepare LB liquid medium, sterilize it at 121 °C for 20 min for later use. The formula includes: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0076] (3) Take 10 g of sieved soil, add it to LB liquid medium, and enrich and culture it at 30 °C and 180 rpm for 48 h.

[0077] (4) Prepare LB solid medium, sterilize it at 121 °C for 20 min for later use. The formula includes: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, 15 g / L purified agar powder.

[0078] (5) Take 10 -2 μL of the bacterial dilution solution with a dilution concentration and spread it on the LB solid plate, with three replicates for each treatment.

[0079] (6) After culturing at 30 °C for 48 h, pick single colonies with phenotypic differences and streak them on LB medium for purification culture.

[0080] 3. Identification Method of Serratia marcescens GZAUS341020

[0081] (1) Morphological identification

[0082] After inoculating the Serratia marcescens GZAUS341020 strain into LB medium and culturing it for 48 h, bright red colonies can be seen on the LB medium. The colonies are round, moist, and have neat edges. The schematic diagram of strain purification culture is as Figure 1 .

[0083] (2) Molecular biological identification

[0084] After culturing the Serratia marcescens GZAUS341020 strain in LB medium for 48 h, it was sent to Guangzhou Aiji Biotechnology Company for 16S sequencing. Using 27F: (5-AGAGTTTGATCC TGGCTCAG-3) and 1492R: (5-GGTTACCTTGTTACGACTT-3) as primers, in the GenBank database of NCBI, the 16S sequence of Serratia marcescens was retrieved by BLAST, identified, and the phylogenetic tree of the strain was constructed. The constructed phylogenetic tree is as shown in Figure 2 .

[0085] 4. Exploration of the biodegradation ability of Serratia marcescens GZAUS341020 under different temperature conditions

[0086] 4.1 Lignin degradation by Serratia marcescens GZAUS341020 at different temperatures

[0087] (1) Strain culture: Add alkaline lignin to LB liquid medium to make the final concentration 50 μg / mL, inoculate the strain according to the inoculation amount of 1%, and culture it at 20 °C, 30 °C, and 40 °C for 48 h respectively;

[0088] (2) Reagent preparation: Take alkaline lignin powder and prepare it into a 40 μg / mL solution with distilled water. If the solubility of lignin in distilled water is low, the pH can be adjusted to 12 to help dissolve it and prepare a lignin standard solution; prepare an 8 mM potassium ferricyanide solution and a 0.1 M ferric chloride solution;

[0089] (3) Standard curve establishment: Take 9 test tubes, configure them according to Table 1, shake the test tubes for 5 min, incubate them at 30 °C for 10 min, and measure the OD 700 value, and establish a standard curve based on the lignin concentration and absorbance value;

[0090] (4) Measurement: Take 1.5 mL of the bacterial liquid, add 100 μL of potassium ferricyanide, and then immediately add 100 μL of ferric chloride, shake the test tube for 5 min, incubate it at 30 °C for 10 min, and use the blank LB medium as a control to measure the OD 700 absorbance value;

[0091] (5) Calculation: Substitute the absorbance value of the sample into the standard curve, look up the value, and calculate the degradation rate according to the following formula: Degradation rate (%) = [(50 - X) / 50] * 100, where X is the value looked up in the standard curve for the sample.

[0092] Table 3 Establishment of the standard curve for lignin concentration and absorbance value

[0093]

[0094] 4.2 Amylase Activity of Serratia marcescens GZAUS341020 at Different Temperatures

[0095] (1) Inoculate into the starch-producing liquid medium at an inoculation amount of 1%, and culture at 20 °C, 30 °C, and 40 °C at 180 r / min for 48 h. Take 12000 r of the culture solution and centrifuge for 3 min to obtain the enzyme solution to be measured;

[0096] (2) Prepare a 1 g / ml maltose solution and establish a standard curve according to Table 2;

[0097] Table 4 Establishment of the Standard Curve of Maltose Concentration and Absorbance Value

[0098]

[0099] (3) Take 1 ml of the enzyme solution and water bath at 40 °C for 5 min, add 1 ml of 1% starch solution pre-incubated at 40 °C, water bath at 40 °C for 10 min, add 2 ml of DNS solution, boil water bath for 5 min, cool to room temperature and make up the volume to 25 ml, and measure the OD value at a wavelength of 540 nm;

[0100] (4) Calculate the amylase activity according to the following formula: U = [(milligrams of maltose × N) / 10] × 1000;

[0101] 4.3 Determination of Lipase Activity of Serratia marcescens GZAUS341020

[0102] (1) Preparation of the enzyme solution to be measured: Culture in LB liquid medium at 30 °C and 180 rpm for 48 h, and collect the culture solution for use;

[0103] (2) Determination: Take a 100 ml Erlenmeyer flask, make a blank A and a sample B, add 4 ml of the substrate and 5 ml of the buffer solution respectively, then add 15 ml of 95% alcohol to A, water bath at 40 °C for 5 min, then add 1 ml of the enzyme solution to each, mix immediately and start timing. After reacting accurately for 15 min, immediately add 15 ml of 95% alcohol to B to terminate the reaction, take out, add two drops of phenolphthalein to each of A and B, and titrate with the sodium hydroxide standard solution until the light red color does not fade for 30 s as the titration end point, and record the volume of the sodium hydroxide standard solution;

[0104] (3) Calculation: The enzyme activity of the lipase preparation is calculated according to the following formula:

[0105]

[0106] Where: X 1is the enzyme activity of the sample, U / ml; V 1 is the volume (mL) of the sodium hydroxide standard solution consumed when titrating the sample; V 2 is the volume (mL) of the sodium hydroxide standard solution consumed when titrating the blank; c is the sodium hydroxide standard solution (mol / L); n 1 is the sample dilution factor;

[0107] 4.4 Cellulase activity of Serratia marcescens GZAUS341020 at different temperatures

[0108] (1) Standard curve plotting: Take 6 stoppered graduated test tubes of 25 mL, add 0.0, 0.4, 0.8, 1.2, 1.6, 2.0 mL of the glucose standard solution of 1.0 mg / ml, add 2.0, 1.6, 1.2, 0.8, 0.4, 0.0 mL of distilled water, add 1.5 mL of DNS reagent, mix well, heat in a boiling water bath for 5 minutes, take out and immediately cool with cold water, make up the volume to 25 mL with water, shake well, measure the absorbance A, use the absorbance as the ordinate and the glucose content as the abscissa to plot the standard curve;

[0109] (2) Preparation of the bacterial liquid to be tested: In the LB liquid medium, culture at 20 °C, 30 °C, 40 °C, 180 rpm for 48 h, centrifuge the bacterial suspension for later use;

[0110] (3) Determination: Take 1.5 mL of the CMC-Na solution and 0.5 ml of the appropriately diluted bacterial liquid in a 25 ml test tube, after incubating in a water bath at 40 °C for 30 min, immediately add 1.5 mL of the DNS color reagent, boil in a boiling water bath for 5 min, take out and immediately cool, make up the volume to 25 ml with water, measure the absorbance Abs at 540 nm.

[0111] (4) Blank sample: First add 1.5 mL of DNS reagent, then add 0.5 mL of the bacterial liquid to be tested and 1.5 mL of the CMC-Na solution in a 25 ml test tube, boil in a boiling water bath for 5 min, cool and make up the volume to 25 ml with water, measure the absorbance Abs at 540 nm. According to A, check the glucose content P from the standard curve;

[0112] (5) Calculation: Calculate the enzyme activity according to the following formula: Enzyme activity (u / g) = P * K * 1000 / 0.5 * 30, where K is the dilution factor.

[0113] 4.5 Experimental conclusion

[0114] (1) The lignin degradation rates of Serratia marcescens GZAUS341020 at 20 °C, 30 °C, and 40 °C were (48.54 ± 0.51)%, (47.25 ± 0.91)%, and (52.02 ± 1.54)%, respectively, as Figure 3 ;

[0115] (2) The amylase activities of Serratia marcescens GZAUS341020 at 20 °C, 30 °C, and 40 °C were (5.89 ± 0.8) U / mL, (23.61 ± 0.42) U / mL, and (31.58 ± 0.55) U / mL, respectively, as Figure 4 ;

[0116] (3) The lipase activities of Serratia marcescens GZAUS341020 at 20 °C, 30 °C, and 40 °C were (34.53 ± 1.11) U / mL, (7.57 ± 1.17) U / mL, and (14.77 ± 1.19) U / mL, respectively, as Figure 5 ;

[0117] (4) The cellulase activities of Serratia marcescens GZAUS341020 at 20 °C, 30 °C, and 40 °C were (45.39 ± 3.67) U / mL, (26.66 ± 0.89) U / mL, and (28.06 ± 2.40) U / mL, respectively, as Figure 6 。

[0118] 5. Growth curve of Serratia marcescens GZAUS341020

[0119] (1) Activate the strain on the LB plate;

[0120] (2) Pick single colonies and transfer them to 9 conical flasks containing 100 mL of LB liquid medium respectively;

[0121] (3) Take three conical flasks as a group and place them in different shakers respectively;

[0122] (4) Set the temperatures of the incubator to 20 °C, 30 °C, and 40 °C respectively, the rotation speed to 200 rpm, and culture with constant temperature oscillation;

[0123] (5) Using the LB liquid medium as a control, detect the OD 600 value every 2 h and continuously monitor for 24 h;

[0124] (6) According to the monitored values, draw the growth curve, as Figure 7 。

[0125] 6. Gene sequence of Serratia marcescens GZAUS341020

[0126]

Claims

1. A strain of Serratia marcescens ( Serratia marcescens ) GZAUS341020, characterized in that, The Serratia marcescens ( Serratia marcescens ) GZAUS341020, deposited in Guangdong Microbiological Culture Collection Center, and its deposit number is GDMCC NO: 64617.

2. The Serratia marcescens according to claim 1 ( Serratia marcescens ) Application of GZAUS341020, characterized in that, The Serratia marcescens ( Serratia marcescens )Application of GZAUS341020 in the preparation of agricultural straw biodegradation agent.

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