Screening method and application of a Korean Comamonas strain that produces xylanase at low temperature and can degrade cow dung

Abstract

The present invention relates to a screening method and application of a Korean Comamonas strain that can degrade cow dung and produce low-temperature xylanase, the classification name of Korean Comamonas is Comamonas koreensis, the preservation institution is the General Microbiological Center of the China Microbiological Culture Collection Administration, the preservation number of Korean Comamonas is CGMCC No.26059, and the preservation date is November 7, 2022. The present invention separates and screens high-efficiency low-temperature xylanase-producing Korean Comamonas from cow dung and dung in different low-temperature regions, and applies it to the fermentation of livestock and poultry manure under low-temperature conditions in Northeast China, reduces the impact of livestock and poultry manure pollution on the environment in Northeast China, and lays a solid foundation for sustainable agricultural development.

Description

Technical Field

[0001] The invention relates to a screening method for a Korean Comamonas bacterium capable of degrading cow dung and producing xylanase at low temperature and an application thereof, and belongs to the field of microbiology. More specifically, the invention relates to a screening method for a Korean Comamonas bacterium capable of degrading cow dung and producing xylanase at low temperature and an application thereof. Background Art

[0002] Cow dung contains a lot of lignocellulose. Due to the long low temperature period in the Northeast, there are specific low-temperature microbial communities in specific environments. Low-temperature microorganisms refer to microbial groups that have the ability to grow and reproduce in low-temperature environments. They grow and reproduce in various low-temperature environments. There are very rich low-temperature microbial resources in the natural biosphere. Therefore, microbial resources are fully utilized in life and production practices. The growth and reproduction temperature of low-temperature microorganisms can generally be carried out at room temperature, without the use of other energy sources. They are easy to obtain and widely distributed. Therefore, people have begun to devote themselves to the study of low-temperature microorganisms, which has also attracted the attention of some experts and scholars. So far, there is relatively little development and utilization of low-temperature microorganisms. From the perspective of research progress at home and abroad, most of the research is still on medium and high-temperature microorganisms, and there is little research on low-temperature microbial degradation bacteria.

[0003] So far, most of the microbial species that can secrete xylanase that have been recorded and reported at home and abroad are some fungal flora, such as Trichoderma, Aspergillus, etc. There are very few reports on Korean Comamonas that can produce xylanase at low temperature.

[0004] At present, most of the research has the disadvantages of high production cost, inefficient and unstable enzyme production capacity and enzyme production, and narrow pH adaptability range. Therefore, it is necessary to continue to explore and develop new strains with high activity and wider action range, and further expand new ideas for cattle manure and sewage degradation. Low-temperature bacteria have more advantages and potential in energy saving and cost reduction, and have great development prospects.

[0005] In conclusion, the screening method and application of a Korean Comamonas strain that can degrade cow dung and produce xylanase at low temperature have not been reported. Summary of the invention

[0006] The present invention aims to solve the above problems and provides a screening method and application of a Korean Comamonas bacterium capable of degrading cow dung and producing xylanase at low temperature.

[0007] In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical scheme:

[0008] A strain of Korean Comamonas that can degrade cow dung and produce xylanase at low temperature. The classification name of Korean Comamonas is Comamonas koreensis. The preservation institution is the General Microbiology Center of China Culture Collection of Microorganisms. The preservation number of Korean Comamonas is CGMCC No. 26059. The preservation date is November 7, 2022. The address of the preservation unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The 16S rDNA sequence of Comamonas koreaensis was published in GenBank with the accession number MHAVGUTB014. Its 16S rDNA sequence is:

[0010]

[0011] The biological characteristics of Korean Comamonas are: the colonies on LB culture medium are small, round, neat, opaque, smooth, sticky and easy to pick up, with typical bacterial colony characteristics.

[0012] Isolation method of Korean Comamonas strains:

[0013] Add 5 g of the collected cow dung sample from Northeast China into a 150 mL Erlenmeyer flask containing 50 mL of sterile water, shake at 120 r / min for 30 min, draw 1 mL of the suspension into a Erlenmeyer flask containing enrichment culture medium, and culture in an incubator (15°C) with a shake rate of 120 r / min for 15 days. Inoculate into new enrichment culture medium at a 5% (V / V) inoculation rate every 15 days, and subculture for 10 times.

[0014] The composition of the enriched Hutcheson screening medium is KH2PO4 1.0 g / L, NaCl 0.1 g / L, FeCl3·6H2O 0.01 g / L, MgSO4·7H2O 0.3 g / L, NaNO3 2.5 g / L, CaCl2·6H2O 0.1 g / L, xylan 100-1000 mg / L, and the balance is water; the pH of the culture medium is 4.8-7.2.

[0015] Screening of Comamonas strains in Korea:

[0016] a. Initial screening: Spread the enriched culture medium on the plate, dilute it by 10 -8 , 10 -9 , 10 -10 The sample solution was coated on the solid culture medium and the culture dish was placed in a constant temperature incubator at 15°C for 72 hours. The purified strain was inoculated on the xylan Congo red culture medium and cultured at 15°C for 4 days. Preliminary screening was performed based on the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d).

[0017] b. Rescreening: Transfer the primary screened strains into liquid culture medium and culture at 15°C for 3 days. The liquid culture fermentation liquid was heated at 3500 r / min -1 After centrifugation for 8 minutes, the xylanase activity of the supernatant was determined and reselected.

[0018] The advantages of the present invention are:

[0019] 1. The present invention collects low-temperature cow dung and sewage in the Northeast region, enriches it for low-temperature cultivation, and screens out microbial strains that can efficiently degrade cow dung and sewage at low temperatures. The microbial strains can produce efficient xylanase at low temperatures (15°C) to decompose cow dung and sewage, and can be used as a fermentation and composting agent for livestock and poultry dung and sewage in the low-temperature and cold regions of the Northeast.

[0020] 2. The low-temperature xylanase-producing strain of the present invention has great development potential in large-scale production. The strain can not only degrade lignocellulose under low temperature conditions, but also effectively transform nutrients in cow dung. The decomposed organic fertilizer can be applied to the soil to improve soil fertility and enrich the soil.

[0021] 3. The composting bacteria product produced by the present invention has the unique characteristic of decomposing cow dung at low temperature and has low cost. The prepared organic fertilizer can effectively increase soil organic matter, promote seed germination rate, and enhance the drought and cold resistance of crops after application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing changes in L42 xylanase under different carbon source conditions of the present invention.

[0023] Figure 2 This is a graph showing changes in L42 xylanase under different nitrogen source conditions of the present invention.

[0024] Figure 3 This is a graph showing changes in L42 xylanase under different inorganic salt conditions of the present invention.

[0025] Figure 4 It is a graph showing the changes of L42 xylanase under different pH conditions of the present invention.

[0026] Figure 5 This is a graph showing changes in L42 xylanase under different temperature conditions of the present invention.

[0027] Figure 6 This is a diagram of the enzymatic kinetic changes of L42 xylanase under liquid culture conditions at 15°C of the present invention.

[0028] Figure 7 A diagram showing the effect of the low-temperature xylanase-producing Comamonas koreana of the present invention on the temperature of cow dung composting.

[0029] Figure 8 This is a diagram showing the effect of the low-temperature xylanase-producing Comamonas koreana of the present invention on lignin in cow dung compost.

[0030] Fig. 9 This is a diagram showing the effect of the low-temperature xylanase-producing Comamonas koreana of the present invention on cellulose in cow dung compost. DETAILED DESCRIPTION

[0031] The following is attached to the instruction manual Figure 1-9 The present invention is further described in detail with reference to the accompanying drawings and embodiments.

[0032] A screening method and application of a Korean Comamonas strain that produces xylanase at low temperature, including the Korean Comamonas (L42) strain. The preservation institution is the General Microbiology Center of the China Microbiological Culture Collection Administration. The Korean Comamonas preservation number is CGMCC No.26059, and the preservation date is November 7, 2022.

[0033] Isolation method of strains:

[0034] Add 5 g of the collected cow dung sample from Northeast China into a 150 mL Erlenmeyer flask containing 50 mL of sterile water, shake at 120 r / min for 30 min, draw 1 mL of the suspension into a Erlenmeyer flask containing enrichment culture medium, and culture in an incubator (15°C) with a shake rate of 120 r / min for 15 days. Inoculate into new enrichment culture medium at a 5% (V / V) inoculation rate every 15 days, and subculture for 10 times.

[0035] A method for screening a Korean Comamonas strain that can degrade cow dung and produce xylanase at low temperature.

[0036] The composition of the enriched Hutcheson screening medium is KH2PO4 1.0 g / L, NaCl 0.1 g / L, FeCl3·6H2O 0.01 g / L, MgSO4·7H2O 0.3 g / L, NaNO3 2.5 g / L, CaCl2·6H2O 0.1 g / L, xylan 100-1000 mg / L, and the balance is water; the pH of the culture medium is 4.8-7.2.

[0037] Screening of Comamonas strains in Korea:

[0038] a. Initial screening: Spread the enriched culture medium on the plate, dilute it by 10 -8 , 10 -9 , 10 -10 The sample solution was coated on the solid culture medium and the culture dish was placed in a constant temperature incubator at 15°C for 72 hours. The purified strain was inoculated on the xylan Congo red culture medium and cultured at 15°C for 4 days. Preliminary screening was performed based on the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d).

[0039] b. Rescreening: Transfer the primary screened strains into liquid culture medium and culture at 15°C for 3 days. The liquid culture fermentation liquid was heated at 3500 r / min -1 After centrifugation for 8 minutes, the xylanase activity of the supernatant was determined and reselected.

[0040] Optimization of fermentation conditions of strains:

[0041] a Effects of different carbon sources on the growth and xylanase activity of strain L42

[0042] Liquid culture medium was prepared with (glucose, maltose, sucrose, corn flour, glycerol, lactose, fructose, mannitol, citric acid, soluble starch) carbon sources as the carbon sources in the culture medium. 1 mL of L42 seed culture medium was inoculated into the culture medium (1000 mg / L inorganic salt liquid xylan powder fermentation medium), and after shaking culture at 28°C and 120 rpm for 2 days, centrifuged at 13000 rpm for 15 minutes, and the xylanase content in the supernatant was determined. At the same time, the concentration of bacterial suspension in the culture medium was determined to screen the carbon source.

[0043] b Effects of different nitrogen sources on the growth and xylanase activity of strain L42

[0044] The nitrogen source liquid culture solution was prepared with (peptone, yeast extract, soybean powder, casein, urea, ammonium sulfate, potassium nitrate, beef extract) nitrogen source as the nitrogen source in the culture medium. The rest of the method was the same as above, the xylanase content in the supernatant was determined, and the concentration of the bacterial suspension in the culture solution was determined at the same time to screen the nitrogen source.

[0045] c Effects of different inorganic salts on the growth and xylanase of strain L42

[0046] MgSO4, NaCl, KH2PO4 are used as inorganic salts in the culture medium, and liquid culture solutions with different inorganic salts are prepared. The rest of the method is the same as above, and the xylanase content in the supernatant is determined, and the concentration of the bacterial suspension in the culture solution is determined at the same time to screen the inorganic salts.

[0047] d Effects of different initial pH on the growth and xylanase activity of strain L42

[0048] Prepare liquid culture medium with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. The rest of the method is the same as above, the content of xylanase in the supernatant is determined, and the concentration of bacterial suspension in the culture medium is determined at the same time to screen the initial pH value.

[0049] e Effects of different temperatures on the growth and xylanase of strain L42

[0050] Prepare h liquid culture medium. Take 1mL L42 seed culture medium and inoculate it into liquid culture medium, place it in a shaking incubator at 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, and follow the same method as above. Measure the xylanase content in the supernatant, and at the same time measure the concentration of bacterial suspension in the culture medium, and select the temperature.

[0051] The Korean Comamonas strain of the present invention reaches a xylanase peak in 96 hours in terms of enzymatic kinetics, and the optimum carbon source is corn flour, the optimum nitrogen source is peptone, the optimum inorganic salt is magnesium sulfate, the optimum pH is 5-6, and the optimum fermentation temperature is 15-25° C. A high content of xylanase is produced when the pH value is between 3-10 and the fermentation temperature is between 10° C. and 40° C., indicating that the bacterium has good tolerance to both temperature and pH, and can be further developed into a functional strain for industrial production of xylanase.

[0052] Example 1

[0053] CK: 1 ton of cow dung is adjusted to 50% moisture content

[0054] L42: Add 6kg of bacterial agent L42 to CK

[0055] Composting time: 2021.3.13-2021.4.25.

[0056] Composting location: Shenyang.

[0057] Specific process: For each compost pile of 1000kg, the cow dung is neatly stacked parallel to the ground. When it rises by 20cm, a small amount of inoculant is sprinkled on the surface and it is tamped down to be compacted. The pile height is maintained at 1.5 meters and the outer surface is covered with agricultural plastic film. The pile is stacked for a total of 45 days.

[0058] Indicators: Determine the temperature of cow dung composting and the lignin and cellulose content of straw at different periods.

[0059] The results showed that under the condition of an average spring temperature of 6°C, by adding low-temperature xylanase-producing Korean Comamonas, the average temperature of cow dung compost reached 47.3°C, which was 50.7% higher than the control, and the lignin content and cellulose content were reduced by 31.5% and 36.9% respectively. This indicates that low-temperature xylanase-producing Korean Comamonas can increase the compost temperature and accelerate the decomposition of lignin and cellulose content in straw.

[0060] Example 2

[0061] The experiment was conducted in Zhangzhuang Village, Cainiu Town, Tieling City, Liaoning Province (E123°35′58.86〞, N 42°21′12.17〞) in December 2020. The area has a semi-humid monsoon continental climate and is a typical dry farming area in the Liaobei Plain. The soil type of the experimental site is brown soil, and the physical and chemical nutrient status of the tillage layer is shown in Table 1. The customary planting method is mainly one-season spring corn, and the spring rotary tillage of 12-15cm is carried out for consecutive years.

[0062] Table 1 Basic physical and chemical properties of cultivated soil

[0063]

[0064] The experiment was a field plot experiment with four treatments: ① no fertilizer control (no fertilizer, CK); ② conventional planting, N, P2O5, K2O dosage of 180, 75, 90 kg hm -2 (FP); ③ Cow dung was composted at low temperature and then tilled back into the field (9000kg hm -2 ), the dosage of N, P2O5, and K2O is 180, 75, and 90 kg hm -2 (SFPM).

[0065] The test fertilizers were urea (N 46%), diammonium phosphate (N 18%, P2O5 46%), potassium sulfate (K2O 50%) and superphosphate (P2O5 12%), and the application methods of each treatment were the same; the test corn variety was Tieyan 58.

[0066] Each treatment was repeated three times, and the plot was 10 m long, 3.48 m wide, and 35 m in area. 2 The two treatments of no fertilizer control and conventional planting were rotary tilled in spring and managed in the field in the same way. The cow dung was evenly spread in the field before spring sowing after being decomposed by the L42 strain at low temperature, and then rotary tilled and ridged by a machine and applied to the tillage layer. The corn was sown at equal intervals in each treatment, with plant and row spacing of 27 cm and 58 cm respectively. The field was managed in the same way and harvested in September.

[0067] result

[0068] 1. Changes in corn yield under different treatments;

[0069] As shown in Table 2, the increase in corn grain yield and straw yield after returning cow dung to the field after low-temperature composting reached statistically significant differences. Compared with the FP treatment, the yield of the SFPM treatment after returning cow dung to the field increased by 28.72% and the straw biomass increased by 17.75%. It can be seen that after returning cow dung to the field after low-temperature composting, corn yield and straw biomass showed an increasing trend.

[0070] Table 2 Effect of returning straw to fields on corn yield from 2017 to 2020

[0071]

[0072] Note: Different letters indicate significant differences among different treatments (P<0.05).

[0073] 2. Changes in soil organic matter, nitrogen, phosphorus and potassium content in different treatments;

[0074] Low-temperature composting of cow dung helps to increase the organic matter content, as shown in Table 3. The organic matter content of the SFPM treatment was 19.12 g kg-1, which was significantly different from that of the FP treatment.

[0075] Low-temperature composting of cow dung also affects the accumulation of macronutrients in the soil. The total nitrogen, total phosphorus, and total potassium contents of the soil increased in the year of SFPM application, and the total nitrogen content was 1.18 g kg -1 , total phosphorus content is 0.62g kg -1 , total potassium content is 22.3g kg -1 , which was significantly different from conventional fertilization FP.

[0076] Table 3 Effect of returning straw to fields on soil nutrient content

[0077]

[0078] 3. Distribution of aggregates of different particle sizes between treatments;

[0079] The distribution of soil aggregates in different treatments is shown in the table. In the 0-40cm soil layer, >2mm and 0.25-1mm aggregates accounted for the highest proportion in each treatment, and the conventional fertilization FP treatment accounted for the lowest proportion of 48.8% and 47.88% respectively, and the rest of the treatments accounted for more than 50%. The proportion of aggregates in the SFPM treatment 0-20cm soil layer was greater than the sum of the two particle sizes of the 20-40cm soil layer, and the different levels were 65.12% and 61.64%; the percentage of <250um in the FP treatment was higher than that in other treatments, and the different levels were 18.14% and 18.61%.

[0080] Table 4 Mass percentage of soil aggregates in the 0-40 cm tillage layer

[0081]

[0082] In summary, field experiments have shown that low-temperature composting of cow dung can significantly increase soil organic matter, improve soil fertility, increase soil aggregate content, and increase crop yield and biomass. It is an effective way to treat cow dung.

Claims

1. A strain of Comamonas koreaensis that can degrade cow dung and produce xylanase at low temperature, characterized in that: The classification name of Comamonas in Korea is Comamonas koreensis The preservation institution is the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation number of Korean Comamonas is CGMCC No.26059, and the preservation date is November 7, 2022.

2. The Korean Comamonas bacteria capable of degrading cow dung and producing xylanase at low temperature according to claim 1, characterized in that: The biological characteristics of Korean Comamonas are: the colonies on LB culture medium are small, round, neat, opaque, smooth, sticky and easy to pick up, with typical bacterial colony characteristics.

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