CN118530916A - Method for screening and breeding high-temperature fungus compost fungus - Google Patents

Abstract

The present invention discloses a method for screening and breeding thermophilic composting bacteria, belonging to the field of microbial technology, and specifically comprising: (1) gradient enrichment and domestication, selecting a bacterial sample, inoculating the bacterial sample into an LB culture medium at an inoculation rate of 10% (v/v), and sequentially performing enrichment and domestication at three temperature levels; (2) coating, respectively dissolving the bacterial sample after enrichment and domestication at three temperatures in physiological saline, fully mixing, coating on an LB culture medium, culturing at 50°C, separating and purifying the strain, and obtaining thermophilic bacteria; (3) testing and verification, testing the growth of the obtained thermophilic bacteria under different temperature conditions; the thermophilic bacteria selected and bred by the method have a tolerance of 80°C and maintain high functional activity.

Description

A method for screening and breeding high-temperature composting bacteria

Technical Field

The invention relates to a method for screening and breeding bacteria, in particular to a method for screening and breeding high-temperature compost bacteria, and belongs to the technical field of microorganisms.

Background Art

Thermotolerant bacteria and facultative thermophiles can grow below 30℃, and the maximum growth temperature is 45-55℃ and 50-65℃ respectively; the minimum growth temperature of obligate thermophiles and extreme thermophiles is 40℃, and the optimum growth temperature is higher than 65℃; the maximum growth temperature of extreme thermophiles is higher than 70℃. At the same time, these thermophiles have the characteristics of fast metabolism, high activity, short generation time, high thermal stability of enzymes, and creating high temperature conditions to kill pathogens. They play an immeasurable role in the degradation of organic matter and the killing of pathogenic microorganisms during the high temperature period of composting. Studies have shown that inoculating thermodegradable bacteria in compost can effectively shorten the composting cycle and improve the quality of composting, creating favorable conditions for the industrial production of organic waste composting to better achieve reduction, harmlessness, stabilization, and resource utilization, and has a high application prospect. Based on the many shortcomings of traditional composting technology, such as low fermentation temperature, long fermentation cycle, incomplete harmlessness, and serious odor pollution, it has become a bottleneck restricting the comprehensive promotion and application of this technology. Therefore, the screening of thermophiles and the study of their enzymatic properties have high research value and great significance.

There are many reported application methods of thermophilic composting bacteria, including a method for aerobic composting of sludge using thermophilic bacteria (above 55°C) and white rot fungi (Plantariomyces chrysosporium) reported in patent (CN106905006A); a method for producing a composite ultra-high temperature composting agent by synergistic fermentation of three extreme thermophilic bacteria, the temperature of which can reach 70-80°C reported in patent (CN107937303A); a method for high temperature solid fermentation of soybean meal using thermotolerant bacteria (Bacillus stearothermophilus) reported in patent (CN108935945A), the optimal growth temperature of which is 55-60°C; a technical method for screening target strains using a constant temperature 70°C water bath reported in patent (CN109517754A); a filter mud thermophilic composting agent LC and its application reported in patent (CN110093296A), including Bacillus licheniformis (Bacillus licheniformis)GBIF-3, during the fermentation and decomposition process, the lignocellulase activity (the sum of filter paper enzyme activity, cellulase activity and xylanase activity) in the crude enzyme solution was determined, and the maximum value was: 50U; Patent (CN110819554A) reported an extreme thermophilic bacteria and its application in high-temperature composting fermentation, which is a mutant obtained by mutagenesis, domestication, gene recombination or natural mutation. During the high-temperature composting fermentation process, esterase, cellulase, alkaline amylase and alkaline protease are added to promote the compost to quickly enter the high-temperature stage. The breeding technology used is the traditional inefficient mutant acquisition method; Patent (CN112322498A) reported a method for preparing a high-temperature bacterial agent for efficient composting of kitchen waste, which involves the screening of a specific and efficient culture medium. The screened strain can degrade cellulose, lignin and protein with high-efficiency functional strains. The strain is a thermophilic Thermus thermophilus UTM802 with a temperature tolerance of 65-70℃.

Based on the defects and shortcomings of existing microbial agents, the existing strains have low temperature and narrow tolerance range, low enzyme activity, and traditional inefficient breeding techniques and methods. Therefore, developing a screening and breeding method for high-temperature bacterial compost that can overcome the above defects has become a technical problem that needs to be urgently solved by technical personnel in this field.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for screening and breeding thermophilic composting bacteria. The method is simple and easy to implement. The thermophilic bacteria screened and bred by the method have a tolerance of 50-80°C, up to 80°C, and maintain high functional activity.

In order to solve the above technical problems, the present invention provides a method for screening and breeding thermophilic composting bacteria, which specifically comprises the following steps:

(1) Gradient enrichment and domestication

Select bacterial samples, inoculate them into LB medium at a 10% (v/v) inoculation rate, and perform enrichment and acclimatization at three temperature levels in sequence;

① Place in a shaker/water bath/oven at 60°C, culture and observe continuously, measure OD600 every 24 hours, for 3-5 times in a row;

② Transfer the bacterial sample cultured in step ① to fresh LB medium at a 10% (v/v) inoculation volume, place it in a shaker/water bath/oven at 70°C, culture and observe continuously, and measure OD600 every 24 hours for 3-5 times;

③ Transfer the bacterial sample cultured in step ② to fresh LB medium at a 10% (v/v) inoculation volume, place it on a shaker/water bath/oven at 80°C, culture and observe continuously, and measure OD600 every 24 hours for 3-5 times;

(2) Coating

The three temperature-enriched and domesticated bacterial samples were dissolved in physiological saline, mixed thoroughly, spread on LB medium, cultured at 50°C, and the strains were separated and purified to obtain thermophilic bacteria;

(3) Testing and verification

The obtained thermophilic bacteria were tested for growth under different temperature conditions.

The technical solution further defined in the present invention is:

Furthermore, in the aforementioned method for screening and breeding high-temperature composting bacteria, during the culture process of the strain sample in step ③ on a shaker/water bath/oven at 80°C, it is necessary to replenish sterile water to the starting scale line every 24 hours.

Technical effect: Water is easy to evaporate at 80℃, and adding sterile water can keep the culture medium components stable, making the bacterial turbidity data reliable and stable.

In the above-mentioned method for screening and breeding thermophilic composting bacteria, characterizing the growth performance of the thermophilic bacteria obtained includes the following steps:

(1) Plate activation: The obtained thermophilic bacteria were activated separately in a clean bench and cultured on a plate at 50°C for 2 days;

(2) Preparation of seed solution: LB medium was placed in a conical flask, and each strain was aseptically inoculated on a clean bench. The culture was shaken at 50°C for 2 days to obtain the prepared seed solution.

(3) Liquid temperature test: LB medium was placed in a conical flask, and the seed liquid was inoculated into fresh LB medium at a 10% (v/v) inoculum, in parallel with each other; the flasks were placed in ovens at 60°C, 70°C, and 80°C, respectively, and cultured and observed continuously. The OD600 was measured every 24 hours for 7 consecutive days, and the culture was observed and recorded continuously;

(4) Plate temperature test: For thermophilic bacteria, the growth of the plates was evaluated at 60°C, 70°C, and 80°C.

In the above-mentioned method for screening and breeding thermophilic composting bacteria, the antagonistic behavior of the thermophilic bacteria obtained is characterized, including the following steps:

(1) Plate activation and seed solution preparation;

(2) Antagonism test: The thermophilic bacteria were subjected to antagonism test, including single strain, combination of two strains, combination of three strains, and combination of four strains were inoculated into LB culture medium, and the two-by-two cross method was used, and the streaking culture was observed on a 50°C plate;

Among them, the starting OD600 of the thermophilic strain was adjusted to 1 to ensure the consistency of the initial strain inoculation concentration.

In the above-mentioned method for screening and breeding thermophilic composting bacteria, characterization of the thermophilic bacteria obtained for their resistance to foreign bacteria is carried out, including the following steps:

(1) Plate activation and seed solution preparation;

(2) Antibacterial test: 1) Activate the glycerol tube strains and culture them on plates at 50°C; 2) Inoculate the strains with primary seed solution for 24 hours at 50°C and LB, and measure their starting OD600; 3) Inoculate the sterilized group and non-sterilized group at 10% of the inoculation amount at 80°C and LB, and measure their OD600 and viable count at 4 and 7 days; 4) Obtain and record experimental data;

The sterilization group sterilized the culture medium at 121°C for 30 min; the non-sterile group used natural culture medium;

The starting OD600 of the thermophilic strain was adjusted to 1 to ensure a consistent initial strain inoculation concentration.

In the above-mentioned method for screening and breeding thermophilic composting bacteria, characterizing the enzyme activity of the thermophilic bacteria obtained includes the following steps:

(1) Plate activation: The obtained thermophilic strains were activated separately in a clean bench and cultured on a plate at 50°C for 2 days;

(2) Preparation of seed solution: LB medium was placed in a conical flask, and each strain was aseptically inoculated on a clean bench. The culture was shaken at 50°C for 2 days to obtain the prepared seed solution.

(3) Cellulase and ligninase activity plate test: The seed solution obtained in step (2) was diluted to OD600 = 1, and wells were punched with an Oxford cup. 200ul/well was taken, respectively, and the wells were placed in culture medium B2 and M5 at 50°C for static observation and continuous culture observation and recording;

(4) The three ligninase activities of thermophilic bacteria, Lac, MnP, and Lip, were detected in a liquid culture medium with alkaline lignin as the substrate. Samples were taken on the second day and a series of enzyme activity tests were performed and recorded.

In the above-mentioned method for screening and breeding thermophilic composting bacteria, the obtained thermophilic bacteria are subjected to ARTP breeding and high-throughput screening, which comprises the following steps:

(1) Preparation of seed solution: plate activation and seed solution preparation;

(2) Centrifugal washing: The seed solution in step (1) is washed 1-2 times with 0.85% saline. The number of ARTP-induced colonies is calculated based on the observation on the hemocytometer and controlled at 106-108 CFU/mL. The centrifugal conditions are: 2000 rpm, 2 min/time;

(3) ARTP mutagenesis: Dilute the washed spore suspension 10 times with 10% glycerol and resuspend it. Apply it on the iron sheet until it is completely covered. At the same time, pipette 0.85% saline into a centrifuge tube for subsequent shaking and elution.

(4) Mutagenesis conditions: The time was set to 0s, 30s, 60s, 90s, 120s, and 180s, respectively; 2 parallels per group, power 100w, gas volume 10SLM; after the mutagenesis, the elution was shaken for ≥1min, all the liquid was washed out, and the plates were coated with LB (0s), B2, and M5, respectively, and placed in a 37°C incubator for culture, and the induced lethality was observed and calculated;

(5) High-throughput screening: Screening of high-yield mutant strains was carried out on the screening plate M5, and the mutant strains were screened by high-throughput screening using a high-throughput screening instrument. The growth rate was measured by an ELISA instrument, and the enzyme activity was measured and characterized.

In the aforementioned screening and breeding method of thermophilic composting bacteria, the B2 formula is: sodium carboxymethylcellulose 10 g/L, KH ₂ PO ₄ 2 g/L, MgSO ₄ ·7H ₂ O 0.5 g/L, MnSO ₄ 0.5 g/L, Congo red 0.4 g, add distilled water to 1000 mL, and adjust the pH value to 6.0 with acetic acid;

The formula of M5 is: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, aniline blue 0.1g/L, add distilled water to 1000mL, pH natural.

In the above-mentioned screening and breeding method of thermophilic composting bacteria, the formula of LB culture medium is: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and the pH of LB culture medium is controlled at 7.0.

The beneficial effects of the present invention are:

The present invention will use gradient domestication, combined with liquid bacteria turbidity and plate temperature testing, and multiple methods for breeding to obtain strains with stable temperature tolerance, wide adaptability and high application performance. The ARTP and high-throughput breeding technology used in the present invention greatly improve the mutation rate and positive mutant bacteria, and the target strains obtained have superior performance and the method is efficient.

The present invention provides a method for screening and breeding high-temperature composting bacteria, including strain screening technology, characterization of growth performance, antagonism, resistance to foreign bacteria and enzyme activity of target strains, and ARTP breeding and high-throughput screening of the selected high-activity strains. The strains screened by the present invention not only have a tolerance of 50-80°C and are thermophilic up to 80°C, but also have efficient and rich lignin series enzyme activities, including cellulase activity, laccase (lac), and lignin peroxidase (LiP).

The ARTP breeding and high-throughput screening adopted in the present invention obtained mutant strains with excellent performance, and the lac enzyme activities of the mutant strains were respectively increased by 116.88%, 176.41%, 114.98%, 97.58%, 125.49%, 128.93% and 126.02% relative to the starting strains.

The ARTP breeding carried out in the present invention uses active energy-rich particles in a normal-pressure room-temperature plasma source using helium as the working gas, which damages the genetic material of the strain and induces the biological cells to initiate the SOS repair mechanism. The high fault tolerance rate of SOS repair will produce a rich variety of mismatch sites, and ultimately stabilize the inheritance to form mutant strains.

The high-throughput screening instrument used in the present invention performs high-throughput screening on mutant strains, and combines with an ELISA instrument to characterize their growth conditions, and has the advantages of rapidity, high efficiency, high sensitivity, high specificity, and high signal-to-noise ratio.

The high-efficiency strain obtained by the present invention is suitable for high-temperature composting and maturation processes of high-temperature paint livestock and poultry manure, straw compost, garden waste or greening waste, and can be prepared as a solid microbial high-temperature composting inoculant, which has low cost and can avoid the inactivation of active microorganisms in the inoculant during the high-temperature fermentation period. It has a wide temperature application range when used in composting.

The high-efficiency strain obtained by the present invention has the characteristics of fast metabolism, high activity, short generation time, high thermal stability of enzymes, and the ability to kill pathogens under high temperature conditions. It plays an immeasurable role in the degradation of organic matter and the killing of pathogenic microorganisms during the high temperature period of composting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the growth of thermophilic bacteria on a 70° C. plate according to an embodiment of the present invention;

FIG2 is a graph showing the growth of four thermophilic bacteria at 60° C. according to an embodiment of the present invention;

FIG3 is a graph showing the growth of four bacterial strains at 70° C. according to an embodiment of the present invention;

FIG4 is a graph showing the growth of four bacterial strains at 80° C. according to an embodiment of the present invention;

FIG5 is a diagram showing the liquid antagonistic growth of strains in an embodiment of the present invention;

FIG6 is a diagram showing the antagonistic growth of strains on a plate according to an embodiment of the present invention;

FIG7 is a diagram showing the live bacteria count in an anti-bacteria experiment according to an embodiment of the present invention;

FIG8 is a diagram showing the characterization of enzyme activity on a plate of a strain in an embodiment of the present invention;

FIG9 is a graph showing the lethality at different mutagenesis times in an embodiment of the present invention;

FIG. 10 is a graph showing the growth of the dominant strain and the enzyme activity assay results in an embodiment of the present invention.

DETAILED DESCRIPTION

The present invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

Embodiment 1:

The present embodiment provides a method for screening and breeding thermophilic composting bacteria, which specifically includes the following steps:

(1) Gradient enrichment and domestication

The bacterial sample G1 was selected. The source of G1 was the compost sample of Jiangsu Environmental Engineering Taihu Ecological Technology Co., Ltd., which was obtained by aerobic fermentation with crop straw, garden waste and cyanobacteria mud as raw materials.

The bacterial sample G1 was inoculated into LB medium (liquid volume 100mL/250mL) at a 10% (v/v) inoculation volume, and enriched and acclimated at three temperature levels in sequence;

① Place in a shaker/water bath/oven at 60°C, culture and observe continuously, measure OD600 every 24 hours, for 3-5 times in a row (depending on the growth situation);

② Transfer the bacterial sample cultured in step ① to fresh LB medium at a 10% (v/v) inoculation volume, place it in a shaker/water bath/oven at 70°C, culture and observe continuously, and measure OD600 every 24 hours for 3-5 times (depending on the growth situation);

③ Transfer the bacterial sample cultured in step ② to fresh LB medium at a 10% (v/v) inoculation volume, place it in a shaker/water bath/oven at 80°C, culture and observe continuously, and measure OD600 every 24 hours for 3-5 times (depending on the growth situation);

The OD600 values of the G1 acclimation results at each temperature after gradient enrichment acclimation are shown in Table 1;

(2) Coating

The bacterial samples after enrichment and acclimation at three temperatures were dissolved in physiological saline, mixed thoroughly, spread on LB medium, cultured at 50°C, and strains with different colony morphology were selected for separation and purification. Eight high-temperature strains were obtained and are to be verified.

(3) Testing and verification

The obtained thermophilic bacteria were verified on a plate at 70°C as shown in Figure 1;

The obtained thermophilic strains were further inoculated into LB liquid culture medium (liquid volume 100 mL/250 mL), and cultured continuously for 2 days on a shaker/water bath/oven at 80°C. The OD600 was measured and the experimental results were observed and recorded as shown in Table 2.

In this embodiment, the medium formula of LB is: tryptone 10g/L, yeast extract 5g/L, sodium chloride (NaCl) 10g/L, pH 7.0.

During the culture process of the strain in a shaker/water bath/oven at 80°C, sterile water needs to be added to the starting liquid level, i.e. the starting scale line, every 24 hours.

Table 1 OD600 values of sample G1 temperature acclimation results

Table 2 Verification of thermophilic strain liquid at 80℃

Result analysis:

1) It can be seen from Table 1 that through the enrichment and domestication at different temperatures, such as 60°C, 70°C, and 80°C, it can be seen that there are high-temperature resistant strains in the strains in the sample; by gradually increasing the temperature, the miscellaneous bacteria are eliminated and sorted, and the enriched and domesticated bacterial community at 80°C is sorted.

2) Through gradient dilution and coating screening, 8 strains that can grow at a high temperature of 50°C were initially obtained; the 8 thermophilic bacteria obtained were further tested and rescreened under temperature conditions of 70°C on a plate as shown in Figure 1. It was found that GW-80-6 grew at 50°C and could hardly grow at 70°C, so it was initially eliminated; other strains could grow at 50-80°C.

3) The results of the temperature test of 80℃ for the rescreened strains GW-80-1, GW-80-2, GW-80-3, GW-80-4, GW-80-5, GW-80-7 and GW-80-8 are shown in Table 2. It can be seen that the OD600 of strains GW-80-3, GW-80-5 and GW-80-8 are relatively high, reaching between 0.3 and 0.5, indicating that the three strains grow well at a high temperature of 80℃ and are used as backup strains for further experiments.

Embodiment 2:

(1) Based on the fact that the existing 21 strains can be purchased from the China General Microbiological Culture Collection Center (CGMCC), China Center of Industrial Culture Collection (CICC), China Center for Type Culture Collection (CCTCC), American Type Culture Collection (ATCC), and Agricultural Culture Collection of China (ACCC), see Table 3 for details;

The purchased strains were subjected to plate and liquid temperature tests; the detection indicators were plate observation and OD600 determination.

Table 3 Sources of thermophilic bacteria samples

The present embodiment provides a method for screening and breeding thermophilic composting bacteria, which specifically includes the following steps:

(1) Strain revival and initial screening: Thaw the above strain glycerol tube naturally, apply 100ul to LB plate, culture at 50℃ for 2-3 days, observe its growth, and perform initial screening, as shown in Table 4;

(2) Temperature test and rescreening: The strains obtained from the initial screening were transferred to liquid LB medium and cultured in an 80°C water bath. Their growth was observed, tested and recorded, as shown in Table 5.

Table 4 Growth of thermophilic strains on 50℃ plates

Table 5 Growth of thermophilic strains in 80℃ liquid

Result analysis:

According to Table 4, the initial screening showed that the strains with good growth were GXGD-YZ-438 and GXGD-YZ-900. According to Table 5, the strains that grew well at a high temperature of 80°C after rescreening showed the following order of high temperature effect: GXGD-YZ-438>GXGD-YZ-900>GXGD-YZ-822>GXGD-YZ-441>GXGD-YZ-1028.

GXGD-YZ-438, GXGD-YZ-900, and GXGD-YZ-822 were comprehensively screened and used as backup strains for further experiments.

Embodiment 3:

This example provides a characterization of the growth performance of the thermophilic strains obtained in Examples 1 and 2, specifically:

(1) Plate activation: Activate the above four bacterial strains GXGD-YZ-900, GXGD-YZ-822, GXGD-YZ-438, and GW-80-5 separately on a clean bench and culture them on a plate at 50°C for 2 days (depending on the growth conditions).

(2) Preparation of seed solution: 40 ml of LB medium was placed in a 100 ml Erlenmeyer flask, and each strain was aseptically inoculated on a clean bench. The culture was shaken at 50°C for 2 days to obtain the prepared seed solution.

(3) Liquid temperature test: 200 ml LB medium is placed in a 500 ml triangular bottle. The seed liquid is inoculated into a new LB medium at a 10% (v/v) inoculation rate. Each group is parallel. Place in an oven at 60°C, 70°C, and 80°C (check the temperature or add water regularly). Continuously cultivate and observe. Measure OD600 every 24 hours for 7 consecutive days (depending on the growth situation). Continuously cultivate and observe and record as shown in Figures 2, 3, and 4.

(4) Plate temperature test: The growth characteristics of the four bacterial strains at 60°C, 70°C, and 80°C were evaluated as shown in Table 6.

(1) The results of the liquid growth analysis are shown in Figures 2, 3, and 4;

1) According to Figure 2, it can be seen that the four strains can grow well at 60℃. Among them, GXGD-YZ-438 reached a peak of 2.5 on the third day, and there was a fluctuation in the later flow of nutrient solution, and then it was able to continue the rapid growth process; among them, GXGD-YZ-900 reached a peak of 0.55 on the fourth day, and there was a fluctuation in the later flow of nutrient solution, and there was a small increase in the later period, but the increase was stable; among them, GXGD-YZ-822 reached a peak of about 1.0 on the second day, and there was a fluctuation in the later flow of supplementary solution, and then it reached a peak of about 0.9 again on the fourth day, and then it was able to continue to grow; among them, WG-80-5 grew slowly in the first 5 days, reached a peak of 2.3 on the sixth day, and there was a fluctuation in the later flow of supplementary solution, and then there was the potential for continued growth. Comprehensively speaking, the growth of the four strains at 60℃ was GXGD-YZ-438>WG-80-5>GXGD-YZ-822>GXGD-YZ-900.

2) According to Figure 3, it can be seen that the growth of the four strains at 70°C is significantly improved compared to that at 60°C. The growth activity of strains WG-80-5 and GXGD-YZ-900 is significantly improved, and the growth activity of strains GXGD-YZ-822 and GXGD-YZ-438 is not fully activated. Considering that the temperature startup process of the strains should be increased step by step from 50°C, 60°C, 70°C, and 80°C. Among them, GXGD-YZ-438 reached a peak value of 0.4 on the second day, and there was a fluctuation in the later flow-added supplement, but the bacterial concentration OD600 fluctuated between 0.25 and 0.4 from day 1 to day 7; among them, GXGD-YZ-900 reached a peak value of about 2.75 on the fourth day, and there was a fluctuation in the later flow-added supplement, and there was a small increase in the later period, but the increase was stable. The growth of this strain at 70°C was significantly better than that at 60°C; among them, the bacterial concentration OD600 of GXGD-YZ-822 fluctuated between 0.25 and 0.42 from day 1 to day 7, and there was a fluctuation in the later flow-added supplement, with a slight growth trend; among them, WG-80-5 was in a rapid growth trend from day 1 to day 7, reaching a peak value of 4.5, and there was a fluctuation in the later flow-added supplement, and then there was the potential for continued growth. The growth conditions of the four strains under 70℃ were as follows: WG-80-5>GXGD-YZ-900≥GXGD-YZ-822≈GXGD-YZ-438.

3) According to Figure 4, it can be seen that the growth of the four strains at 80°C is significantly inhibited compared with 60°C and 70°C. Considering that the temperature starting process of the strains should be gradually increased from 50°C, 60°C, 70°C, and 80°C, it will be beneficial to activate the activity of high-temperature strains. Among them, the bacterial concentration OD600 of GXGD-YZ-438 on the 1st to 7th day fluctuated between 0.18 and 0.25; among them, the bacterial concentration OD600 of GXGD-YZ-900 on the 1st to 7th day fluctuated between 0.20 and 0.25; among them, the bacterial concentration OD600 of GXGD-YZ-822 on the 1st to 7th day fluctuated between 0.18 and 0.25, and there was a fluctuation in the later flow-added supplementary liquid, and there may be a certain growth range in the later period; among them, the bacterial concentration OD600 of GXGD-YZ-822 on the 1st to 7th day fluctuated between 0.18 and 0.25, and there was a fluctuation in the later flow-added supplementary liquid, and there may be a certain growth range in the later period; it reached a peak value of 0.4 on the 2nd day, and there was a fluctuation in the later flow-added supplementary liquid, but the bacterial concentration OD600 on the 1st to 7th day fluctuated between 0.25 and 0.4; among them, WG-80-5 reached a peak value of about 0.4 on the 5th day, and there was a fluctuation in the later flow-added supplementary liquid, and there is still potential for continued growth in the later period. The growth conditions of the four strains under 80℃ were as follows: WG-80-5>GXGD-YZ-900≥GXGD-YZ-822≈GXGD-YZ-438.

(2) The growth conditions of the four strains at 60℃, 70℃ and 80℃ are shown in Table 6.

Table 6 Growth of 4 strains under different temperature conditions

Summary: According to Table 6, the growth characteristics of four thermophilic bacteria were investigated under the temperature conditions of 60℃, 70℃ and 80℃. It was found that under the condition of 60℃, the growth conditions of the four strains were GXGD-YZ-438≈WG-80-5> GXGD-YZ-822>GXGD-YZ-900; under the conditions of 70℃ and 80℃, the growth conditions were consistent with WG-80-5> GXGD-YZ-900≥GXGD-YZ-822≈GXGD-YZ-438.

Embodiment 4:

This example characterizes the antagonism of the high-temperature strains obtained in Examples 1 and 2, specifically:

Plate activation and seed solution preparation were the same as in Example 3;

Antagonism test: Antagonism tests were conducted on the four strains WG-80-5, GXGD-YZ-900, GXGD-YZ-822, and GXGD-YZ-438 in pairs, three-strain combinations, and four-strain combinations;

Specifically, group 1: individual bacterial strains were inoculated into LB medium;

Group 2: 4 bacterial strains were mixed and inoculated into LB medium;

Group 3: two different strains of bacteria were inoculated into LB medium;

Group 4: three different strains of bacteria were inoculated into LB medium;

The experimental results are shown in FIG5 (the abbreviated strain numbers in FIG5 correspond to 4 strains according to the digital numbers). The 4 strains were subjected to the cross-cross method and streaked on a 50°C plate for observation, as shown in FIG6 .

Among them, the starting OD600 of the four bacterial strains was adjusted to 1 to ensure that the initial strain inoculation concentration was consistent.

Result analysis:

1) According to the liquid growth concentration in Figure 5, it can be seen that the two-by-two combinations of 438+80-5, the three strain combinations of 438+822+80-5, 822+900+80-5, and 900+80-5+438 are significantly higher than the single strain, and the composite effect of the four strains is not good. Among them, strains YZ-438 and GW-80-5 have good high temperature synergy.

2) According to Figure 6, the four thermophilic bacteria were crossed in pairs and antagonism experiments were carried out at 50°C. The results showed that the strain combination YZ-822 was weakly antagonistic to YZ-80-5, YZ-438, and YZ-900, and no antagonism was observed between the other strains, further confirming some of the results in Figure 5.

Embodiment 5:

This example characterizes the resistance of the high-temperature strains obtained in Examples 1 and 2 to other bacteria, specifically:

(1) Plate activation and seed solution preparation are the same as in Example 3;

(2) Antibacterial test: Antibacterial experiments were conducted on four thermophilic bacteria (GXGD-YZ-900, GXGD-YZ-822, GXGD-YZ-438, and GW-80-5);

1) The 4 obtained thermophilic bacteria were frozen in glycerol tubes, and the glycerol tube strains were activated on plates (50°C) for anti-bacteria experiments; 2) The strains were inoculated with primary seed solution, 24h (1d), 50°C, LB, and their starting OD600 was measured respectively; 3) The sterilization group and non-sterilization group were inoculated with 10% inoculation volume, 80°C, LB, and their OD600 and viable count were measured at 4d and 7d respectively; 4) The experimental data obtained are shown in Table 7 and Figure 7;

Among them, the sterilization group sterilized the culture medium at a high temperature of 121°C for 30 minutes; the non-sterile group used natural culture medium;

Among them, the starting OD600 of the four bacterial strains was adjusted to 1 to ensure the consistency of the initial strain inoculation concentration;

Among them, the number of live bacteria can be drawn according to the reference line and then diluted in a gradient manner. The main purpose is to observe the proportion of target bacteria and non-target bacteria in order to evaluate the antibacterial performance.

Table 7 Bacterial concentration and viable count of 4 strains of thermophilic bacteria in the antibacterial experiment

Result analysis:

According to Table 7, the 4 groups of bacteria were subjected to anti-bacteria experiments, and the base materials were subjected to sterilized group and non-sterilized group experiments. It was found that the OD600 of the two groups of experiments was not much different. The live bacteria were counted and it was found that the live bacteria counts of the sterilized group and the non-sterilized group were basically close, among which there were a few foreign bacteria in the non-sterilized group. Among them, the strain GXGD-YZ-438 had a stronger ability to resist foreign bacteria, while the strain GXGD-YZ-900 had a weaker ability to resist foreign bacteria.

Embodiment 6:

This example screens and characterizes the enzyme activity of the thermophilic strains obtained in Examples 1 and 2, specifically:

(1) Plate activation: Activate the above four strains of bacteria GW-80-5, GXGD-YZ-438, GXGD-YZ-822, and GXGD-YZ-900 on a clean bench and culture them on a plate at 50°C for 2 days (depending on the growth conditions);

(2) Preparation of seed solution: 40 ml of LB medium was placed in a 100 ml Erlenmeyer flask, and each strain was aseptically inoculated on a clean bench. The culture was shaken at 50°C for 2 days to obtain the prepared seed solution.

(3) Cellulase and ligninase activity plate test: The seed solution obtained in step (2) was diluted with sterile water to OD600 = 1, and wells were punched with an Oxford cup. 200ul/well was taken, respectively, and the wells were placed in culture medium B2 and M5 at 50°C for static observation and continuous culture observation and recording, as shown in Table 8 and Figure 8;

(4) The activity of three ligninases (Lac, MnP, Lip) in the four thermophilic bacteria GXGD-YZ-438, GXGD-YZ-900, GXGD-YZ-822, and GW-80-5 was detected in liquid culture medium with alkaline lignin as substrate. The data are as follows. Samples were taken on the second day and a series of enzyme activity tests were performed, as shown in Table 9.

Table 8 Characterization of enzyme activity system of four thermophilic bacteria

Table 9 Results of the determination of three ligninase activities in four strains

Result analysis:

1) According to Table 8 and Figure 8, the main lignin-related enzyme systems were systematically characterized under 50°C conditions for 2-3 consecutive days of observation. The qualitative characterization results of the plate color development are shown in Table 8. Among them, the strain GXGD-YZ-822 has a richer enzyme system, including cellulase activity, laccase (lac), and lignin peroxidase (LiP); the strain GXGD-YZ-438 has a relatively high specific activity of cellulase activity and lignin enzyme activity; the strain GXGD-YZ-900 mainly has laccase (lac) and lignin peroxidase (LiP). It is preliminarily concluded that the comprehensive enzyme activity of strain GXGD-YZ-438 is a relatively high strain.

2) The main lignin-related enzyme systems of the four strains were characterized by liquid system according to Table 9, which was basically consistent with the results of qualitative characterization by plate colorimetry, mainly laccase (lac) and lignin peroxidase (LiP) enzyme activities. Based on the analysis of the plate and liquid experimental results, strain GXGD-YZ-438 was selected as the starting strain for ARTP breeding and high-throughput screening.

Embodiment 7:

This example provides ARTP breeding and high-throughput screening of efficient and active functional strains. The high-temperature strain GXGD-YZ-438 with the highest enzyme activity obtained in Example 6 was subjected to ARTP and high-throughput screening to obtain 1-2 strains with excellent performance, specifically:

(1) Preparation of seed solution: Plate activation and seed solution preparation are the same as in Example 3;

(2) Centrifugal washing: The seed solution in step (1) is washed 1-2 times with 0.85% saline. The number of ARTP-induced colonies is calculated based on the observation on the blood cell counting plate and controlled at 10 ⁶ -10 ⁸ CFU/mL. The preferred centrifugal conditions are: 2000 rpm, 2 min/time;

(3) ARTP mutagenesis: Dilute the washed spore suspension 10 times with 10% glycerol and resuspend it. Then take 15ul and apply it on the iron sheet until it is completely covered (200 CFU/mL/iron sheet can be calculated by reverse calculation). At the same time, draw 200ul of 0.85% saline into a 2ml centrifuge tube for subsequent shaking and elution.

(4) Mutagenesis conditions: The time was set to 0s, 30s, 60s, 90s, 120s, and 180s, respectively; 2 parallels per group, power 100w, gas volume 10SLM; after the mutagenesis, the elution was shaken for ≥1min, all the liquid was washed out, and the plates were coated with LB (0s), B2, and M5, respectively, and placed in a 37°C incubator for culture. Observation was performed at appropriate times, and the induced lethality was calculated as shown in Figure 9;

(5) High-throughput screening: A mutagenesis time of 90 s was selected, and high-yield mutant strains were screened on the screening plate M5. The mutant strains obtained on the screening plate M5 were subjected to high-throughput screening using a high-throughput screening instrument, as shown in Table 10. Combined with the determination of their growth rate using an ELISA instrument, the growth rate and enzyme activity determination results are shown in Figure 10.

Table 10 Growth of high-yield mutant strains under high-throughput screening conditions

Result analysis:

1) According to Figure 9, the optimal mutagenesis time of strain GXGD-YZ-438 was tested and it was determined that the optimal mutagenesis time was 90s, the lethality was 87.95%, the starting colonies were 100-200, and the mutant strains were 10-20, which basically met the experimental requirements.

Under the optimal mutagenesis conditions of 100W power and 90s, an M5 high enzyme activity mutant strain experiment was carried out to obtain 300-500 mutant strains, and the colony size and enzyme activity transparent zone formed were subjected to high-throughput screening. High-throughput screening of high-yield mutant strains was carried out on a 96-well plate as shown in Table 10, and the data are the growth of the strain OD600.

2) Among the 90 selected mutant strains (A1-A3 were blank controls and B1-B3 were starting strains), 32 strains with OD600 greater than the average starting strain (1.0935) and greater than 1.15 were screened, and their lac enzyme activities were measured. The specific data are shown in Figure 10. According to the characterization of strain growth and enzyme activity in Figure 10, 7 strains with high growth rate and enzyme activity were screened out, namely B5, C4, D5, D6, D9, G8, and H12, with enzyme activities of (68±6.03) U/mL, (86.93±9.23) U/mL, (67.61±0.67) U/mL, (62.14±10.31) U/mL, (70.92±17.17) U/mL, (72.00±3.77) U/mL, and (71.08±10.89) U/mL, respectively; compared with the enzyme activity of the starting strains (B1-B3) (31.45±5.34) U/mL, their enzyme activities were increased by 116.88%, 176.41%, 114.98%, 97.58%, 125.49%, 128.93%, and 126.02%, respectively.

The LB medium formula in Example 1-7 is: tryptone 10 g/L, yeast extract 5 g/L, sodium chloride (NaCl) 10 g/L, pH 7.0;

The formula of B2 medium is: sodium carboxymethylcellulose 10g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ ·7H ₂ O 0.5g/L, MnSO ₄ 0.5g/L, Congo red 0.4g, add distilled water to 1000mL, adjust the pH to 6.0 with acetic acid, and divide into 200mL/500mL;

The formula of M5 medium is: LB, 0.1g/L aniline blue, distilled water to 1000mL, natural pH;

Among them, B2 is the cellulase activity identification medium; M5 is the ligninase activity identification medium.

The present invention provides a method for screening and breeding high-temperature composting bacteria. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims (9)

1. A method for screening and breeding high-temperature composting bacteria, characterized in that it specifically comprises the following steps: (1) Gradient enrichment and domestication Select bacterial samples, inoculate them into LB medium at a volume ratio of 10%, and perform enrichment and acclimatization at three temperature levels in sequence; ① Place in a shaker/water bath/oven at 60°C, culture and observe continuously, measure OD600 every 24 hours, for 3-5 times in a row; ② Transfer the bacterial sample cultured in step ① to fresh LB medium at a volume ratio of 10%, place it in a shaker/water bath/oven at 70°C, culture and observe continuously, and measure OD600 every 24 hours for 3-5 times; ③ Transfer the bacterial sample cultured in step ② to fresh LB medium at a volume ratio of 10%, place it on a shaker/water bath/oven at 80°C, culture and observe continuously, and measure OD600 every 24 hours for 3-5 times; (2) Coating The three temperature-enriched and domesticated bacterial samples were dissolved in physiological saline, mixed thoroughly, spread on LB medium, cultured at 50°C, and the strains were separated and purified to obtain thermophilic bacteria; (3) Testing and verification The obtained thermophilic bacteria were tested for growth under different temperature conditions. 2. The method for screening and breeding high-temperature composting bacteria according to claim 1 is characterized in that: during the culture process of the strain sample in step ③ on a shaker/water bath/oven at 80°C, sterile water needs to be supplemented to the starting scale line every 24 hours. 3. The method for screening and breeding thermophilic composting bacteria according to claim 1, characterized in that the growth performance of the thermophilic bacteria obtained is characterized, comprising the following steps: (1) Plate activation: The obtained thermophilic bacteria were activated separately in a clean bench and cultured on a plate at 50°C for 2 days; (2) Preparation of seed solution: LB medium was placed in a conical flask, and each strain was aseptically inoculated on a clean bench. The culture was shaken at 50°C for 2 days to obtain the prepared seed solution. (3) Liquid temperature test: LB medium was placed in a conical flask, and the seed liquid was inoculated into fresh LB medium at a volume ratio of 10%, with each group in parallel; the flasks were placed in ovens at 60°C, 70°C, and 80°C, respectively, and cultured and observed continuously. The OD600 was measured every 24 hours for 7 consecutive days, and the culture was observed and recorded continuously; (4) Plate temperature test: For thermophilic bacteria, the growth of the plates was evaluated at 60°C, 70°C, and 80°C. 4. The method for screening and breeding thermophilic composting bacteria according to claim 1, characterized in that the antagonistic characteristics of the thermophilic bacteria obtained are characterized, comprising the following steps: (1) Plate activation and seed solution preparation; (2) Antagonism test: The thermophilic bacteria were subjected to antagonism test, including single strain, combination of two strains, combination of three strains, and combination of four strains were inoculated into LB culture medium, and the two-by-two cross method was used, and the streaking culture was observed on a 50°C plate; Among them, the starting OD600 of the thermophilic strain was adjusted to 1 to ensure the consistency of the initial strain inoculation concentration. 5. The method for screening and breeding thermophilic composting bacteria according to claim 1, characterized in that the thermophilic bacteria obtained are characterized for their resistance to foreign bacteria, comprising the following steps: (1) Plate activation and seed solution preparation; (2) Antibacterial test: 1) Activate the glycerol tube strains and culture them on plates at 50°C; 2) Inoculate the strains with primary seed solution for 24 hours at 50°C and LB, and measure their starting OD600; 3) Inoculate the sterilized group and non-sterilized group at 10% of the inoculation amount at 80°C and LB, and measure their OD600 and viable count at 4 and 7 days; 4) Obtain and record experimental data; The sterilization group sterilized the culture medium at 121°C for 30 min; the non-sterile group used natural culture medium; The starting OD600 of the thermophilic strain was adjusted to 1 to ensure a consistent initial strain inoculation concentration. 6. The method for screening and breeding thermophilic composting bacteria according to claim 1, characterized in that the enzyme activity of the thermophilic bacteria obtained is characterized, comprising the following steps: (1) Plate activation: The obtained thermophilic strains were activated separately in a clean bench and cultured on a plate at 50°C for 2 days; (2) Preparation of seed solution: LB medium was placed in a conical flask, and each strain was aseptically inoculated on a clean bench. The culture was shaken at 50°C for 2 days to obtain the prepared seed solution. (3) Cellulase and ligninase activity plate test: The seed solution obtained in step (2) was diluted to OD600 = 1, and wells were punched with an Oxford cup. 200ul/well was taken, respectively, and the wells were placed in culture medium B2 and M5 at 50°C for static observation and continuous culture observation and recording; (4) The three ligninase activities of thermophilic bacteria, Lac, MnP, and Lip, were detected in a liquid culture medium with alkaline lignin as the substrate. Samples were taken on the second day and a series of enzyme activity tests were performed and recorded. 7. The method for screening and breeding thermophilic composting bacteria according to claim 1, characterized in that: the obtained thermophilic bacteria are subjected to ARTP breeding and high-throughput screening, comprising the following steps: (1) Preparation of seed solution: plate activation and seed solution preparation; (2) Centrifugal washing: The seed solution in step (1) is washed 1-2 times with 0.85% saline. The number of ARTP-induced colonies is calculated based on the observation on the hemocytometer and controlled at 106-108 CFU/mL. The centrifugal conditions are: 2000 rpm, 2 min/time; (3) ARTP mutagenesis: Dilute the washed spore suspension 10 times with 10% glycerol and resuspend it. Apply it on the iron sheet until it is completely covered. At the same time, pipette 0.85% saline into a centrifuge tube for subsequent shaking and elution. (4) Mutagenesis conditions: The time was set to 0s, 30s, 60s, 90s, 120s, and 180s, respectively; 2 parallels per group, power 100w, gas volume 10SLM; after the mutagenesis, the elution was shaken for ≥1min, all the liquid was washed out, and the plates were coated with LB (0s), B2, and M5, respectively, and placed in a 37°C incubator for culture, and the induced lethality was observed and calculated; (5) High-throughput screening: Screening of high-yield mutant strains was carried out on the screening plate M5, and the mutant strains were screened by high-throughput screening using a high-throughput screening instrument. The growth rate was measured by an ELISA instrument, and the enzyme activity was measured and characterized. 8. The method for screening and breeding high-temperature composting bacteria according to claim 6 or 7, characterized in that: the B2 formula is: sodium carboxymethyl cellulose 10g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ ·7H ₂ O 0.5g/L, MnSO ₄ 0.5g/L, Congo red 0.4g, add distilled water to 1000mL, and adjust the pH value to 6.0 with acetic acid; The M5 formula is: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, aniline blue 0.1g/L, distilled water is added to 1000mL, and the pH is natural. 9. The method for screening and breeding thermophilic composting bacteria according to any one of claims 1 to 7, characterized in that the formula of the LB culture medium is: peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, and the pH of the LB culture medium is controlled to 7.0.