CN118530916A - Method for screening and breeding high-temperature fungus compost fungus - Google Patents
Method for screening and breeding high-temperature fungus compost fungus Download PDFInfo
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Abstract
The invention discloses a screening and breeding method of high temperature fungus compost fungus, which belongs to the technical field of microorganisms and specifically comprises the following steps: (1) Gradient enrichment and domestication, namely selecting a strain sample, inoculating the strain sample into an LB culture medium according to 10% (v/v) of inoculum size, and sequentially carrying out enrichment and domestication at three temperature levels; (2) Coating, namely respectively dissolving three temperature enriched and domesticated strain samples in normal saline, fully and uniformly mixing, coating on an LB culture medium, culturing at 50 ℃, and separating and purifying strains to obtain high-temperature bacteria; (3) Testing and verifying, namely testing the growth condition of the obtained high-temperature bacteria under different temperature conditions; the high Wen Junnai receptor screened by the method reaches 80 ℃ and keeps high functional activity.
Description
Technical Field
The invention relates to a screening and breeding method of strains, in particular to a screening and breeding method of high-temperature fungus compost bacteria, and belongs to the technical field of microorganisms.
Background
The thermophilic bacteria and the facultative thermophilic bacteria can grow even when the temperature is lower than 30 ℃, and the highest growth temperature is 45-55 ℃ and 50-65 ℃ respectively; the minimum growth temperature of the obligate thermophilic bacteria and the extreme thermophilic bacteria is 40 ℃, and the optimal growth temperature is higher than 65 ℃; the maximum growth temperature of the extreme thermophiles is higher than 70 ℃. Meanwhile, the high-temperature bacteria have the characteristics of fast metabolism, high activity, short generation time, high enzyme thermal stability, capability of killing pathogenic bacteria under high-temperature conditions, and the like, and have immeasurable effects on degradation of organic matters and killing pathogenic microorganisms in a high-temperature period of the compost. Research shows that the inoculation of high-temperature degrading bacteria in the compost can effectively shorten the composting period and improve the composting quality, creates favorable conditions for better realization of reduction, innocuity and stabilization of organic waste composting and industrial production of resource utilization, and has very high application prospect. Based on the defects of lower fermentation temperature, long fermentation period, incomplete harmless treatment, serious odor pollution and the like of the traditional composting technology, the technology has become the bottleneck for limiting the comprehensive popularization and application of the technology. Therefore, the screening of the thermophilic bacteria and the research on the enzymatic properties thereof have high research value and great significance.
The application methods of the high-temperature fungus compost bacteria reported at present are numerous, wherein patent (CN 106905006A) reports a method for carrying out sludge aerobic composting by utilizing the combined action of high-temperature fungus (more than 55 ℃) and white rot fungus (Phanerochaete chrysosporium); 3 extreme thermophilic bacteria reported in the patent (CN 107937303A) are used for producing the composite ultra-high temperature composting microbial inoculum through synergistic fermentation, and the temperature can reach 70-80 ℃; the patent (CN 108935945A) reports a method for carrying out high-temperature solid state fermentation on soybean meal by using high-temperature resistant bacteria (bacillus stearothermophilus), and the optimal growth temperature of the strain is 55-60 ℃; the technical method reported in the patent (CN 109517754A) adopts a water bath with the constant temperature of 70 ℃ to screen target strains; the patent (CN 110093296A) reports a mud high-temperature composting microbial inoculum LC and application thereof, wherein the mud high-temperature composting microbial inoculum comprises bacillus licheniformis (Bacillus licheniformis) GBIF-3, and the lignocellulose activity (the sum of the filter paper enzyme activity, the cellulose enzyme activity and the xylanase activity) in crude enzyme liquid is measured in the fermentation and decomposition process, and the highest value is: 50U; an extreme thermophilic bacterium reported in the patent (CN 110819554A) and the application thereof in high-temperature composting fermentation are mutants obtained through mutagenesis, domestication, gene recombination or natural mutation, and in the high-temperature composting fermentation process, esterase, cellulase, alkaline amylase and alkaline protease are added to promote composting to enter a high-temperature stage rapidly, and the adopted breeding technology method is a traditional low-efficiency mutant obtaining method; a preparation method of high Wen Junji for kitchen waste efficient composting reported in patent (CN 112322498A) relates to screening of a specific efficient culture medium, and the screened strain can degrade cellulose, lignin and protein efficient functional strains, and is thermophilic thermus UTM802 with temperature tolerance of 65-70 ℃.
Based on the defects and shortcomings of the existing microbial agents, the existing strains are low in temperature, narrow in tolerance range, low in enzyme activity and low in efficiency in the traditional breeding technical method, so that the research and development of a method for screening and breeding high-temperature bacterial compost capable of overcoming the defects is a technical problem to be solved urgently by the technicians in the field.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects of the prior art and providing a screening and breeding method of high-temperature fungus compost fungus, which is simple and easy to implement, and the high Wen Junnai receptor degree screened and bred by the method is 50-80 ℃ and reaches 80 ℃ at most, so that the high functional activity is maintained.
In order to solve the technical problems, the invention provides a method for screening and breeding high-temperature fungus compost, which specifically comprises the following steps:
(1) Gradient enrichment domestication
Selecting a strain sample, inoculating the strain sample into an LB culture medium according to 10% (v/v) of inoculum size, and sequentially carrying out enrichment domestication at three temperature levels;
① Placing on a shaker/water bath/oven at 60deg.C, continuously culturing and observing, and measuring OD600 every 24h for 3-5 times;
② Transferring the strain sample cultured in the step ① into fresh LB culture medium according to 10% (v/v) of inoculation amount, placing the culture medium on a shaking table/water bath/oven at 70 ℃, continuously culturing and observing, and measuring OD600 every 24 hours for 3-5 times continuously;
③ Transferring the strain sample cultured in the step ② into fresh LB culture medium according to 10% (v/v) of inoculation amount, placing the culture medium on a shaking table/water bath/oven at 80 ℃, continuously culturing and observing, and measuring OD600 every 24 hours for 3-5 times continuously;
(2) Coating
Respectively dissolving three temperature enriched and domesticated strain samples in normal saline, fully and uniformly mixing, coating on an LB culture medium, culturing at 50 ℃, and separating and purifying strains to obtain high-temperature bacteria;
(3) Testing and verification
And testing the growth condition of the obtained high-temperature bacteria under different temperature conditions.
The technical scheme of the invention is as follows:
Further, in the above-mentioned method for screening and breeding the high temperature fungus compost fungus, in the culturing process of the strain sample in step ③ on the shaker/water bath/oven at 80 ℃, sterile water needs to be supplemented to the starting scale line every 24 hours.
The technical effect is that the water is volatile at 80 ℃, and the sterile water is supplemented to well keep the components of the culture medium stable, so that the bacteria turbidity data is reliable and stable.
In the above-mentioned method for screening and breeding high temperature fungus compost fungus, the method for characterizing growth performance of the obtained high temperature fungus includes the following steps:
(1) Plate activation: respectively completing strain activation of the obtained high-temperature bacteria on an ultra-clean workbench, and culturing for 2d at the temperature of 50 ℃ of a flat plate;
(2) Preparing seed liquid: filling LB culture medium into the triangular flask, completing aseptic inoculation of each strain on an ultra-clean workbench, and carrying out shake culture at 50 ℃ for 2d to obtain prepared seed liquid;
(3) Liquid temperature test: filling LB culture medium into the triangular flask, inoculating the seed liquid into fresh LB culture medium according to 10% (v/v) of inoculation amount, wherein each group is parallel; placing in oven at 60deg.C, 70deg.C and 80deg.C respectively, continuously culturing and observing, measuring OD600 every 24h, continuously culturing and observing for 7 days, and recording;
(4) Plate temperature test: for high temperature strains, the plate growth condition of the strain is characterized and evaluated at 60 ℃, 70 ℃ and 80 ℃.
In the above-mentioned method for screening and breeding high temperature fungus compost fungus, the antagonism condition characterization is carried out on the obtained high temperature fungus, including the following steps:
(1) Plate activation and seed liquid preparation;
(2) Antagonism test: the method comprises the steps of carrying out antagonism experiments on a high-temperature fungus combination, including a single plant combination, a two-plant combination, a three-plant combination and a four-plant combination, respectively inoculating the combination into an LB culture medium, carrying out a two-by-two crisscross method, and carrying out streak culture observation on a 50 ℃ plate;
wherein, the starting OD600 of the high-temperature strain is adjusted to be 1, so as to ensure the consistency of the inoculation concentration of the initial strain.
In the above-mentioned method for screening and breeding high temperature fungus compost fungus, the method for characterizing the antibacterial condition of the obtained high temperature fungus includes the following steps:
(1) Plate activation and seed liquid preparation;
(2) Antibacterial test: 1) Respectively activating glycerol tube strains to plates for culturing at 50 ℃; 2) Inoculating the strain with first-stage seed solution, and respectively measuring its original OD600 at 50deg.C and LB; 3) Respectively inoculating a sterilization group and a non-sterilization group according to 10% of inoculation amount, and respectively measuring OD600 and viable count at 80 ℃ and LB and 4d and 7 d; 4) Obtaining experimental data and recording;
the sterilization group is to sterilize the culture medium material at high temperature 121 ℃ for 30min; the non-sterilization group is a natural culture medium material;
The starting OD600 of the high-temperature strain is adjusted to be 1, so that the inoculation concentration of the initial strain is consistent.
In the above-mentioned method for screening and breeding high temperature fungus compost fungus, the method for characterizing the enzyme activity energy of the obtained high temperature fungus comprises the following steps:
(1) Plate activation: respectively completing strain activation of the obtained high-temperature strain on an ultra-clean workbench, and culturing for 2d at the temperature of 50 ℃ of a flat plate;
(2) Preparing seed liquid: filling LB culture medium into the triangular flask, completing aseptic inoculation of each strain on an ultra-clean workbench, and performing shake culture at 50 ℃ for 2d to obtain prepared seed liquid;
(3) Cellulose and lignin enzyme activity plate test conditions: diluting the seed liquid obtained in the step (2) to OD 600=1, punching by using oxford cups, respectively sucking 200 ul/hole, standing and observing at 50 ℃ in culture mediums B2 and M5, continuously culturing and observing and recording;
(4) The enzyme activities of 3 kinds of lignin of the thermophilic bacteria are Lac, mnP, lip, the enzyme activities of the thermophilic bacteria are detected in a liquid culture medium taking alkaline lignin as a substrate, and a series of enzyme activity detection and recording are carried out after sampling in the 2 nd step.
In the above-mentioned method for screening and breeding high temperature fungus compost fungus, ARTP breeding and high throughput screening are carried out on the obtained high temperature fungus, which comprises the following steps:
(1) Preparing seed liquid: plate activation and seed liquid preparation;
(2) And (3) centrifugal washing: washing the seed solution in the step (1) with 0.85% physiological saline for 1-2 times, and carrying out inverse calculation on the number of ARTP mutagenized colonies according to the observation of a blood cell counting plate, wherein the centrifugation conditions are as follows: 2000rpm,2 min/time;
(3) Art mutagenesis: diluting the washed spore suspension by 10 times with 10% glycerol for resuspension, sucking and coating on an iron sheet, and fully coating; and simultaneously sucking 0.85% physiological saline into the centrifuge tube for later shaking and eluting;
(4) Mutagenesis conditions: the time is respectively set to 0s, 30s, 60s, 90s, 120s and 180s; 2 parallel gas flow 10SLM with power of 100 w; after mutagenesis is finished, shake-eluting for more than or equal to 1min, washing out all liquid, coating a flat plate to LB (0 s), B2 and M5 respectively, placing in a 37 ℃ incubator for culture, observing and calculating the mutagenesis mortality;
(5) High throughput screening: screening the high-yield mutant strain on a screening plate M5, carrying out high-throughput screening on the mutant strain by adopting a high-throughput screening instrument, measuring the growth speed of the mutant strain by combining with an enzyme-labeling instrument, and carrying out measurement condition characterization on the enzyme activity of the mutant strain.
In the screening and breeding method of the high temperature fungus compost fungus, the formula B2 comprises the following steps: 10g/L, KH 2PO42g/L、MgSO4·7H2O 0.5g/L、MnSO4 0.5.5 g/L of sodium carboxymethylcellulose, 0.4g of Congo red and 1000mL of distilled water, and adjusting the pH value to 6.0 by acetic acid;
the formula of M5 is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride and 0.1g/L aniline blue, distilled water to 1000mL, and the pH is natural.
In the screening and breeding method of the high temperature fungus compost fungus, the formula of the LB culture medium is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, and pH7.0 of LB medium.
The beneficial effects of the invention are as follows:
The invention combines gradient domestication, liquid bacteria turbidity and plate temperature test, breeds by multiple methods to obtain strain with stable temperature tolerance, wider adaptability and higher application performance, the ARTP and high-throughput breeding technology adopted in the invention greatly improves mutation rate and positive mutant bacteria, and the obtained target strain has excellent performance and high efficiency.
The invention provides a screening and breeding method of high-temperature fungus compost, which comprises the steps of strain screening technical method, developing growth performance, antagonism condition, anti-microbial condition and enzyme activity condition characterization on target strains, and developing ARTP breeding and high-throughput screening work on the bred high-activity strains, wherein the screened strains have tolerance of 50-80 ℃ and heat resistance and thermophilic temperature of up to 80 ℃ and also have high-efficiency and abundant lignin series enzyme activities including cellulase activity, laccase (lac) and lignin peroxidase (LiP).
The ARTP breeding and high-throughput screening adopted by the invention obtain mutant strains with excellent performance, and the lac enzyme activities of the mutant strains are respectively improved by 116.88%, 176.41%, 114.98%, 97.58%, 125.49%, 128.93% and 126.02% relative to that of the original strain.
The ARTP breeding developed by the invention adopts a normal pressure room temperature plasma source which adopts helium as working gas, is rich in active energy particles, damages genetic materials of strains, induces biological cells to start an SOS repair mechanism, repairs SOS repair at a high fault tolerance rate, generates mismatch sites with rich types, and finally inherits stably to form mutant strains.
The high-throughput screening instrument is used for high-throughput screening of mutant strains, and the mutant strains are combined with the enzyme-labeled instrument to perform shape characterization on growth conditions, so that the method has the advantages of high speed, high efficiency, high sensitivity, high specificity and high signal to noise ratio.
The high-efficiency strain obtained by the invention is suitable for the high-temperature composting process of high-temperature lacquer livestock manure, straw compost, garden garbage or greening garbage, and the like, can be prepared into a solid microorganism high-temperature composting inoculant, has low cost, can avoid the inactivation of active microorganisms in the inoculant in a high-temperature fermentation period, and has wide temperature application range when being applied to composting.
The high-efficiency strain obtained by the invention has the characteristics of quick metabolism, high activity, short generation time, high enzyme thermal stability, capability of killing pathogenic bacteria under high-temperature conditions, and the like, and has immeasurable effects on degradation of organic matters and killing pathogenic microorganisms in a high-temperature period of the compost.
Drawings
FIG. 1 is a graph showing growth of a plate at 70℃for a thermophilic bacterium according to an embodiment of the present invention;
FIG. 2 is a graph showing growth conditions of 4 high temperature bacteria at 60℃in the example of the present invention;
FIG. 3 is a graph showing the growth of 4 strains at 70℃in the example of the present invention;
FIG. 4 is a graph showing the growth of 4 strains at 80℃in the example of the present invention;
FIG. 5 is a graph showing the growth of liquid antagonism between strains according to an embodiment of the present invention;
FIG. 6 is a graph showing the growth of plate antagonism between strains according to an embodiment of the present invention;
FIG. 7 is a diagram showing the count of live bacteria in an antibacterial experiment according to an embodiment of the present invention;
FIG. 8 is a diagram showing the characterization of the activity of a strain plate in an embodiment of the invention;
FIG. 9 is a graph showing mortality at various mutagenesis times in the examples of the present invention;
FIG. 10 is a graph showing the growth of the dominant strain and the measurement of the enzyme activity thereof in the example of the present invention.
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
Example 1:
The screening and breeding method of the high-temperature fungus compost fungus provided by the embodiment specifically comprises the following steps:
(1) Gradient enrichment domestication
Selecting a strain sample G1, wherein the source of the strain sample G1 is a compost sample of Jiangsu cycloworker Taihu lake ecological technology limited company, and the strain sample is obtained by taking crop straws, garden greening garbage and blue algae mud as raw materials to cooperatively perform aerobic fermentation;
Inoculating the strain sample G1 into LB culture medium (100 mL/250mL of liquid loading amount) according to 10% (v/v) inoculum size, and sequentially carrying out enrichment domestication at three temperature levels;
① Placing on a shaker/water bath/oven at 60deg.C, continuously culturing and observing, and measuring OD600 every 24h, and continuously 3-5 times (depending on growth condition);
② Transferring the strain sample cultured in the step ① into fresh LB culture medium according to 10% (v/v) inoculum size, placing the culture medium on a shaking table/water bath/oven at 70 ℃, continuously culturing and observing, and measuring OD600 at intervals of 24 hours for 3-5 times (depending on growth conditions);
③ Transferring the strain sample cultured in the step ② into fresh LB culture medium according to 10% (v/v) inoculum size, placing the culture medium on a shaking table/water bath/oven at 80 ℃, continuously culturing and observing, and measuring OD600 at intervals of 24 hours for 3-5 times (depending on growth conditions);
OD600 values of the domesticated results of the G1 temperatures after gradient enrichment and domestication are shown in table 1;
(2) Coating
Respectively dissolving three temperature enriched and domesticated strain samples in normal saline, fully and uniformly mixing, coating on an LB culture medium, culturing at 50 ℃, and selecting strains with different colony morphological characteristics for separation and purification to obtain 8 high-temperature strains to be verified;
(3) Testing and verification
The plate verification of the obtained thermophilic bacteria at 70 ℃ is shown in figure 1;
the obtained high temperature strains were further inoculated into LB liquid medium (liquid loading amount 100mL/250 mL), cultured continuously on shaker/water bath/oven at 80℃for 2d, OD600 was measured and observed and experimental results were recorded as shown in Table 2.
In this example, the culture medium formulation for LB was: peptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, pH7.0.
In the process of culturing the strain on a shaking table/water bath/oven at 80 ℃, sterile water needs to be supplemented every 24 hours until the position of the starting liquid level is the starting scale mark.
TABLE 1 OD600 value case of sample G1 temperature domestication results
TABLE 2 verification of high temperature Strain fluid at 80℃
Analysis of results:
1) As can be seen from Table 1, the strain in the sample can be seen to have a high temperature resistant strain by enriching and acclimating at different temperatures, such as 60 ℃, 70 ℃ and 80 ℃; and (3) eliminating and sorting the mixed bacteria by increasing the temperature step by step, and sorting the enriched domesticated bacteria at 80 ℃.
2) 8 Strains capable of growing at the high temperature of 50 ℃ are preliminarily obtained through gradient dilution, coating and screening; and the obtained 8 high-temperature bacteria are further subjected to temperature test under the flat plate condition of 70 ℃ for re-screening, as shown in figure 1, GW-80-6 is found to grow at 50 ℃, growth can hardly occur at 70 ℃, and preliminary elimination is carried out; other strains can grow at 50-80 ℃.
3) The temperature test conditions of the re-screened strains GW-80-1, GW-80-2, GW-80-3, GW-80-4, GW-80-5, GW-80-7 and GW-80-8 at high temperature and 80 ℃ are shown in Table 2, and the OD600 of the strains GW-80-3, GW-80-5 and GW-80-8 is higher and reaches between 0.3 and 0.5, which indicates that the 3 strains grow well at the high temperature and 80 ℃ and are used as standby strains for further experiments.
Example 2:
(1) Based on the existing 21 strains, the strain can be obtained from the collection center of China general microbiological culture collection center (CHINA GENERAL Microbiological Culture Collection Center, CGMCC), china industry microbiological culture collection center (CHINA CENTER of Industrial Culture Collection, CICC), china center for type culture collection (CHINA CENTER for Type Culture Collection, CCTCC), american center for type culture collection (AMERICAN TYPE Culture Collection, ATCC) and China center for agricultural microbiological culture collection (Agricultural Culture Collection of China, ACCC), and the specific details are shown in table 3;
Performing a plate and liquid temperature test on the purchased strain; the detection index is plate observation and OD600 measurement.
TABLE 3 Thermomyces sample Source
The screening and breeding method of the high-temperature fungus compost fungus provided by the embodiment specifically comprises the following steps:
(1) And (3) strain reviving and primary screening: naturally thawing the strain glycerol tube, sucking 100ul of the strain glycerol tube, coating on an LB plate, culturing at 50 ℃ for 2-3d, observing the growth condition, and performing primary screening as shown in table 4;
(2) Temperature test and re-screening: the strain obtained by the primary screening was transferred to a liquid LB medium, put into a water bath at 80 ℃ for cultivation, and the growth condition was observed, detected and recorded as shown in Table 5.
TABLE 4 growth of high temperature strains on plates at 50 ℃
TABLE 5 growth of high temperature strains in 80℃liquid
Analysis of results:
From Table 4, the growth of the primary screen shows that the strains with good growth are GXGD-YZ-438 and GXGD-YZ-900; according to Table 5, the strain which grows well at a high temperature of 80℃was rescreened with the order of high temperature effect of 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 selected as well, and the 3 strains were used as backup strains for further experiments.
Example 3:
this example provides a characterization of the growth performance of the high temperature strains obtained in examples 1 and 2, specifically:
(1) Plate activation: the above 4 strains of bacteria GXGD-YZ-900, GXGD-YZ-822, GXGD-YZ-438 and GW-80-5 were subjected to strain activation on an ultra-clean bench, and cultured at 50℃for 2 days (depending on the growth conditions).
(2) Preparing seed liquid: filling 40ml LB culture medium in 100ml triangular flask, completing aseptic inoculation of each strain on an ultra-clean workbench, and performing shake culture at 50 ℃ for 2d to obtain prepared seed liquid;
(3) Liquid temperature test: 500ml of the flask was filled with 200ml of LB medium, and the seed solution was inoculated into a new LB medium in an inoculum size of 10% (v/v), each set being parallel. Placing in oven at 60deg.C, 70deg.C and 80deg.C (periodically checking temperature or supplementing water), continuously culturing and observing, measuring OD600 every 24h, continuously culturing and observing for 7 days (according to growth condition), and recording as shown in figure 2, figure 3 and figure 4;
(4) Plate temperature test: the plate growth of the 4 strains was evaluated at 60℃and 70℃and 80℃as shown in Table 6.
(1) Analysis of liquid growth condition results is shown in fig. 2,3 and 4;
1) According to FIG. 2, it can be seen that the 4 strains can grow well at 60 ℃. Wherein GXGD-YZ-438 reaches a peak value of 2.5 on the 3 rd day, and the late-stage fed-batch nutrient solution has fluctuation, and then the rapid growth process can be continued; wherein GXGD-YZ-900 reaches a peak value of 0.55 on the 4 th day, the fed-batch nutrient solution in the later period has fluctuation, the later period has small increase, but the increase is stable; wherein GXGD-YZ-822 reaches about 1.0 peak value on day 2, the late fed-batch supplementing liquid has fluctuation, then reaches about 0.9 peak value again on day 4, and can continue to grow; wherein WG-80-5 slowly increases in the first 5 days, peaks at 2.3 on day 6, and late feed with make-up has fluctuations, followed by the potential to continue to increase. The growing condition of the comprehensive 4 strains is GXGD-YZ-438> WG-80-5> GXGD-YZ-822> GXGD-YZ-900 at 60 ℃.
2) According to FIG. 3, it can be seen that the growth conditions of the 4 strains at 70 ℃ are significantly improved relative to those of the strains WG-80-5 and GXGD-YZ-900 at 60 ℃, the growth activity capacities of the strains GXGD-YZ-822 and GXGD-YZ-438 are not fully activated, and the temperature start process of the strains is preferably improved step by step from 50 ℃,60 ℃, 70 ℃, 80 ℃ in consideration of the temperature start process of the strains. Wherein GXGD-YZ-438 reaches a peak value of 0.4 on the 2 nd day, the late-stage fed-batch supplementing liquid has fluctuation, but the bacterial concentration OD600 on the 1 st to 7 th days fluctuates by 0.25 to 0.4; wherein GXGD-YZ-900 reaches a peak value of about 2.75 on the 4 th day, the fed-batch supplementary liquid in the later period has fluctuation, the later period has small increase, but the increase is stable, and the growth condition of the strain at 70 ℃ is obviously better than that of the strain at 60 ℃; wherein, the OD600 fluctuation of the bacterial concentration of GXGD-YZ-822 in days 1-7 is 0.25-0.42, and the late-stage fed-batch supplementary solution has fluctuation and has small increasing trend; wherein WG-80-5 is in a rapid growth trend all the time on days 1-7, and reaches peak value 4.5 at the highest, and the late fed-batch supplementing liquid has fluctuation, and then the potential of continuous growth exists. The growing condition of the comprehensive 4 strains is WG-80-5> GXGD-YZ-900 is more than or equal to GXGD-YZ-822 approximately equal to GXGD-YZ-438 at 70 ℃.
3) According to FIG. 4, it can be seen that the growth condition of the 4 strains at 80 ℃ is obviously inhibited relative to 60 ℃ and 70 ℃, and the temperature starting process of the strains is considered to be preferably improved from 50 ℃, 60 ℃, 70 ℃ and 80 ℃ step by step for domestication, which is beneficial to activating the activity capability of the high-temperature strains. Wherein the bacterial concentration OD600 fluctuation of GXGD-YZ-438 at 1-7 days is 0.18-0.25; wherein the bacterial concentration OD600 fluctuation of GXGD-YZ-900 at 1-7 days is 0.20-0.25; wherein, the OD600 fluctuation of the bacterial concentration of GXGD-YZ-822 in days 1-7 is 0.18-0.25, the late-stage fed-batch supplementary solution has fluctuation, and the late stage can have a certain increase amplitude; wherein, the OD600 fluctuation of the bacterial concentration of GXGD-YZ-822 in days 1-7 is 0.18-0.25, the late-stage fed-batch supplementary solution has fluctuation, and the late stage can have a certain increase amplitude; the peak value reaches 0.4 on the 2 nd day, and the late fed-batch supplementing liquid has fluctuation, but the bacterial concentration OD600 fluctuation is 0.25-0.4 on the 1 st to 7 th days; wherein WG-80-5 reaches about 0.4 peak value on day 5, the late fed-batch supplementary liquid has fluctuation, and the late still has the potential to continue to grow. The growing condition of the comprehensive 4 strains is WG-80-5> GXGD-YZ-900 is more than or equal to GXGD-YZ-822 approximately equal to GXGD-YZ-438 at 80 ℃.
(2) The growth of the 4 strains at 60℃and 70℃and 80℃was summarized in Table 6.
Table 6 4 shows growth conditions of strains under different temperature conditions
The small knot: according to the conditions of the growth characteristics of 4 strains of high temperature bacteria at 60 ℃, 70 ℃ and 80 ℃ in Table 6, the growth conditions of 4 strains of bacteria at 60 ℃ are GXGD-YZ-438 approximately equal to WG-80-5> GXGD-YZ-822> GXGD-YZ-900; at 70 ℃ and 80 ℃, the growth conditions are identical and are WG-80-5> GXGD-YZ-900 is more than or equal to GXGD-YZ-822 apprxeq GXGD-YZ-438.
Example 4:
the antagonism of the high temperature strain obtained in examples 1 and 2 was characterized in this example, specifically:
Plate activation and seed liquid preparation were the same as in example 3;
antagonism test: performing pairwise combination, three-strain combination and four-strain combination antagonism experiments on 4 strains of bacteria WG-80-5, GXGD-YZ-900, GXGD-YZ-822 and GXGD-YZ-438;
Specifically, group 1: the single strain is respectively inoculated into LB culture medium;
group 2:4 strains of bacteria are mixed and then inoculated into an LB culture medium;
group 3: every two different strains are inoculated into LB culture medium;
group 4: every three different strains are inoculated into LB culture medium;
the experimental results are shown in FIG. 5 (the abbreviated strain numbers in FIG. 5 correspond to 4 strains according to the number), and the 4 strains were subjected to a two-by-two crisscross method and observed on a 50℃plate by streak culture, as shown in FIG. 6.
Wherein, the starting OD600 of the 4 strains is adjusted to be 1, so as to ensure that the inoculation concentration of the initial strains is consistent.
Analysis of results:
1) As can be seen from the liquid growth concentrations in FIG. 5, the combination of the two strains 438+80-5 and the combination of the 3 strains 438+822+80-5, 822+900+80-5 and 900+80-5+438 are significantly higher than that of the single strain, and the 4 strains have poor compounding effect. Wherein the strain YZ-438 and GW-80-5 have better high-temperature synergistic effect.
2) According to FIG. 6, the 4 strains of high temperature bacteria are streaked by a two-to-two crisscross method, and antagonism experiments are carried out at 50 ℃, so that the results show that the strain combination strains YZ-822 and YZ-80-5, YZ-438 and YZ-900 have weak antagonism, the antagonism phenomenon is not found among other strains, and the partial result phenomenon of FIG. 5 is further demonstrated.
Example 5:
the present example characterizes the antibacterial conditions of the high temperature strains obtained in examples 1 and 2, in particular:
(1) Plate activation and seed liquid preparation were the same as in example 3;
(2) Antibacterial test: performing an antibacterial experiment on 4 strains of high temperature bacteria (GXGD-YZ-900, GXGD-YZ-822, GXGD-YZ-438 and GW-80-5);
1) The obtained 4 high-temperature bacteria are frozen in glycerol tubes, and the glycerol tube strains are respectively activated on a flat plate (50 ℃) when an anti-microbial experiment is carried out; 2) Inoculating the strain with first-stage seed solution, and measuring its original OD600 at 50deg.C and LB for 24 hr (1 d); 3) Respectively inoculating a sterilization group and a non-sterilization group according to 10% of inoculation amount, and respectively measuring OD600 and viable count at 80 ℃ and LB and 4d and 7 d; 4) The experimental data were obtained as shown in table 7, fig. 7;
Wherein the sterilization group is to sterilize the culture medium material at high temperature 121 ℃ for 30 min; the non-sterilization group is a natural culture medium material;
Wherein, the starting OD600 of the 4 strains is adjusted to be 1, so as to ensure the consistency of the inoculation concentration of the initial strains;
the number of living bacteria can be marked by reference lines, and then the bacteria are diluted in a gradient way, so that the main purpose is to observe the ratio situation of target bacteria to non-target bacteria so as to evaluate the antibacterial performance.
TABLE 7 bacterial concentration and viable count for antibacterial experiments on 4 strains of hyperthermophiles
Analysis of results:
Performing an antibacterial test on 4 groups of bacteria according to table 7, performing a sterilizing group and a non-sterilizing group test on the base material, finding that the OD600 of the 2 groups of tests is not different, counting viable bacteria, and finding that the viable bacteria numbers of the sterilizing group and the non-sterilizing group are basically similar, wherein a small amount of mixed bacteria exists in the non-sterilizing group; wherein the strain GXGD-YZ-438 has stronger antibacterial ability and GXGD-YZ-900 has weaker antibacterial ability.
Example 6:
The enzyme activity of the high temperature strain obtained in examples 1 and 2 can be screened and characterized in this example, specifically:
(1) Plate activation: respectively activating the 4 strains of bacteria GW-80-5, GXGD-YZ-438, GXGD-YZ-822 and GXGD-YZ-900 on an ultra-clean workbench, and culturing for 2d at the temperature of a flat plate 50 ℃ according to the growth condition;
(2) Preparing seed liquid: filling 40ml LB culture medium in 100ml triangular flask, completing aseptic inoculation of each strain on an ultra-clean workbench, and performing shake culture at 50 ℃ for 2d to obtain prepared seed liquid;
(3) Cellulose and lignin enzyme activity plate test conditions: diluting the seed solution obtained in the step (2) to OD 600=1 with sterile water, punching with oxford cup, respectively sucking 200 ul/hole, standing and observing in culture mediums B2 and M5 at 50 ℃, continuously culturing and observing and recording, as shown in table 8 and figure 8;
(4) The enzyme activities of 3 types of lignin enzyme activities (Lac, mnP, lip) in 4 strains of hyperthermophiles GXGD-YZ-438, GXGD-YZ-900, GXGD-YZ-822 and GW-80-5 were measured in a liquid medium using alkaline lignin as a substrate as follows, and samples were taken at 2d and serial enzyme activity measurements were performed as shown in Table 9.
Table 8 characterization of the enzyme Activity System for 4 thermophilic bacteria
Table 9 results of measurement of 3 Lignin enzyme Activity liquids of 4 strains of bacteria
Analysis of results:
1) Through the table 8 and the figure 8, the observed results of continuous 2-3 days at 50 ℃ show that the lignin related main enzyme system is systematically characterized, and the qualitative characterization results through plate color development are shown in the table 8, wherein the enzyme system is richer strain GXGD-YZ-822, including cellulase activity, laccase (lac) and lignin peroxidase (LiP); wherein the bacterial strain GXGD-YZ-438 has higher cellulase activity and lignin enzyme activity; wherein the strain GXGD-YZ-900 mainly comprises laccase (lac) and lignin peroxidase (LiP), and the strain GXGD-YZ-438 with relatively high comprehensive enzyme activity is obtained in the first step.
2) The liquid system characterization is carried out on the lignin related main enzyme system of 4 strains through the table 9, the liquid system characterization is basically consistent with the qualitative characterization result of the plate color development, the enzyme activities of laccase (lac) and lignin peroxidase (LiP) are mainly adopted, the analysis of the plate and liquid experimental results is synthesized, and the strain GXGD-YZ-438 is preferably selected as a starting strain, and the ARTP breeding and high-throughput screening work is carried out.
Example 7:
The present example provides art breeding and high throughput screening of high efficiency active functional strains, and for the highest enzyme activity high temperature strain GXGD-YZ-438 obtained in example 6, art and high throughput screening work was performed to obtain 1-2 strains with excellent performance, specifically:
(1) Preparing seed liquid: plate activation and seed liquid preparation were the same as in example 3;
(2) And (3) centrifugal washing: washing the seed solution in the step (1) with 0.85% physiological saline for 1-2 times, and performing inverse calculation to obtain ARTP mutagenesis colony number according to the observation of a blood cell counting plate, wherein the colony number is controlled at 10 6-108 CFU/mL, and the centrifugation condition is preferable: 2000rpm,2 min/time;
(3) Art mutagenesis: re-suspending the washed spore suspension by 10 times diluted with 10% glycerol, and washing 15ul of the spore suspension to be coated on an iron sheet, wherein the spore suspension is fully coated (200 CFU/mL/iron sheet can be reversely calculated); and simultaneously sucking 200ul of 0.85% physiological saline into a 2ml centrifuge tube for later shake elution;
(4) Mutagenesis conditions: the time is respectively set to 0s, 30s, 60s, 90s, 120s and 180s; 2 parallel gas flow 10SLM with power of 100 w; after mutagenesis is finished, shake-eluting for more than or equal to 1min, washing out all the liquid, coating the liquid on a flat plate to LB (0 s), B2 and M5 respectively, placing the flat plate in a 37 ℃ incubator for culture, observing at proper time, and calculating the mutagenesis lethality as shown in figure 9;
(5) High throughput screening: the high-yield mutant strain is screened on a screening plate M5 by selecting 90s of mutagenesis time, the mutant strain obtained on the screening plate M5 is screened in high throughput by adopting a high throughput screening instrument, the growth rate is measured by combining an enzyme-labeling instrument, and the growth rate and the enzyme activity measurement condition are represented in figure 10.
TABLE 10 growth of high-yielding mutant strains under high-throughput screening conditions
Analysis of results:
1) According to the experiment of the optimal mutagenesis time of the strain GXGD-YZ-438 in FIG. 9, the optimal mutagenesis time is 90s, the lethality is 87.95%, the number of starting bacterial colonies is 100-200, the number of mutant strains is 10-20, and the basic number of the experiment requirement is basically met.
Under the conditions of optimal mutagenesis condition power of 100w and 90s, carrying out M5 high-enzyme activity mutant strain experiments to obtain 300-500 mutant strains, carrying out high-flux screening on the formed bacterial colony sizes and enzyme activity transparent rings, and carrying out high-flux screening on high-yield mutant strains on a 96-well plate, wherein the data are shown in Table 10, and the growth conditions of the strain OD 600.
2) The 90 mutants selected (wherein A1-A3 are blank control and B1-B3 are starting strain) were screened for 32 strains having OD600 greater than the average starting strain (1.0935) and greater than 1.15, and lac enzyme activity was measured, and specific data are shown in FIG. 10. According to the strain growth condition and the enzyme activity condition characterization in FIG. 10, 7 strains with high growth speed and high enzyme activity are selected as B5, C4, D5, D6, D9, G8 and H12, and the enzyme activities are （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、（71.08±10.89）U/mL; U/mL relative to the enzyme activities (31.45 +/-5.34) of the original strain (B1-B3), and are respectively improved by 116.88%, 176.41%, 114.98%, 97.58%, 125.49%, 128.93% and 126.02%.
The LB medium formulations in examples 1-7 were: peptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, pH7.0;
The formula of the B2 culture medium is as follows: 10g/L, KH 2PO42g/L、MgSO4·7H2O 0.5g/L、MnSO4 0.5.5 g/L sodium carboxymethylcellulose, 0.4g Congo red, adding distilled water to 1000mL, adjusting pH to 6.0 with acetic acid, and sub-packaging to 200mL/500mL;
The formula of the M5 culture medium is as follows: LB,0.1g/L aniline blue, distilled water to 1000mL, and natural pH;
Wherein B2 is a cellulase activity identification medium; m5 is lignin enzyme activity identification medium.
The invention provides a screening and breeding method of high-temperature fungus compost fungus, and the method and the way for realizing the technical scheme are more specific, the above is only the preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.
Claims (9)
1. The method for screening and breeding the high-temperature fungus compost fungus is characterized by comprising the following steps:
(1) Gradient enrichment domestication
Selecting a strain sample, inoculating the strain sample into an LB (LB) culture medium according to an inoculum size of 10% by volume, and sequentially carrying out enrichment domestication at three temperature levels;
① Placing on a shaker/water bath/oven at 60deg.C, continuously culturing and observing, and measuring OD600 every 24h for 3-5 times;
② Transferring the strain sample cultured in the step ① into fresh LB culture medium according to the inoculum size of 10% by volume, placing the culture medium on a shaking table/water bath/oven at 70 ℃, continuously culturing and observing, and measuring OD600 every 24 hours for 3-5 times continuously;
③ Transferring the strain sample cultured in the step ② into fresh LB culture medium according to the inoculum size of 10% by volume, placing the culture medium on a shaking table/water bath/oven at 80 ℃, continuously culturing and observing, and measuring OD600 every 24 hours for 3-5 times continuously;
(2) Coating
Respectively dissolving three temperature enriched and domesticated strain samples in normal saline, fully and uniformly mixing, coating on an LB culture medium, culturing at 50 ℃, and separating and purifying strains to obtain high-temperature bacteria;
(3) Testing and verification
And testing the growth condition of the obtained high-temperature bacteria under different temperature conditions.
2. The method for screening and breeding the high-temperature fungus compost fungus according to claim 1, which is characterized in that: in the culture process of the strain sample in the step ③ on a shaking table/water bath/oven at 80 ℃, sterile water needs to be supplemented to the starting scale mark every 24 hours.
3. The method for screening and breeding the high-temperature fungus compost fungus according to claim 1, which is characterized in that: and carrying out growth performance condition characterization on the obtained high-temperature bacteria, wherein the method comprises the following steps of:
(1) Plate activation: respectively completing strain activation of the obtained high-temperature bacteria on an ultra-clean workbench, and culturing for 2d at the temperature of 50 ℃ of a flat plate;
(2) Preparing seed liquid: filling LB culture medium into the triangular flask, completing aseptic inoculation of each strain on an ultra-clean workbench, and carrying out shake culture at 50 ℃ for 2d to obtain prepared seed liquid;
(3) Liquid temperature test: filling LB culture medium into the triangular flask, inoculating seed liquid into fresh LB culture medium according to the inoculum size of 10% by volume ratio, wherein each group is parallel; placing in oven at 60deg.C, 70deg.C and 80deg.C respectively, continuously culturing and observing, measuring OD600 every 24h, continuously culturing and observing for 7 days, and recording;
(4) Plate temperature test: for high temperature strains, the plate growth condition of the strain is characterized and evaluated at 60 ℃, 70 ℃ and 80 ℃.
4. The method for screening and breeding the high-temperature fungus compost fungus according to claim 1, which is characterized in that: carrying out antagonism condition characterization on the obtained thermophilic bacteria, and comprises the following steps:
(1) Plate activation and seed liquid preparation;
(2) Antagonism test: the method comprises the steps of carrying out antagonism experiments on a high-temperature fungus combination, including a single plant combination, a two-plant combination, a three-plant combination and a four-plant combination, respectively inoculating the combination into an LB culture medium, carrying out a two-by-two crisscross method, and carrying out streak culture observation on a 50 ℃ plate;
wherein, the starting OD600 of the high-temperature strain is adjusted to be 1, so as to ensure the consistency of the inoculation concentration of the initial strain.
5. The method for screening and breeding the high-temperature fungus compost fungus according to claim 1, which is characterized in that: carrying out characterization of antibacterial conditions on the obtained thermophilic bacteria, and comprises the following steps:
(1) Plate activation and seed liquid preparation;
(2) Antibacterial test: 1) Respectively activating glycerol tube strains to plates for culturing at 50 ℃; 2) Inoculating the strain with first-stage seed solution, and respectively measuring its original OD600 at 50deg.C and LB; 3) Respectively inoculating a sterilization group and a non-sterilization group according to 10% of inoculation amount, and respectively measuring OD600 and viable count at 80 ℃ and LB and 4d and 7 d; 4) Obtaining experimental data and recording;
the sterilization group is to sterilize the culture medium material at high temperature 121 ℃ for 30min; the non-sterilization group is a natural culture medium material;
The starting OD600 of the high-temperature strain is adjusted to be 1, so that the inoculation concentration of the initial strain is consistent.
6. The method for screening and breeding the high-temperature fungus compost fungus according to claim 1, which is characterized in that: and carrying out enzyme activity energy condition characterization on the obtained thermophilic bacteria, wherein the method comprises the following steps of:
(1) Plate activation: respectively completing strain activation of the obtained high-temperature strain on an ultra-clean workbench, and culturing for 2d at the temperature of 50 ℃ of a flat plate;
(2) Preparing seed liquid: filling LB culture medium into the triangular flask, completing aseptic inoculation of each strain on an ultra-clean workbench, and performing shake culture at 50 ℃ for 2d to obtain prepared seed liquid;
(3) Cellulose and lignin enzyme activity plate test conditions: diluting the seed liquid obtained in the step (2) to OD 600=1, punching by using oxford cups, respectively sucking 200 ul/hole, standing and observing at 50 ℃ in culture mediums B2 and M5, continuously culturing and observing and recording;
(4) The enzyme activities of 3 kinds of lignin of the thermophilic bacteria are Lac, mnP, lip, the enzyme activities of the thermophilic bacteria are detected in a liquid culture medium taking alkaline lignin as a substrate, and a series of enzyme activity detection and recording are carried out after sampling in the 2 nd step.
7. The method for screening and breeding the high-temperature fungus compost fungus according to claim 1, which is characterized in that: carrying out ARTP breeding and high-throughput screening on the obtained thermophilic bacteria, and comprises the following steps:
(1) Preparing seed liquid: plate activation and seed liquid preparation;
(2) And (3) centrifugal washing: washing the seed solution in the step (1) with 0.85% physiological saline for 1-2 times, and carrying out inverse calculation on the number of ARTP mutagenized colonies according to the observation of a blood cell counting plate, wherein the centrifugation conditions are as follows: 2000rpm,2 min/time;
(3) Art mutagenesis: diluting the washed spore suspension by 10 times with 10% glycerol for resuspension, sucking and coating on an iron sheet, and fully coating; and simultaneously sucking 0.85% physiological saline into the centrifuge tube for later shaking and eluting;
(4) Mutagenesis conditions: the time is respectively set to 0s, 30s, 60s, 90s, 120s and 180s; 2 parallel gas flow 10SLM with power of 100 w; after mutagenesis is finished, shake-eluting for more than or equal to 1min, washing out all liquid, coating a flat plate to LB (0 s), B2 and M5 respectively, placing in a 37 ℃ incubator for culture, observing and calculating the mutagenesis mortality;
(5) High throughput screening: screening the high-yield mutant strain on a screening plate M5, carrying out high-throughput screening on the mutant strain by adopting a high-throughput screening instrument, measuring the growth speed of the mutant strain by combining with an enzyme-labeling instrument, and carrying out measurement condition characterization on the enzyme activity of the mutant strain.
8. The method for screening and breeding the thermophilic bacteria compost bacteria according to claim 6 or 7, which is characterized in that: the formula of the B2 is as follows: 10g/L, KH 2PO4 2g/L、MgSO4·7H2O 0.5g/L、MnSO4 0.5.5 g/L of sodium carboxymethylcellulose, 0.4g of Congo red and 1000mL of distilled water, and adjusting the pH value to 6.0 by acetic acid;
the formula of the M5 is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride and 0.1g/L aniline blue, distilled water to 1000mL, and the pH is natural.
9. The method for screening and breeding the high-temperature fungus compost fungus according to any one of claims 1 to 7, which is characterized in that: the formula of the LB culture medium is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, and pH7.0 of LB medium.
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