Domestication method of low-temperature-resistant straw degrading bacteria, separation and purification method and application thereof
By using exogenous amino acid induction and cyclic acclimatization methods, the problem of low straw degradation efficiency at low temperatures was solved, and highly efficient cold-resistant strains were screened out, achieving efficient straw degradation under low-temperature conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG UNIV OF TECH
- Filing Date
- 2026-05-29
- Publication Date
- 2026-07-24
AI Technical Summary
Existing technologies struggle to efficiently degrade straw under low-temperature conditions. Traditional methods suffer from long screening cycles, limited degradation activity, poor environmental adaptability, and high technical barriers.
By combining exogenous amino acid induction with systematic cyclic acclimatization, the low-temperature adaptability and degradation activity of the microbial community were enhanced by gradually reducing the temperature, decreasing the amino acid concentration, and introducing periodic temperature fluctuations.
The breeding cycle was significantly shortened, and cold-resistant strains with strong genetic stability were screened out. These strains have significant low-temperature degradation capabilities and improve straw degradation efficiency.
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Figure CN122445480A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of agricultural microbial technology and waste resource utilization, and in particular to a method for the domestication, separation and purification of low-temperature resistant straw-degrading bacteria and its application. Background Technology
[0002] In northern my country, the autumn harvest is often followed by a sharp drop in temperature, with minimum temperatures reaching 0–10℃, significantly inhibiting the biodegradation of straw. Under these low-temperature conditions, the enzyme activity of conventional microbial agents decreases drastically, cell membrane fluidity weakens, and microbial metabolism tends to stagnate, resulting in slow straw decomposition during winter and consequently affecting subsequent agricultural arrangements. Currently, the main approaches to obtaining highly efficient low-temperature degrading strains still face significant bottlenecks: First, strains directly isolated from cold environments such as high mountains and polar regions often suffer from long screening cycles, limited degradation activity, and poor environmental adaptability; second, physical or chemical mutagenesis methods are highly random, with low positive mutation rates and a tendency to introduce undesirable traits; third, genetic engineering methods are limited by high technical barriers, relevant regulations, and difficulties in practical application. While traditional microbial domestication methods can improve substrate affinity through continuous passage on straw substrates, their evolutionary rate is slow under low-temperature stress, making it difficult to effectively enrich cold-resistance-related physiological mechanisms.
[0003] Therefore, developing a highly efficient, targeted, and safe breeding method for low-temperature resistant straw-degrading bacteria to obtain microbial agents that can maintain high straw degradation activity at low temperatures has important application value and practical significance. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for the domestication, isolation, and purification of low-temperature resistant straw-degrading bacteria that has a short breeding cycle, strong targeting, and significant effects, as well as its applications. This domestication method actively enhances the low-temperature adaptability and degradation activity of the microbial community by combining exogenous amino acid induction with systematic cyclic domestication.
[0005] This invention discloses a method for domesticating low-temperature resistant straw-degrading bacteria, and the technical solution adopted is as follows: A method for acclimatizing a low-temperature resistant straw-degrading bacterium, used to obtain the aforementioned low-temperature resistant straw-degrading bacterium, includes the following steps: S1, collect samples from a low-temperature environment to obtain bacterial samples; S2, pre-treat the straw to obtain pre-treated straw; S3, organic nutrient components, phosphate buffer components, alkaline earth metal salts, transition metal salts and trace element salts are mixed, dissolved and brought to a fixed volume in deionized water and then sterilized to obtain a basic culture medium with a natural pH value. S4, the pretreated straw is added to the basic culture medium at a set concentration as the sole carbon source, and after stirring and sterilization, the finished culture medium is obtained. The inoculum sample is mixed evenly and added to the finished culture medium in proportion. Low-temperature shaking culture is carried out to obtain the initial microbial community. S5, add amino acids to the finished culture medium, stir and disperse, then adjust the volume, and sterilize to obtain the amino acid induction culture medium, wherein the pH of the amino acid induction culture medium is the natural value; S6. The initial bacterial population was subjected to multiple rounds of low-temperature acclimatization culture. After each round of acclimatization, the degradation efficiency of the bacterial population was detected by the straw weight loss rate as an indicator. During the acclimatization process, a gradient increasing selection pressure was applied by gradually reducing the culture temperature, gradually reducing and eventually eliminating the addition of amino acids, and gradually reducing the concentration of straw carbon sources. Periodic temperature fluctuation stress was introduced during the cooling phase. When the increase in straw weight loss rate was lower than a preset threshold for two consecutive rounds, the acclimatization was terminated, thus obtaining a stable low-temperature resistant straw-degrading bacterium. The low-temperature resistant straw-degrading bacterium was deposited at the China Center for Type Culture Collection (CCTCC) on April 27, 2026, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The accession number is CCTCC NO: M2026814. The taxonomic name of the low-temperature resistant straw-degrading bacterium is *Brucea buddinga* X4-8. (Aureobasidium sp. X4-8) .
[0006] The entire acclimatization process described above is carried out in a low-temperature constant temperature incubator. In this invention, low temperature refers to 0-10℃.
[0007] Preferably, the components of the basal culture medium include: yeast extract, peptone, KH2PO4, K2HPO4, MgSO4·7H2O, CaCl2·2H2O, FeCl3·6H2O, MnSO4·H2O, ZnSO4·7H2O, and CoCl2·6H2O. The components in the basal culture medium are in the following mass ratio: yeast extract: peptone: KH2PO4: K2HPO4: MgSO4·7H2O: CaCl2·2H2O: FeCl3·6H2O: MnSO4·H2O: ZnSO4·7H2O: CoCl2·6H2O = 0.5:1:2:0.5:0.5:0.3:0.01:0.0016:0.0014:0.0020.
[0008] Preferably, the amino acid in S5 is selected from one or more of proline, glutamic acid, glycine, and arginine, and the total concentration of the amino acid is 0.5 g / L to 5.0 g / L. More preferably, the total concentration of the amino acid is 1.0 g / L to 2.0 g / L.
[0009] The amino acids added to the amino acid induction medium are selected from one or more of proline, glutamic acid, glycine, and arginine. The addition of these amino acids aims to utilize their known physiological functions in microbial low-temperature adaptation (e.g., proline as a compatible solute, glutamic acid participating in nitrogen metabolism and stress signaling) to enhance the low-temperature activity of the bacterial community through multiple pathways. The total concentration of added amino acids is generally 0.5 g / L-5.0 g / L. Concentrations below 0.5 g / L have no significant induction effect, while concentrations above 5.0 g / L may inhibit microbial growth due to excessive osmotic pressure or metabolic interference. A concentration range of 1.0 g / L-2.0 g / L can better balance the induction effect and the growth stress on the bacterial community. For example, in the examples below, a total concentration of 1.0 g / L of added proline and glutamic acid (0.5 g / L each) achieved good results. Those skilled in the art can adjust the types or combinations of amino acids according to the actual domestication effect. The domestication and screening method provided by this invention is not only applicable to corn straw, but its principle is also applicable to straws of various crops such as rice and wheat. By changing the type of straw in the induction and screening medium, highly efficient low-temperature degrading strains targeting specific straws can be screened out.
[0010] Preferably, in S6, the volume ratio for each passage is 1:5 to 1:20, and the culture time for a single round is 7 to 14 days. The periodic temperature fluctuation involves periodically switching between two set high and low target temperatures, with a switching cycle of 12 to 24 hours. This volume ratio is based on conventional passage practices in liquid microbial culture, aiming to maintain the bacterial population density while introducing appropriate growth competition. Each culture round typically lasts 7 to 14 days, and the culture endpoint can be determined by observing the turbidity of the culture or monitoring changes in pH value, ensuring that the bacterial population completes its main growth cycle. Both the high and low target temperatures mentioned above are low-temperature ranges.
[0011] Furthermore, this invention also discloses a method for isolating and purifying low-temperature resistant straw-degrading bacteria obtained through the above-mentioned domestication method. The specific technical solution is as follows: A method for isolating and purifying low-temperature resistant straw-degrading bacteria obtained based on the above domestication method includes the following steps: The culture medium obtained after acclimatization was serially diluted to obtain diluted bacterial solutions; The diluted bacterial solution was spread onto a low-temperature straw degradation screening plate medium and cultured at low temperature until single colonies grew. Observe and pick positive colonies that have a transparent hydrolysis zone around them; The selected positive colonies were streaked and purified to obtain purified low-temperature resistant straw-degrading strains.
[0012] Preferably, the low-temperature straw degradation screening plate medium is sodium carboxymethyl cellulose solid medium, which comprises: 10 g / L sodium carboxymethyl cellulose, 1.5 g / L KH₂PO₄, 3 g / L yeast extract, 5 g / L peptone, 0.2 g / L MgSO₄·7H₂O, 0.5 g / L NaCl, and 15-20 g / L agar powder. Positive colonies with a transparent hydrolysis zone around them are observed and selected using the Congo red staining method. Specifically, after cultivation, staining is performed with 1 mg / mL Congo red solution for 30 minutes, followed by destaining with 1 mol / L NaCl solution for 30 minutes.
[0013] Finally, the present invention also discloses the application of a low-temperature resistant straw-degrading bacterium obtained by the above-mentioned domestication method. The low-temperature resistant straw-degrading bacterium is used to degrade crop straw under low-temperature conditions, wherein the low-temperature conditions are 0-10℃, and the crop straw includes one or more of corn straw, rice straw, and wheat straw.
[0014] The application employs the above technical solution and has at least the following beneficial effects: 1. This invention combines exogenous amino acid induction with cyclic acclimation. Amino acids, acting as signaling molecules and protectants, actively stimulate the cold tolerance potential of the bacterial community, laying the foundation for subsequent high-pressure screening and significantly shortening the breeding cycle.
[0015] 2. Unlike traditional isothermal steady-state acclimatization, this invention introduces periodic temperature fluctuations (i.e., a combination of cooling and heating) during the acclimatization process to simulate the diurnal temperature variation in nature, which can effectively screen out superior strains that can adapt to complex environmental changes and have stronger genetic stability.
[0016] 3. This invention constructs a gradient-increasing selection pressure through multiple dimensions such as synchronous or stepwise temperature reduction, periodic temperature fluctuation, reduction of amino acid concentration, and restriction of carbon source, ensuring that the final strain has stable and efficient low-temperature degradation performance.
[0017] 4. The strains obtained by the method of this invention have a significant ability to degrade straw at low temperatures, and the activities of sodium carboxymethyl cellulose enzyme and filter paper enzyme are both at high levels, providing a powerful microbial resource for solving the problem of straw return to the field in cold regions.
[0018] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a schematic diagram of a method for domesticating low-temperature resistant straw-degrading bacteria according to the present invention; Figure 2 This is a schematic diagram of a method for isolating and purifying low-temperature resistant straw-degrading bacteria according to the present invention; Figure 3 This is a plate dilution comparison diagram of a low-temperature resistant straw-degrading bacteria before and after domestication according to the present invention; Figure 4 This is a comparison image of the low-temperature resistant straw-degrading bacteria before and after domestication according to the present invention. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0022] The low temperature mentioned in this invention refers to a temperature range of 0-10℃, which is the typical low temperature environment in northern my country after the autumn harvest and during the winter straw return to the field. The periodic temperature fluctuation stress mentioned in this invention refers to setting two target temperatures, high and low, within the above-mentioned low temperature range and periodically alternating between them to simulate the diurnal temperature variation in nature, so as to enhance the strain's adaptability to low temperature environmental fluctuations and improve its low temperature stability and degradation activity.
[0023] First, please see Figures 1 to 2 This invention discloses a method for the domestication and isolation / purification of low-temperature resistant straw-degrading bacteria. The domestication method specifically includes: Initial inoculum collection: Inoculum is collected from samples that have been exposed to low temperatures for an extended period to obtain inoculum samples. These samples can be cold spring soil, permafrost, alpine meadow soil, or low-temperature decomposed straw piles, etc.
[0024] Straw pretreatment: One or more types of corn straw, rice straw, and wheat straw (i.e., straw or straw mixture) are naturally dried, crushed, sieved, washed to remove impurities, and then dried again to obtain pretreated straw. The pretreated straw is sterilized before use. Specifically, after natural drying, the straw or straw mixture is crushed and passed through a 40-mesh sieve. It is then repeatedly washed with deionized water to remove soluble impurities and dried for later use. Before use, the pretreated straw is placed in a culture container and sterilized by autoclaving at 121°C for 20 minutes. This sterilization condition is a standard method for microbiological experiments, effectively sterilizing the straw and having an acceptable impact on the main structural components, without affecting the accuracy of subsequent screening.
[0025] Preparation of basal culture medium: Yeast extract, peptone, KH₂PO₄, K₂HPO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, FeCl₃·6H₂O, MnSO₄·H₂O, ZnSO₄·7H₂O, and CoCl₂·6H₂O were mixed, dissolved thoroughly in deionized water, and then brought to a final volume and sterilized to obtain the basal culture medium. The pH of this basal culture medium is its natural value. The preferred mass ratio of yeast extract, peptone, KH₂PO₄, K₂HPO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, FeCl₃·6H₂O, MnSO₄·H₂O, ZnSO₄·7H₂O, and CoCl₂·6H₂O is 0.5:1:2:0.5:0.5:0.3:0.01:0.0016:0.0014:0.0020.
[0026] Preparation of finished culture medium: Pretreated straw is used as the sole carbon source and added to the basal culture medium at a set concentration. After sterilization, the basal culture medium with straw as the sole carbon source is obtained, which is the finished culture medium.
[0027] Initial microbial community preparation: The collected bacterial samples were mixed evenly and added to the finished culture medium in proportion. The mixture was then cultured under low temperature with shaking to obtain the initial microbial community.
[0028] Preparation of amino acid induction medium: Take a separate commercial culture medium, add specific amino acids to it, stir to fully disperse the straw and amino acids, add deionized water to fully dissolve and bring to a final volume, then sterilize to obtain the amino acid induction medium. The amino acids are selected from one or more of proline, glutamic acid, glycine, and arginine, and the total concentration of added amino acids is controlled at 0.5 g / L - 5.0 g / L, preferably 1.0 g / L - 2.0 g / L. Maintain the natural pH value throughout the process; no additional acid-base adjustment is required.
[0029] Cyclic domestication implementation: This invention involves at least three rounds of domestication. Domestication begins from the second round, while gradually reducing the culture temperature (e.g., from 10°C to 7°C, then to 4°C), decreasing the concentration of added amino acids (e.g., from 2.0 g / L to 1.0 g / L, then to 0.5 g / L, until no amino acids are added at all), and decreasing the concentration of the carbon source straw (e.g., from 20 g / L to 10 g / L) to apply stronger selection pressure and screen out strains that already possess strong resistance to cold degradation. Specifically: First round of acclimatization: The initial bacterial population was inoculated into an amino acid-induced culture medium and cultured statically at low temperature and constant temperature to obtain the first round of culture solution. During this process, the bacterial population was cultured at low temperature and constant temperature until it entered the early stage of the stationary phase, activating the cold-resistant metabolic pathways of the bacterial population and enriching cold-resistant mutants.
[0030] The purpose of the first round of domestication was not to pursue degradation efficiency, but to allow the bacterial community to utilize exogenous amino acids as "protective agents" or "signaling molecules" under low-temperature stress, thereby activating its cold-resistance-related metabolic pathways and promoting the growth and accumulation of cold-resistant mutants. Culture continued until the bacterial community entered the early stage of stationary growth.
[0031] Second round of acclimatization: A certain amount of the first round culture medium was added to the amino acid induction medium and incubated at low temperature to obtain the second round culture medium. Periodic temperature fluctuations were introduced in the second round of acclimatization, but the temperature in the second round of acclimatization was not higher than that in the first round of acclimatization. In addition, the total concentration of amino acids added to the amino acid induction medium used in the second round of acclimatization was lower than that in the amino acid induction medium used in the first round of acclimatization.
[0032] The third round of acclimatization: A certain amount of the second-round culture medium was added to the amino acid induction medium and incubated at low temperature to obtain the third-round culture medium. Periodic temperature fluctuations were also performed in the third round of acclimatization, but the temperature in the third round was not higher than that in the second round. Furthermore, the total concentration of amino acids added to the amino acid induction medium used in the third round of acclimatization was lower than that used in the amino acid induction medium used in the second round of acclimatization.
[0033] The nth round of acclimatization: Take a certain amount of the (n-1)th round culture solution and add it to the finished culture medium. Incubate at low temperature and constant temperature to obtain the nth round culture solution. The amount of amino acids added to the culture medium used in the nth round is 0, and the concentration of added straw can be kept unchanged (i.e., the same as the straw concentration in the culture medium used in the (n-1)th round).
[0034] The (n+1)th round of domestication: Take a certain amount of the culture solution from the nth round, add it to the finished culture medium, and incubate at low temperature and constant temperature to obtain the (n+1)th round of culture solution. The amount of amino acids added to the finished culture medium used in the (n+1)th round is 0, and the straw concentration in the culture medium used in the (n+1)th round of domestication must be lower than the straw concentration in the culture medium used in the nth round of domestication.
[0035] As can be seen, the domestication method for the low-temperature resistant straw-degrading bacteria of this invention involves at least three rounds of domestication. Starting from the second round, the culture temperature is gradually reduced (e.g., from 10°C to 7°C, then to 4°C), the concentration of added amino acids is gradually reduced (e.g., from 2.0 g / L to 1.0 g / L, then to 0.5 g / L until no more amino acids are added), and the concentration of the carbon source straw is gradually reduced (e.g., from 20 g / L to 10 g / L). These processes can be carried out simultaneously or separately to apply stronger selection pressure and screen out strains that already possess strong cold-resistant degradation capabilities. During each round of temperature reduction, periodic temperature fluctuation stress is introduced. This fluctuation design aims to simulate the alternating environment of mild daytime temperatures and cold nighttime temperatures to activate the cold stress response of the bacterial community and improve its adaptability. Specifically, it involves alternating high and low temperatures within the target cooling range. For example, in the round of domestication where the temperature drops from 10°C to 7°C, the culture system is switched between 10°C and 7°C every 12 hours for 7 days.
[0036] The aforementioned acclimatization cycle can be repeated multiple times until the low-temperature degradation performance of the microbial community stabilizes. A criterion for determining performance stability is that after two consecutive rounds of acclimatization, the increase in straw weight loss is below a set threshold (e.g., 5%). In practice, the number of acclimatization rounds is typically 3-5.
[0037] In addition, after each round of acclimatization, the straw weight loss rate is measured using the culture medium obtained from the acclimatization solution. This rate is used as the core indicator to determine the degradation efficiency of the microbial community. When the increase in straw weight loss rate after two consecutive rounds of acclimatization is lower than a preset value, it indicates that the low-temperature degradation performance of the microbial community has stabilized, and the acclimatization cycle can be terminated. For further precise characterization, the amount of cellulose reducing sugar produced can be measured using the DNS method, and the lignin residue rate can be measured using the Paraná method. When the changes in these indicators tend to be stable, they can be used as auxiliary criteria for judgment. The total number of acclimatization cycles in the method of this invention is typically 3-5 rounds, which can be flexibly adjusted in conjunction with the above quantitative judgment criteria.
[0038] The isolation and purification method specifically includes: after the (n+1)th round of acclimatization, taking the (n+1)th round of culture medium, and sequentially performing serial dilution, plating culture, positive colony screening, purification culture, and strain preservation. Specific procedures are as follows: Serial dilution of bacterial culture: Take an appropriate amount of the (n+1)th round of culture medium and perform serial dilutions using sterile physiological saline (preferably 10⁻¹⁰). -1 -10 -6 (Gradient), mix thoroughly to prepare diluted bacterial solution; Low-temperature plating culture: Take 0.1-0.2 mL of bacterial suspension diluted with different gradients and spread it evenly on low-temperature straw degradation screening plate medium (containing Congo red staining agent for identification of cellulose degradation zone). Place the plate in a constant temperature incubator at 4℃ and invert it for 7 days until clear single colonies grow on the plate. Initial screening of positive colonies: Observe the transparent hydrolysis zone around a single colony on the plate, and select single colonies with obvious cellulose hydrolysis zones (these colonies are positive colonies with the ability to degrade straw at low temperatures) as target colonies for later use; Single colony purification culture: The selected positive target colonies were continuously streaked on the above-mentioned low-temperature straw degradation screening medium using the streak plate method for purification. The culture was inverted at 4℃, and the streak plate method was repeated 2-3 times until pure single colonies with consistent morphology, size, and color were obtained, thus obtaining the purified low-temperature resistant straw-degrading bacteria. The preservation unit for this low-temperature resistant straw-degrading bacteria is the China Center for Type Culture Collection (CCTCC); the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit date is April 27, 2026; the accession number is CCTCC NO: M2026814; the taxonomic name of the low-temperature resistant straw-degrading bacteria is *Bratrichia coli* X4-8. (Aureobasidium sp. X4-8).
[0039] Preservation of purified strains: The obtained purified strains were preserved using two different methods to meet both long-term and short-term use requirements. (1) Long-term preservation: Inoculate the pure strain into a sterile glycerol tube, adjust the final glycerol concentration to 20% (v / v), mix thoroughly, and then freeze in an ultra-low temperature freezer at -80℃; (2) Short-term preservation: Inoculate the pure strain into slant culture medium, store at 4°C, and transfer once a month to ensure the activity of the strain.
[0040] Example (1) Obtaining the initial source of bacteria: Frozen soil samples were collected in Altay City, Xinjiang, and bacteria were collected from these frozen soil samples to obtain the source samples.
[0041] (2) Straw pretreatment: Take straw (corn straw is used in this embodiment), dry it naturally, crush it through a 40-mesh sieve, wash it repeatedly with deionized water to remove soluble impurities, and dry it for later use. Before use, place the pretreated straw in a culture container and sterilize it with high-pressure steam at 121°C for 20 minutes.
[0042] (3) Preparation of basal culture medium: Accurately weigh the following per 1L volume: 0.5g yeast extract, 1g peptone, 2g potassium dihydrogen phosphate (KH₂PO₄), 0.5g dipotassium hydrogen phosphate (K₂HPO₄), 0.5g magnesium sulfate heptahydrate (MgSO₄·7H₂O), 0.3g calcium chloride dihydrate (CaCl₂·2H₂O), 0.01g ferric chloride hexahydrate (FeCl₃·6H₂O), 0.0016g manganese sulfate monohydrate (MnSO₄·H₂O), and 0.0014g zinc sulfate heptahydrate (ZnSO₄·7H₂O). g, cobalt chloride hexahydrate (CoCl2·6H2O) 0.0020 g, place the above components in a container, add deionized water and stir thoroughly until completely dissolved, add deionized water to make up to 1000 mL, the culture medium is at its natural pH, no additional pH adjustment is required, autoclave at 121℃ for 20 minutes, and then cool to room temperature for use.
[0043] Preparation of finished culture medium: Take 1L of the previously prepared basal culture medium and add 20g of pretreated corn stalks as the sole carbon source to obtain the finished culture medium. In subsequent acclimatization rounds, the straw concentration of the finished culture medium should be adjusted according to the acclimatization requirements, and the preparation method is the same as above.
[0044] (4) Initial microbial community preparation: Take 10g of the above frozen soil sample and add it to an Erlenmeyer flask containing 100mL of finished culture medium with 20g / L of pretreated corn straw as the sole carbon source. Place it in a constant temperature shaking incubator at 10℃ and 150rpm for 7 days to enrich the initial mixed microbial community for later use.
[0045] (5) Preparation of amino acid induction medium: Take another 1L of the prepared finished medium and add 0.5g of proline and 0.5g of glutamic acid, so that the total amino acid concentration is 1.0g / L. Stir thoroughly to disperse the straw and amino acids evenly. Add deionized water to make up to 1000mL. After dispensing, autoclave at 121℃ for 20 minutes and cool to room temperature for later use. In subsequent acclimatization rounds, the amino acid concentration and straw concentration of the amino acid induction medium are adjusted according to the acclimatization requirements, and the preparation method is the same as above.
[0046] (6) Reincarnation and domestication In this embodiment, the acclimatization was carried out in a low-temperature constant-temperature incubator with a subculture ratio of 1:10 (volume ratio). Each round of cultivation lasted 14 days. The straw weight loss rate was used as the core evaluation index for degradation efficiency. Increasing selection pressure was applied by gradually lowering the cultivation temperature, reducing / eliminating amino acid addition, and adjusting the straw carbon source concentration. At the same time, periodic temperature fluctuation stress was introduced. A total of 5 rounds of acclimatization were carried out in this experiment. The specific operations are as follows: First round of acclimatization: Take 20 mL of the above initial bacterial culture and inoculate it into a 200 mL Erlenmeyer flask containing amino acid induction medium (where the corn straw concentration is 20 g / L and the total concentration of proline and glutamic acid is 1.0 g / L). Incubate at 10℃ for 14 days to obtain the first round of culture medium. The straw weight loss rate was measured to be 30.63%. Second round of acclimatization: 20 mL of the first round culture medium was transferred to 200 mL of amino acid induction medium (containing 20 g / L corn straw and 0.5 g / L total proline and glutamic acid). Periodic temperature fluctuation stress was introduced, alternating the culture temperature between 7°C and 10°C every 12 hours for 7 days, followed by constant temperature culture at 7°C for 7 days, for a total culture time of 14 days, yielding the second round culture medium. The straw weight loss rate was measured to be 32.24%. The third round of acclimatization: 20 mL of the second round culture medium was transferred to 200 mL of amino acid induction medium (containing 20 g / L corn straw and a total concentration of 0.25 g / L proline and glutamic acid). Periodic temperature fluctuation stress was introduced, alternating the culture temperature between 4℃ and 7℃ every 12 hours for 7 days, followed by constant temperature culture at 4℃ for 7 days, for a total culture time of 14 days, yielding the third round culture medium. The straw weight loss rate was measured to be 35.37%. Fourth round of acclimatization: 20 mL of the third round culture medium was transferred to 200 mL of the finished culture medium (containing 20 g / L corn straw, with no added amino acids), and cultured at 4℃ for 14 days to obtain the fourth round culture medium. The straw weight loss rate was measured to be 34.89%. Fifth round of acclimatization: 20 mL of the fourth round culture medium was transferred to 200 mL of finished culture medium (containing 10 g / L corn straw, with no added amino acids), and cultured at 4℃ for 14 days to obtain the fifth round culture medium. The straw weight loss rate was measured to be 35.91%. Please refer to the table below. The straw weight loss rate was measured for the acclimatized microbial community in each round. Calculations showed that the increase in straw weight loss rate in the fourth round relative to the third round and in the fifth round relative to the fourth round was less than 5%, indicating that the low-temperature straw degradation performance of the microbial community had stabilized. The cycle of acclimatization was terminated, and a stable, low-temperature resistant straw-degrading microbial community was obtained after acclimatization.
[0047] Table 1. Degradation rate of straw microbial community under low-temperature cycling acclimatization (7) Isolation and purification: After the fifth round of domestication, the fifth round of culture medium was taken to obtain the stable domesticated bacterial population, which was diluted and spread, and cultured at 4℃ for 7 days. Single colonies were picked for purification, and a total of 110 strains of low-temperature resistant straw-degrading bacteria were obtained.
[0048] Experiment on the transparent zone of low-temperature resistant straw-degrading bacteria obtained after domestication The 110 low-temperature resistant straw-degrading bacterial strains obtained above were inoculated on sodium carboxymethyl cellulose solid medium and cultured at 4℃ for 72 h. After culture, the cultures were stained with 1 mg / mL Congo red solution for 30 minutes, followed by destaining with 1 mol / L NaCl solution for 30 minutes. The diameter of the transparent hydrolysis zone around the colonies was observed and measured. For the sodium carboxymethyl cellulose solid medium (1 L as an example), 10 g of sodium carboxymethyl cellulose, 1.5 g of KH4PO4, 3 g of yeast extract, 5 g of peptone, 0.2 g of MgSO4·7H2O, 0.5 g of NaCl, and 15-20 g of agar powder were mixed, dissolved thoroughly in deionized water, brought to a final volume, and sterilized. The pH of the medium was left at its natural value and no further adjustment was required.
[0049] Please see Figure 3 and Figure 4 The results showed that 28 of the strains exhibited hydrolysis zones under 4℃ and 72h culture conditions, indicating that these strains possess the ability to rapidly produce cellulase at low temperatures.
[0050] The microbial communities and strains obtained through low-temperature cycle screening were subjected to a 14-day straw fermentation experiment at 4℃.
[0051] At 4℃, the bacterial communities before and after domestication were spread on sodium carboxymethyl cellulose plates at the same dilution and cultured. The results showed that the number of bacteria after domestication was significantly higher than that of the undomesticated bacteria.
[0052] After inoculation with low-temperature microorganisms, the culture medium showed obvious turbidity. At the same time, the volume of the straw substrate decreased and the structure became looser. Compared with the original microorganisms that had not undergone low-temperature cycle screening, the straw weight loss rate of the microorganisms that underwent low-temperature cycle screening increased to 35.91%.
[0053] The 110 strains obtained from the cyclic screening were subjected to degradation tests on corn straw at 4℃. After 14 days, the highest weight loss rate of single-strain degradation of straw reached 16.15%, the highest sodium carboxymethyl cellulose enzyme activity was 4.06 IU / ml, and the highest filter paper enzyme activity was 15.97 IU / ml.
[0054] The results showed that the bacterial communities and strains obtained through low-temperature cycle screening were able to effectively utilize corn straw as a carbon source for growth and reproduction at 4℃, and had the ability to degrade straw components.
[0055] The types, combinations, and concentrations of amino acids used in this embodiment are designed based on their general promoting effect on microbial low-temperature metabolism. Those skilled in the art will understand that, without departing from the principles of this invention, other types of amino acids (such as glycine or arginine), different combinations, or adjustments to the specific concentration within the total concentration range of 0.5 g / L-5.0 g / L described in this invention can be attempted, and the effect can be confirmed by measuring the degradation efficiency of the acclimatized microbial community (such as straw weight loss rate).
[0056] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for domesticating low-temperature resistant straw-degrading bacteria, characterized in that, Includes the following steps: S1, collect samples from a low-temperature environment to obtain bacterial samples; S2, pre-treat the straw to obtain pre-treated straw; S3, organic nutrient components, phosphate buffer components, alkaline earth metal salts, transition metal salts and trace element salts are mixed, dissolved and brought to a final volume in deionized water and then sterilized to obtain a basic culture medium with a natural pH value. S4, the pretreated straw is added to the basic culture medium at a set concentration as the sole carbon source, and after stirring and sterilization, the finished culture medium is obtained. The inoculum sample is mixed evenly and added to the finished culture medium in proportion. Low-temperature shaking culture is carried out to obtain the initial microbial community. S5, add amino acids to the finished culture medium, stir and disperse, then adjust the volume, and sterilize to obtain the amino acid induction culture medium, wherein the pH of the amino acid induction culture medium is the natural value; S6, the initial microbial community is subjected to multiple rounds of low-temperature acclimatization culture. After each round of acclimatization, the degradation efficiency of the microbial community is detected by the straw weight loss rate as an indicator. During the acclimatization process, a gradient increasing selection pressure is applied by gradually reducing the culture temperature, gradually reducing and eventually eliminating the addition of amino acids, and gradually reducing the straw concentration. Periodic temperature fluctuation stress is introduced during the cooling phase. When the increase in straw weight loss rate is lower than a preset threshold for two consecutive rounds, the acclimatization is terminated, thus obtaining stable low-temperature resistant straw-degrading bacteria. The low-temperature resistant straw-degrading bacteria was deposited on April 27, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China; accession number: CCTCC NO: M2026814; the taxonomic name of the low-temperature resistant straw-degrading bacteria is *Brucea buddinga* X4-8. (Aureobasidium sp. X4-8) .
2. The domestication method according to claim 1, characterized in that, The components of the basal culture medium include: yeast extract, peptone, KH2PO4, K2HPO4, MgSO4·7H2O, CaCl2·2H2O, FeCl3·6H2O, MnSO4·H2O, ZnSO4·7H2O, and CoCl2·6H2O. The mass ratio of each component in the basal culture medium is: yeast extract: peptone: KH2PO4: K2HPO4: MgSO4·7H2O: CaCl2·2H2O: FeCl3·6H2O: MnSO4·H2O: ZnSO4·7H2O: CoCl2·6H2O = 0.5:1:2:0.5:0.5:0.3:0.01:0.0016:0.0014:0.0020.
3. The domestication method according to claim 1, characterized in that, The amino acid in S5 is selected from one or more of proline, glutamic acid, glycine, and arginine, and the total concentration of the amino acid is 0.5 g / L to 5.0 g / L.
4. The domestication method according to claim 3, characterized in that, The total concentration of added amino acids is 1.0 g / L to 2.0 g / L.
5. The domestication method according to claim 1, characterized in that, In S6, the volume ratio of each passage is 1:5 to 1:20, and the culture time is 7 to 14 days. The periodic temperature fluctuation is a periodic switching between two set high and low target temperatures, with a switching cycle of 12 to 24 hours.
6. The domestication method according to claim 1, characterized in that, The low temperature is 0-10℃.
7. A method for isolating and purifying low-temperature resistant straw-degrading bacteria obtained based on the domestication method described in claim 1, characterized in that, Includes the following steps: The culture medium obtained after the domestication period was completed was serially diluted to obtain diluted bacterial solutions; The diluted bacterial solution was spread onto a low-temperature straw degradation screening plate medium and cultured at low temperature until single colonies grew. Observe and pick positive colonies that have a transparent hydrolysis zone around them; The selected positive colonies were streaked and purified to obtain purified low-temperature resistant straw-degrading bacteria.
8. The separation and purification method according to claim 7, characterized in that, The low-temperature straw degradation screening plate culture medium is sodium carboxymethyl cellulose solid culture medium, which comprises: sodium carboxymethyl cellulose 10 g / L, KH2PO4 1.5 g / L, yeast extract 3 g / L, peptone 5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L and agar powder 15-20 g / L; Positive colonies that produce a transparent hydrolysis zone around their colonies were observed and selected using the Congo red staining method. Specifically, after the culture was completed, the colonies were stained with 1 mg / mL Congo red solution for 30 minutes, followed by destaining with 1 mol / L NaCl solution for 30 minutes.
9. The application of a low-temperature resistant straw-degrading bacteria, characterized in that, The low-temperature resistant straw-degrading bacteria are used to degrade crop straw under low-temperature conditions, namely 0-10℃, and the crop straw includes one or more of corn straw, rice straw, and wheat straw.