Straw leavening agent suitable for microfiltration membrane fermentation system and application of straw leavening agent

Abstract

The invention discloses a straw leavening agent suitable for a microfiltration membrane fermentation system and application of the straw leavening agent, and relates to the technical field of biology. The straw leavening agent comprises a microorganism combination. The microbial composition is prepared from bacillus subtilis (Bacillus subtilis) 1JN2, streptomyces globisporus (Streptomyces globisporus) XS2301 and myroides odortorius (Myroides odortorius) YW-1, and the microbial composition is prepared from bacillus subtilis (Bacillus subtilis) 1JN2, streptomyces globisporus (Streptomyces globisporus) XS2301 and myroides odortorius (Myroides odortorius) YW-1. The straw fermenting agent can improve the humification index in the straw composting process, remarkably shorten the composting period of crop straw and effectively reduce volatilization of irritant gas such as ammonia gas and hydrogen sulfide in the straw composting process. The straw leavening agent provided by the invention not only can efficiently treat crop straws, but also has the dual advantages of environmental protection and agricultural yield increase, and provides important technical support for agricultural sustainable development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a straw fermenting agent applicable to a microporous membrane fermentation system and its application. Background Art

[0002] With the progress of society and the change of residents' lifestyles, the amount of straw directly used as feed, fuel, etc. in the traditional way is decreasing, resulting in a large surplus of straw.

[0003] Currently, the utilization of straw mainly includes the following five aspects: fertilizer utilization, feed utilization, energy utilization, raw material utilization, and substrate utilization. Among them, fertilizer utilization is the main one (47.20%), supplemented by feed utilization (17.99%) and energy utilization (11.79%). The utilization rates of raw material utilization (2.47%) and substrate utilization (2.23%) are relatively low. In the process of fertilizer utilization of straw, direct field return and composting are the main methods. Direct field return of straw has the advantages of simple operation and high mechanization level, but it is prone to adverse effects such as poor soil ventilation, yellow seedlings and dead seedlings, and the spread of pests and diseases. Fertilizer utilization refers to composting straw in different ways, which has the advantages of convenient operation, simple technology, and the ability to kill pests and diseases. However, traditional compost fermentation has problems such as long fermentation time, poor degradation effect, and high labor costs.

[0004] Currently, in the process of straw fermentation, the selection of fermenting agents lacks pertinence and adaptability, resulting in problems such as long fermentation time and incomplete fermentation. In addition, a large amount of irritating gases will be generated during traditional compost fermentation, causing environmental pollution. Therefore, the research and development of new straw fertilizer utilization technologies, especially the analysis of microbial diversity during straw fermentation, and on this basis, the development of suitable and efficient fermenting agents, combined with a pollution-free biological fermentation system, has good application prospects in the fertilizer utilization of straw. Summary of the Invention

[0005] The purpose of the present invention is to provide a straw fermenting agent applicable to a microporous membrane fermentation system and its application to solve the problems existing in the above-mentioned prior art. This straw fermenting agent can increase the humification index of straw, effectively accelerate the composting process of crop straw, and effectively reduce the volatilization of irritating gases, reducing environmental pollution.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a microbial combination for straw fermentation, including Bacillus subtilis 1JN2, Streptomyces globisporus XS2301, and Myroides odoratiminus YW-1;

[0008] The preservation numbers of the Bacillus subtilis 1JN2, the Streptomyces globisporus XS2301, and the Myroides odoratiminus YW-1 in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms are CGMCC No.9759, CGMCC No.29725, and CGMCC No.20620 respectively.

[0009] The present invention also provides the application of the above microbial combination in preparing a straw fermenting agent applicable to a microfiltration membrane fermentation system.

[0010] The present invention also provides a straw fermenting agent applicable to a microfiltration membrane fermentation system, and the active ingredient includes the above microbial combination.

[0011] Further, the quantity ratio of the Bacillus subtilis 1JN2, the Streptomyces globisporus XS2301, and the Myroides odoratiminus YW-1 is 4:3:3.

[0012] The present invention also provides a preparation method of the above straw fermenting agent, including the step of uniformly mixing the bacterial suspension of Bacillus subtilis 1JN2, the bacterial suspension of Streptomyces globisporus XS2301, and the bacterial suspension of Myroides odoratiminus YW-1 to obtain the straw fermenting agent.

[0013] Further, the bacterial suspension of Bacillus subtilis 1JN2 is obtained by fermenting and culturing the Bacillus subtilis 1JN2 to obtain bacterial cells and then resuspending them;

[0014] The bacterial suspension of Streptomyces globisporus XS2301 is obtained by fermenting and culturing the Streptomyces globisporus XS2301 to obtain bacterial cells and then resuspending them;

[0015] The bacterial suspension of Myroides odoratiminus YW-1 is obtained by fermenting and culturing the Myroides odoratiminus YW-1 to obtain bacterial cells and then resuspending them.

[0016] Further, the temperature for fermenting and culturing the Bacillus subtilis 1JN2 is 28°C and the time is 20 hours;

[0017] The temperature for fermenting and culturing the Streptomyces globisporus XS2301 is 28°C and the time is 24 hours;

[0018] The temperature for fermenting the pseudo-odor aroma fungus YW-1 is 30° C. and the time is 20 hours.

[0019] The present invention also provides application of the straw fermentation agent in straw composting fermentation.

[0020] The invention also provides a straw composting and fermenting method, comprising the steps of spraying the straw fermentation agent on the straw to perform composting and fermentation.

[0021] Furthermore, the composting fermentation is carried out using a microporous membrane fermentation system.

[0022] The present invention discloses the following technical effects:

[0023] The present invention develops a straw fermentation agent suitable for a microporous membrane fermentation system, including Bacillus subtilis 1JN2, Streptomyces globosporus XS2301 and pseudo-odor-like fungus YW-1. The straw fermentation agent has the following advantages:

[0024] (1) Improving the humification index: Through the synergistic effect of Bacillus subtilis 1JN2, Streptomyces sphaerocephala XS2301 and Pseudomonas pseudoodorifera YW-1, the humification index of straw composting was significantly improved, thereby promoting the conversion of organic matter into stable humus and improving the quality of fertilizer.

[0025] (2) Promote seed germination: The straw biofertilizer prepared using the fermentation agent can effectively promote the germination rate of rice seeds, improve the soil microecological environment, provide good basic conditions for the growth of crops, and has obvious agricultural application value.

[0026] (3) Accelerate composting speed: Compared with traditional methods, this fermentation agent can significantly shorten the composting cycle of crop straw, accelerate the decomposition of organic matter, improve resource utilization efficiency, and provide an efficient solution for agricultural production.

[0027] (4) Reduce the volatilization of irritating gases: During the composting process, the fermentation agent can effectively reduce the volatilization of irritating gases such as ammonia and hydrogen sulfide, reduce the harm to the environment and human health, and embody the characteristics of environmental friendliness.

[0028] In summary, the straw fermentation agent provided by the present invention can not only efficiently process crop straw, but also has the dual advantages of environmental protection and agricultural production increase, and provides important technical support for achieving sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0030] Figure 1 It is the DGGE map of bacteria during the fermentation process of the microporous membrane fermentation system; among them, 1-3 are samples before fermentation, and 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, and 22-24 are samples collected 1-7 weeks after the start of fermentation respectively;

[0031] Figure 2 It is a statistical chart of the germination rates of rice seeds in the inoculant treatment group and the blank control group;

[0032] Figure 3 It is a statistical chart of the humification index in the inoculant treatment group and the blank control group. Detailed implementation manners

[0033] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which will be obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention will be obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0037] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0038] Bacillus subtilis 1JN2 in the following examples was deposited at the General Microbiology Center of the China National Center for Culture Collection of Microorganisms on October 13, 2014. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 9759; Streptomyces globisporus XS2301 was deposited at the General Microbiology Center of the China National Center for Culture Collection of Microorganisms on January 22, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29725; Myroides odoratiminus YW-1 was deposited at the General Microbiology Center of the China National Center for Culture Collection of Microorganisms on September 9, 2020. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 20620.

[0039] Example 1

[0040] 1. Analysis of microbial diversity at different times in the microfiltration membrane fermentation system

[0041] The test site was Zhenjiang Jinfeng Biotechnology Co., Ltd. Crop straw samples in the microfiltration membrane fermentation system were collected at different time points, and the total DNA of the samples was extracted. The hypervariable region sequences of 16S rDNA of the samples were amplified using the universal primers GC-338F and 518R. The change rules of bacterial diversity were analyzed by PCR-DGGE, and the dominant bands were sequenced and analyzed.

[0042] DGGE was completed in the DCode mutation detection system. Take 25 μL of the PCR concentrated product for DGGE. The denaturant gradient of the DGGE gel was 30% - 60%, the acrylamide concentration was 8% (100% concentration was defined as 7 mol / L urea and 40% deionized formamide), the electrophoresis buffer was 1×TAE, the voltage was 130 V, and electrophoresis was carried out at 60 °C for 7 h. After electrophoresis, it was stained with silver nitrate for 10 - 15 min, scanned and photographed. The differential bands between different treatment groups and the control group were compared, the gel was cut and recovered, and sequenced and compared.

[0043] The sequencing results were analyzed using DNAstar and Cluster software, and the sequences of the most similar strains were downloaded as reference sequences for the phylogenetic tree. Then, MEGA software was used to construct a phylogenetic tree by the neighbor-joining method (NJ method for short), and 1000 bootstrap datasets were generated by the Bootstrap method to obtain a consensus tree. The DGGE profiles of bacteria during the fermentation process in the microfiltration membrane fermentation system are shown in Figure 1 .

[0044] The results showed that during the fermentation process in this system, Bacillus and Streptomyces were the dominant populations, which appeared 1 week after the start of sample fermentation and continued until 7 weeks after fermentation.

[0045] Example 2

[0046] Detection of enzyme activity produced by fermentation strains:

[0047] According to the detection results of bacterial diversity during the fermentation process in Example 1, Bacillus subtilis 1JN2, Streptomyces globisporus XS2301, and Myroides odoratiminus YW-1 were selected from the laboratory strain resource library, and the enzyme activities produced by each strain were detected using detection plates.

[0048] The strains were inoculated onto a cellulase activity assay plate (10 g of peptone, 10 g of yeast extract, 10 g of sodium carboxymethyl cellulose, 5 g of sodium chloride, 1 g of potassium dihydrogen phosphate, 18 g of agar, made up to 1000 mL, pH = 7.0). After culturing at 28 °C for 48 h, they were stained with 1 g / L of Congo red for 1 h, then the staining solution was poured off, and they were soaked in 1 M NaCl for 1 h. The presence or absence of a clear zone was detected and its inner and outer diameters were measured.

[0049] The strains were cultured on a medium with colloidal chitin as the sole carbon source: (1.0 g of NH4H2PO4, 0.2 g of KCl, 0.2 g of MgSO4·7H2O, 1% (w / v) of colloidal chitin, 20 g of agar, made up to 1000 mL, pH 7.0). After inoculation, they were cultured at 30 °C for 3 days, and the size of the clear zone was measured.

[0050] The results showed that the hydrolysis zone radius of strain XS2301 reached 6 mm on the cellulase activity assay plate, the hydrolysis zone radius of strain 1JN2 reached 7 mm on the cellulose screening plate, and the hydrolysis zone radii of strain YW-1 on the chitin and cellulose screening plates were 9 mm and 6 mm, respectively.

[0051] Example 3

[0052] Preparation of starter culture:

[0053] The strain XS2301 was cultured in beef extract peptone liquid medium at 28 °C and 180 rpm for 24 hours, and then the cells were centrifuged and resuspended to a cell concentration of 10 8 CFU / mL to obtain the XS2301 cell suspension.

[0054] The strain 1JN2 was cultured in LB liquid medium at 28 °C and 180 rpm for 20 hours, and then the cells were centrifuged and resuspended to a cell concentration of 10 8 CFU / mL to obtain the 1JN2 cell suspension.

[0055] The strain YW-1 was cultured in LB liquid medium at 30 °C and 180 rpm for 20 hours, and then the cells were centrifuged and resuspended to a cell concentration of 10 8 CFU / mL to obtain the YW-1 cell suspension.

[0056] The above resuspension was carried out using sterile normal saline.

[0057] The XS2301 cell suspension, 1JN2 cell suspension and YW-1 cell suspension were mixed evenly according to a volume ratio of 4:3:3 to obtain the starter culture.

[0058] Comparative Example 1

[0059] Preparation of starter culture:

[0060] The strain XS2301 was cultured in beef extract peptone liquid medium at 28 °C and 180 rpm for 24 hours, and then the cells were centrifuged and resuspended to a cell concentration of 10 8 CFU / mL to obtain the starter culture.

[0061] Comparative Example 2

[0062] Preparation of starter culture:

[0063] The strain 1JN2 was cultured in LB liquid medium at 28 °C and 180 rpm for 20 hours, and then the cells were centrifuged and resuspended to a cell concentration of 10 8 CFU / mL to obtain the starter culture.

[0064] Comparative Example 3

[0065] Preparation of starter culture:

[0066] The strain YW-1 was cultured in LB liquid medium at 30 °C and 180 rpm for 20 hours, and then the cells were centrifuged and resuspended to a cell concentration of 10 8CFU / mL to obtain the starter culture.

[0067] Example 4

[0068] Fermentation of crop straw by the starter culture and index detection:

[0069] The test site was Zhenjiang Jinfeng Biotechnology Co., Ltd. The fermentation system consisted of an aeration system, a covering system, and a control system. First, corn straw and rice straw were stacked into a strip stack in a weight ratio of 1:1. Each stack was 10 meters long, 2 meters wide, and 1.5 meters high. During the stacking process of the straw, the starter cultures prepared in Example 3 and Comparative Examples 1-3 were used for spraying treatment. 15 L of the starter culture was sprayed on each stack (total straw mass was 500 kg). After stacking, an antibacterial bifunctional nanofiltration membrane CPCM with high-efficiency salt rejection and increased water flux was covered. This polymer microporous membrane material discharged water, leaving 91.6% of the nutrients and organic matter, while killing the pathogenic bacteria in the waste and preventing the diffusion of irritating odor gases, ensuring the timely discharge of carbon dioxide. The aeration system provided the oxygen required for fermentation to the compost pile through the pipes laid at the bottom of the compost pile. During the fermentation process, the instrument data of the Jinfeng intelligent composting system was automatically collected, and the fermentation process was controlled by computer feedback. The ventilation system realized the automatic control of oxygen supply according to the energy consumption in the compost. The temperature, humidity, and oxygen supply in the optimized area were maintained.

[0070] During the fermentation process, natural fermentation without spraying the starter culture was used as the control group.

[0071] At 10 days, 20 days, and 30 days after the start of fermentation, the straw residues in the middle of the stack were collected to detect their promotion of rice seed germination rate (GI) and humification index.

[0072] The method for detecting the germination rate was as follows: 10 g of the compost sample was placed in a stoppered conical flask, 100 mL of distilled water was added, and it was oscillated and extracted at 25 °C for 1 h. After taking it down and standing for 0.5 h, the upper clear liquid was filtered, and the filtrate was collected and shaken well for analysis. One qualitative filter paper was placed in each 9 cm petri dish, and 10 rice seeds of basically the same size and plumpness were evenly placed on it. 10 mL of the test sample extract was added, the petri dish cover was covered, and it was cultured in the dark in an incubator at (25 ± 2) °C for 48 h. The number of germinated seeds was counted, and the main root length was measured one by one with a vernier caliper. The distilled water treatment group was used as the control. GI = (germination rate of the treatment group × average root length) / (germination rate of the control group × average root length) × 100%.

[0073] The method for detecting the humification index was as follows: Extraction and determination of humus (HS), humic acid (HA), and fulvic acid (FA): Take 1 g of the sieved air-dried sample in a 50 mL centrifuge tube, add 20 mL of the mixed extraction solution (0.1 mol·L -1 Sodium hydroxide (NaOH) and 0.1 mol·L-1 Sodium pyrophosphate (Na4P2O7) (V / V = 1:1)) was shaken at room temperature for 30 min, and then centrifuged at 4000 r·min -1 for 20 min. The supernatant was collected, and the above operation was repeated 4 times (until the leaching solution was almost colorless). The 4 supernatants were mixed to obtain the HS solution. Thereafter, the pH was adjusted to 1 with 6 mol·L -1 hydrochloric acid solution (which can be detected with a precision pH test paper), and left to stand at room temperature for 12 h. Then it was centrifuged at 4000 r·min -1 for 15 min. The supernatant was FA, and the precipitate was HA. The contents of HS and FA were determined using a total organic carbon (TOC) analyzer (SHIMADZU, Japan). The content of HA was calculated by subtraction. The ratio of the content of HA to the content of FA was the humification index.

[0074] The results showed that on the 30th day after fermentation treatment, the GI value of the straw residue in the treatment group of Example 3 of the present invention to promote the germination rate of rice seeds reached 93%, that of the natural fermentation group was 35%, and the germination rates of the treatment groups of Comparative Examples 1-3 were much lower than that of the treatment group of Example 3. Only the treatment group of Comparative Example 1 reached 52% ( Figure 2 ); the straw humification index of the treatment group of Example 3 was 64%, that of the natural fermentation group was 21%, and the straw humification indices of the treatment groups of Comparative Examples 1-3 were much lower than that of the treatment group of Example 3. Only the treatment group of Comparative Example 1 reached 40% ( Figure 3 ).

[0075] In summary, the fermentation agent provided by the present invention can effectively accelerate the composting process of crop straw and effectively reduce the volatilization of irritating gases, reducing environmental pollution.

[0076] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A microbial combination for straw fermentation, characterized in that, It includes Bacillus subtilis 1JN2, Streptomyces globisporus XS2301, and Myroides odoratiminus YW-1; The preservation numbers of the Bacillus subtilis 1JN2, Streptomyces globisporus XS2301, and Myroides odoratiminus YW-1 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms are CGMCC No.9759, CGMCC No.29725, and CGMCC No.20620 respectively.

2. Use of a microbial combination as claimed in claim 1 in the preparation of a straw fermenting agent applicable to a microporous membrane fermentation system.

3. A straw fermenting agent applicable to a microporous membrane fermentation system, characterized in that, The active ingredient includes the microbial combination as claimed in claim 1.

4. The straw fermenting agent according to claim 3, characterized in that, The quantity ratio of the Bacillus subtilis 1JN2, Streptomyces globisporus XS2301, and Myroides odoratiminus YW-1 is 4:3:

3.

5. A preparation method of the straw fermenting agent as described in claim 3 or 4, characterized in that, It includes the step of uniformly mixing the bacterial suspension of Bacillus subtilis 1JN2, the bacterial suspension of Streptomyces globisporus XS2301, and the bacterial suspension of Myroides odoratiminus YW-1 to obtain the straw fermenting agent.

6. The preparation method according to claim 5, characterized in that, The bacterial suspension of Bacillus subtilis 1JN2 is obtained by fermenting and culturing the Bacillus subtilis 1JN2 to obtain thalli and then resuspending them; The bacterial suspension of Streptomyces globisporus XS2301 is obtained by fermenting and culturing the Streptomyces globisporus XS2301 to obtain thalli and then resuspending them; The bacterial suspension of Myroides odoratiminus YW-1 is obtained by fermenting and culturing the Myroides odoratiminus YW-1 to obtain thalli and then resuspending them.

7. The preparation method according to claim 6, characterized in that, The temperature for fermenting and culturing the Bacillus subtilis 1JN2 is 28 °C and the time is 20 hours; The temperature for fermenting and culturing the Streptomyces globisporus XS2301 is 28 °C and the time is 24 hours; The temperature for fermenting and culturing the Myroides odoratiminus YW-1 is 30 °C and the time is 20 hours.

8. Use of a straw fermenting agent as claimed in claim 3 or 4 in straw composting fermentation.

9. A method for composting and fermenting straw, characterized in that, It includes the step of spraying the straw fermenting agent as claimed in claim 3 or 4 on the straw and carrying out composting fermentation.

10. The straw composting fermentation method according to claim 9, characterized in that, The composting fermentation is carried out using a microporous membrane fermentation system.

Images