CN110358701B - Paenibacillus polymyxa microbial agent and production method thereof - Google Patents
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Fertilizers (AREA)
- Treatment Of Sludge (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for producing a paenibacillus polymyxa microbial inoculum by taking culture wastewater as a raw material, which can save about 25 percent of energy compared with the traditional mode through fermentation in a raw material opening and continuous feeding mode, has no high-pressure equipment in the production process, and has good safety and high production efficiency. The Paenibacillus polymyxa microbial inoculum produced by the method has wide application, good activity and effective viable count of 8 multiplied by 10 8 The strain/ml, the amount of mixed bacteria is only 4-6%, the product is a high value-added product, and has wide market prospect and extremely remarkable social and ecological benefits.
Description
Technical Field
The invention relates to the field of applied microorganisms, in particular to a paenibacillus polymyxa microbial inoculum and a production method thereof.
Background
The colony house of large-scale plant adopts the clean mode of water washing more, has produced a large amount of water washing excrement piles and a large amount of waste water discharge when clean. The nutrient content in the culture wastewater is higher and the nutrient components are comprehensive, so that the culture wastewater is a valuable fertilizer resource, wherein macroelement nutrients comprise C, H, O, N, P and K; medium nutrient: ca. Mg, S, si; essential nutrient elements of plants: zinc, boron, manganese, molybdenum, copper, iron, chlorine and trace nutrient elements, so that the waste water from the cultivation is wasted. However, the COD of the aquaculture wastewater is up to 8000-20000mg/L, the BOD is up to 2000-20000mg/L, and the wastewater cannot be directly recycled, and the traditional treatment method not only introduces the consumption of PAM, PAC and other chemical reagents and secondary pollution caused by the chemical reagents, but also causes the loss of a large amount of N, P and K elements, thereby being a great waste to resources.
Paenibacillus polymyxa (A), (B)Paenibacillus polymyxa) The biological control agent is widely distributed in nature, is an important biological control bacterium, has antagonistic action on plant pathogenic bacteria, and has a certain control effect on various plant diseases such as plant verticillium wilt, chickpea wilt, rape rot, black pine root rot and the like. Secondly, paenibacillus polymyxa achieves biological control by inducing plants to produce systemic resistance, and insecticidal or bactericidal substances such as antibacterial peptides, bacteriocins, non-ribosomally synthesized lipopeptides, cyclic cationic lipopeptides or non-cyclic cationic lipopeptides and the like. Paenibacillus polymyxa is capable of producing substances that promote plant growth such as indoleacetic acid, ethylene, auxin analogs, and the like. In addition to the biocontrol function, the paenibacillus polymyxa also has the functions of phosphate solubilizing, iron solubilizing and combined nitrogen fixation, can antagonize pathogenic bacteria of human and livestock and promote the growth of animals, and the functions of the paenibacillus polymyxa in the aspects of biological flocculation, microbial degradation, food safety and fruit and vegetable fresh-keeping are also reported in the year.
The production method of the paenibacillus polymyxa is various, and the paenibacillus polymyxa is produced by biomass waste liquid such as lemonade waste liquid, sweet potato waste liquid and the like. Patent CN 103172415 discloses a method for producing paenibacillus polymyxa fertilizer by using excess sludge after sewage treatment as raw material, which needs to be fermented in a totally-closed and absolutely-sterile environment, and has very high requirement on fermentation temperature, and needs microwave heating to 90-120 ℃ to pretreat sludge, and this process has high requirement on equipment, and will consume very large energy, and the batch fermentation yield is low, and the fermentation cost is very high, and the produced product can only be used as fertilizer, and the product is single, and the product added value is low, which is not beneficial to large-scale popularization and application. And the method does not relate to farm wastewater treatment.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a paenibacillus polymyxa microbial inoculum and a production method thereof.
The invention aims to provide a method for producing a paenibacillus polymyxa microbial inoculum by recycling culture wastewater, which specifically comprises the following steps:
1. primarily filtering suspended matters, solid matters and the like in the aquaculture wastewater by using a filtering system;
2. adding paenibacillus polymyxa bacterial liquid into the filtered wastewater according to the volume ratio of 1 8 Uniformly stirring the mixture per ml, discharging the mixture into a clarification tank, standing for 48-72 hours, and performing flocculation precipitation on the wastewater;
3. discarding supernatant, introducing the flocculent precipitate rich in nutrients at the lower layer into a liquid fermentation tank A for fermentation treatment, wherein the fermentation tank A is open, the temperature in the tank is constant at 80 ℃, and the precipitate stays in the fermentation tank A for 30min;
4. introducing the fermented precipitate into a fermentation tank B, cooling to 25-45 deg.C, inoculating live bacteria solution of Paenibacillus polymyxa with viable bacteria number of 2.5-4.5 × 10 into the fermentation tank B according to a volume ratio of 1 8 Stirring uniformly, and then carrying out aeration on the fermentation liquid in the tank B to keep the OD value of the fermentation liquid at 1.5-4mg/L, and standing for 10h;
5. and after the fermentation is finished, the precipitate enters a clarification tank from the tank B, and naturally settles for 12 hours, and the lower-layer precipitate is the paenibacillus polymyxa microbial inoculum.
Preferably, the filtration diameter of the filtration system in step 1 is 80 mesh.
Preferably, the paenibacillus polymyxa added in the step 2 and the step 4 is JP-15, the strain is submitted to the national common microorganism collection center for patent preservation, and the strain preservation number is: CGMCC No.17230.
Preferably, the paenibacillus polymyxa added in step 2 and step 4 is the microbial inoculum produced in step 5.
Preferably, the feeding and discharging in step 3 can be carried out simultaneously.
Preferably, the cooling temperature in step 4 is 30 ℃.
Preferably, the liquid in the microbial inoculum obtained in step 5 can be evaporated or filtered.
The second purpose of the invention is to provide a microbial inoculum produced by the method, the main strain in the microbial inoculum is paenibacillus polymyxa with the content of 94-96 percent and the effective viable count of 8 multiplied by 10 8 One per ml.
The third purpose of the invention is to provide the application of the paenibacillus polymyxa microbial inoculum, and the microbial inoculum can be used as a biological flocculant, a plant growth promoting microbial inoculum, a plant biocontrol microbial inoculum and a fertilizer decomposition microbial inoculum.
The method for producing the paenibacillus polymyxa microbial inoculum provided by the invention takes the culture wastewater as the raw material, fully utilizes the residual nutrients in the culture wastewater, and greatly avoids the waste of water resources of a culture farm and the pollution to the environment.
According to the method for producing the paenibacillus polymyxa microbial inoculum, provided by the invention, the target strain is utilized, the paenibacillus polymyxa has the characteristic of high temperature resistance, can survive in a high-temperature environment of 80 ℃, the culture medium is not completely sterilized, only partial sterilization is carried out under the condition of heating to about 60-80 ℃, raw material feeding is fermented in an open and continuous feeding mode, about 25% of energy can be saved compared with a traditional mode, high-pressure equipment is not required in the production process, the production safety is improved, feeding and discharging are continuously carried out, and the production efficiency is improved.
The paenibacillus polymyxa microbial inoculum provided by the invention has wide application, can be used as a bioflocculant, a plant growth promoting microbial inoculum, a plant biocontrol microbial inoculum and a fertilizer decomposition microbial inoculum to be popularized and applied in multiple fields, and the microbial inoculum has good activity and the effective viable count of 8 multiplied by 10 8 The quantity per ml of the microbial inoculum is only 4-6%, which is far higher than the technical index requirement of national agricultural microbial inoculum product, is a product with high added value, and has wide market prospect and extremely remarkable social benefit and ecological benefit.
Detailed Description
The present invention will be described in detail with reference to specific embodiments. The following examples are presented to aid those skilled in the art in further understanding the invention and are not intended to limit the invention in any manner.
The strain used in the examples below was Paenibacillus polymyxa JP-15 (Paenibacillus polymyxa) (hereinafter referred to as JP-15), the preservation number of the JP-15 strain in China general microbiological culture Collection center is CGMCC No.17230. The address of the depository: west road No.1 hospital No. 3, north jing, chaoyang district, preservation date: 24/01/2019.
The breeding wastewater used in the following examples was collected from a cow farm of agriculture, inc. of Huarui, gansu.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The JP-15 culture medium is prepared according to the following formula: peptone 5.0g, NH 4 Cl0.5g, beef extract 10.0g, yeast powder 5.0g, naCl 5.0g, agar 16.0g, distilled water 1000ml, pH 7.0. Inoculating JP-15 strain into the culture medium, shake-culturing at 30 deg.C until the strain reaches logarithmic phase, stopping culturing, and detecting viable bacteria amount in the strain to be 2.3 × 10 8 And (4) preserving the bacterial liquid at 4 ℃ for later use.
Measuring 100ml of aquaculture wastewater, primarily filtering with 80-mesh filter screen, adding JP-15 bacterial liquid (viable count 2.5-4.5 × 10) 8 Piece/ml) 0.1ml, stirring evenly, and standing for 48-72h for flocculation and precipitation. Discarding the supernatant, placing the flocculent precipitate into a triangular flask, placing in a water bath at 80 ℃, taking out the triangular flask after timing for 30min, and naturally cooling to room temperature. Adding 0.1ml of JP-15 bacterial liquid into a triangular flask, placing the flask into a shaking table, exposing oxygen at 100r/min to keep the OD value of the fermentation liquid at 1.5-4mg/L, and culturing for 10h. And (3) standing and precipitating the cultured triangular flask for 12h, discarding the supernatant, and obtaining the lower precipitate which is the paenibacillus polymyxa microbial inoculum. And (4) measuring the number of the JP-15 viable bacteria and the number of the mixed bacteria in the microbial inoculum. According to the relevant content of technical indexes of agricultural microbial agent products, the technical indexes of the microbial agent are detected, and the experimental results are shown in the following table.
Experimental results show that various indexes of the paenibacillus polymyxa microbial inoculum produced by fermenting the culture wastewater completely meet the technical standard of agricultural microbial inoculum product, wherein the number of the live bacteria of the paenibacillus polymyxa reaches 2.4 multiplied by 10 8 The microbial inoculum has high activity per ml. Effectively realizes the resource utilization of the culture wastewater, optimizes the fermentation process, and reduces the energy consumption and the use amount in the fermentation process, thereby further reducing the production cost and being suitable for large-scale popularization, application and production.
Example 2
The process is consistent with that of the example 1, except that the paenibacillus polymyxa microbial inoculum finally obtained in the example 1 is adopted to replace JP-15 bacterial fluid, various technical indexes of the microbial inoculum are detected according to relevant contents of technical indexes of agricultural microbial inoculum products, and experimental results are shown in the following table.
Experimental results show that the paenibacillus polymyxa microbial inoculum produced by fermentation in the embodiment 1 is used for fermenting culture wastewater, the microbial inoculum meeting the technical standard of agricultural microbial inoculum product can be produced, and the number of effective viable bacteria in the produced microbial inoculum is as high as 8.2 multiplied by 10 8 The bacterial strain/ml is much higher than that of JP-15 bacterial strain directly inoculated in example 1, and the fact that the bacterial strain can be recycled to produce the paenibacillus polymyxa bacterial strain is proved, and the paenibacillus polymyxa bacterial strain produced in a recycling mode has higher biological activity.
Claims (8)
1. A production method of a paenibacillus polymyxa microbial inoculum is characterized by comprising the following steps:
(1) Filtering suspended matters and solid matters in the aquaculture wastewater;
(2) Adding paenibacillus polymyxa JP-15 bacterial liquid into the filtered wastewater in a volume ratio of 1,the number of viable bacteria in the bacterial liquid is 2.5-4.5 × 10 8 And (2) per ml, uniformly mixing, discharging into a clarification tank, standing for 48-72 hours, and flocculating and precipitating wastewater, wherein the strain of Paenibacillus polymyxa JP-15 is submitted to the national common microorganism collection center for patent preservation, and the strain preservation number is as follows: CGMCC No.17230;
(3) Discarding supernatant, introducing the flocculent precipitate rich in nutrients at the lower layer into a liquid fermentation tank A for fermentation at 80 ℃ for 30min;
(4) Introducing the fermented precipitate into a fermentation tank B, cooling to 25-45 deg.C, inoculating Bacillus polymyxa JP-15 viable bacteria solution with viable bacteria number of 2.5-4.5 × 10 according to volume ratio of 1 8 Mixing uniformly, exposing to oxygen to keep OD value of fermentation liquor at 1.5-4mg/L, and fermenting for 10h;
(5) And discharging the fermentation product into a clarification tank, and naturally settling for 12 hours to obtain a lower-layer sediment, namely the paenibacillus polymyxa JP-15 microbial inoculum.
2. The method for producing a paenibacillus polymyxa microbial agent as claimed in claim 1, wherein the diameter of the filter in step (1) is 80 mesh.
3. The method for producing a paenibacillus polymyxa microbial inoculum according to claim 1, wherein the paenibacillus polymyxa added in the step (2) and the step (4) is the microbial inoculum produced in the step (5).
4. The method for producing a paenibacillus polymyxa microbial agent as claimed in claim 1, wherein the feeding and discharging in the fermentation process in step (3) can be performed simultaneously.
5. The method for producing a paenibacillus polymyxa inoculant according to claim 1 wherein the cooling temperature in step (4) is 30 ℃.
6. The method for producing a paenibacillus polymyxa microbial inoculum according to claim 1, wherein the microbial inoculum obtained in step (5) is subjected to evaporation or moisture filtration.
7. A Paenibacillus polymyxa agent produced by the method of any one of claims 1 to 6, wherein the main strain in the agent is Paenibacillus polymyxa JP-15 with a content of 94 to 96% and an effective viable count of 8 x 10 8 Each/ml.
8. The paenibacillus polymyxa microbial inoculum according to claim 7, wherein the microbial inoculum can be used as a bioflocculant, a plant growth promoting microbial inoculum, a plant biocontrol microbial inoculum and a fertilizer decomposition microbial inoculum.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2539804A1 (en) * | 2006-03-07 | 2007-09-07 | Denise Lemay | Integrated apparatus and system for the treatment of waste fluids and drain networks |
| CN102618527A (en) * | 2011-01-27 | 2012-08-01 | 中国科学院生态环境研究中心 | Method for recycling production of paenibacillus polymyxa microbial agent from citric acid wastewater |
| CN103172415A (en) * | 2011-12-26 | 2013-06-26 | 天津鹤岛农业科技有限公司 | Paenibacillus polymyxa microbial fertilizer produced through resource production of surplus sludge |
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| US20070262018A1 (en) * | 2006-05-09 | 2007-11-15 | Michael Richter | Method and product for protecting the residential and commercial aquatic environment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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