CN113969246A - Nitrogen-retaining strain, composite microbial inoculum, preparation method and application of composite microbial inoculum - Google Patents
Nitrogen-retaining strain, composite microbial inoculum, preparation method and application of composite microbial inoculum Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial preparation, and particularly relates to a nitrogen-retaining strain, a composite microbial inoculum, a preparation method of the composite microbial inoculum and application of the composite microbial inoculum. The nitrogen-retaining strain is thermophilic heterotrophic ammonia oxidizing bacteria, a preservation organization is a microorganism strain preservation center in Guangdong province, the strain is classified and named as Aneuribacillus thermoaeophilus J3, the preservation number is GDMCC No:61602, the preservation date is 2021 year 4-month 12-day, the nitrogen-retaining strain is a dominant bacterium in a high-temperature period, the metabolism is vigorous, the adaptability is strong, the ammonia oxidizing efficiency is high, the strain can be rapidly colonized in composts such as biogas residues and the like, the function of promoting decay and retaining nitrogen is exerted, the strain is not limited by the ammonia oxidizing process, a large amount of ammonia can be oxidized into nitrite and nitrate, the volatilization of ammonia is reduced, the content of nitrate nitrogen in the composts is improved, the environmental pollution is reduced, a large amount of nitrogen elements capable of being utilized by the growth of crops can be formed, and the composting quality is improved.
Description
Technical Field
The invention belongs to the technical field of microbial preparation, and particularly relates to a nitrogen-retaining strain, a composite microbial inoculum, a preparation method of the composite microbial inoculum and application of the composite microbial inoculum.
Background
The biogas residue is a semisolid substance remained at the bottom of the biogas digester after biogas fermentation, and contains rich organic matters, humic acid, crude protein, nitrogen, phosphorus, potassium, various trace elements and auxin.
In the biogas residue composting process, organic nitrogen is firstly decomposed by microorganisms and converted into ammonium nitrogen, and is continuously oxidized into nitrite nitrogen and nitrate nitrogen in the subsequent nitrification, and NH is easy to occur due to the change of conditions such as temperature, ventilation and the like in the process3Volatilizes to cause a great deal of nitrogen loss, thereby not only reducing the fertilizer qualityAnd moreover, the method can cause environmental problems such as stink, acid rain, water eutrophication and the like. Therefore, how to reduce NH3Discharge is a problem that must be addressed in the composting process.
Ammonia oxidation is the first step of nitrification and the rate-limiting step, and the high-efficiency ammonia oxidation process is beneficial to reducing the emission of ammonia gas in the high-temperature period of compost and improving the retention of nitrogen, and is of great importance to the conversion of nitrogen in the compost. Ammonia-oxidizing bacteria are indispensable flora in the nitrification process and are the main flora for biological denitrification at present, the ammonia-oxidizing bacteria oxidize ammonia into nitrous acid, and the nitrifying bacteria oxidize the nitrous acid into nitric acid. The growth rate of autotrophic ammonia-oxidizing bacteria is greatly limited by organic matters, a large amount of ammonia is volatilized in the slow growth process, the effects of oxidizing ammonia and reducing nitrogen loss cannot be achieved, and the autotrophic ammonia-oxidizing bacteria have little meaning in practical application; the growth rate of heterotrophic ammonia-oxidizing bacteria is not limited by the ammonia-oxidizing process, ammonia can be oxidized into nitrite and nitrate in a large amount, the content of nitrate nitrogen in compost is improved, and NH is reduced3And (4) volatilizing. Therefore, the invention provides a nitrogen-retaining strain, a composite microbial inoculum, a preparation method and application of the composite microbial inoculum.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects of serious nitrogen loss, limited growth rate of nitrogen oxide bacteria and the like in the ammonia oxidation process in the prior art, so that a nitrogen-retaining strain, a composite microbial inoculum, and a preparation method and application of the composite microbial inoculum are provided.
Therefore, the invention provides the following technical scheme.
The invention provides a nitrogen-protecting strain which is thermophilic heterotrophic ammonia oxidizing bacteria, a preservation organization is Guangdong province microbial strain preservation center, the preservation organization is named as Aneurinibacillus thermoaerophilus J3 in a classification way, the preservation number is GDMCC No. 61602, and the preservation date is 2021, 4 and 12 days.
The invention also provides a composite microbial inoculum which comprises the nitrogen-retaining strain.
The composite microbial inoculum comprises nitrogen-retaining strain powder and decomposed strain powder;
the mass ratio of the nitrogen-retaining strain-containing bacterial powder to the decomposed bacterial powder is 1: (2.5-3.5);
the decomposed bacterium powder is bacillus subtilis and/or bacillus licheniformis.
The content of the decomposed bacteria powder is 2.6 multiplied by 109-5.0×109cfu/g；
The bacterium content of the bacterium powder containing the nitrogen-retaining strain is 4.5 multiplied by 108-7.5×108cfu/g。
The raw materials of the powder containing the nitrogen-retaining strain comprise a culture medium substrate and a growth factor for growth and fermentation of the nitrogen-retaining strain;
the culture medium matrix is at least one of starch, bean cake powder, bran, cassava residue and bean pulp;
the growth factor comprises at least one of a carbon source, a nitrogen source and an inorganic salt;
the mass ratio of the culture medium substrate to the growth factors is (20-35): (3-5).
The bacterial content of the compound bacterial agent is 1.9 multiplied by 109-3.8×109cfu/g。
In addition, the invention provides a preparation method of the composite microbial inoculum, which comprises the step of fermenting the nitrogen-retaining strain.
In the preparation method of the composite microbial inoculum, a culture medium substrate, a growth factor and the nitrogen-retaining strain are mixed, fermented and dried to obtain the powder containing the nitrogen-retaining strain;
mixing the powder of the nitrogen-retaining strain and the powder of the decomposed bacteria to obtain the composite microbial inoculum;
optionally, the fermentation conditions are that the fermentation temperature is 50-55 ℃, the pressure is 0.03-0.05MPa, and the low dissolved oxygen is 1-5%.
The invention also provides the nitrogen-retaining strain, the composite microbial inoculum or the composite microbial inoculum prepared by the method, which has the following application:
(1) application in preparing compost;
(2) the application in the field of ammonia nitrogen oxidation;
(3) the method is applied to ammonia nitrogen sewage purification.
Further, the invention also provides a composting method, wherein composting raw materials are composted by using the nitrogen-retaining strain, the composite microbial inoculum or the composite microbial inoculum prepared by the method;
optionally, the amount of the composite microbial inoculum is 0.05-0.15% of the total mass of the raw materials and the conditioner;
optionally, the composting time is 40-50 days, and the pile is turned once every 2-3 days;
optionally, fresh pig manure biogas residues are used as a composting raw material, wood chips are used as a conditioner, and the C/N of the material is regulated to be 15-30.
Furthermore, the invention provides a method for purifying ammonia nitrogen sewage, which comprises the steps of inoculating the nitrogen-retaining strain, the composite microbial inoculum or the composite microbial inoculum prepared by the method into ammonia nitrogen sewage for culture, and purifying the ammonia nitrogen sewage;
optionally, the temperature of the culture is 52-57 ℃.
The technical scheme of the invention has the following advantages:
1. the invention provides an azotobacter strain, which is thermophilic heterotrophic ammonia oxidizing bacteria, the preservation organization is Guangdong province microorganism strain preservation center, the preservation address is as follows: a59 th floor 5 of Dazhou college No. 100 of Xieli Zhonglu, Guangdong province, which is classified and named Aneuribacillus thermoaeophilus J3, the preservation number is GDMCC No. 61602, the preservation date is 2021 year 4 month 12 days, the nitrogen-retaining strain is separated from a pile sample of the high-temperature period of the biogas residue compost, is a dominant bacterium in the high-temperature period, has high-efficiency ammonia oxidation function, is identified as a thermophilic aerophilic thiamine-degrading bacillus (Aneuribacillus thermoaerophilus), can grow in the temperature range of 45-65 ℃, is vigorous in metabolism, strong in adaptability and high in ammonia oxidation efficiency, can be rapidly colonized in the compost such as the biogas residue and the like, plays a role in promoting decay and retaining nitrogen, is not limited by the ammonia oxidation process, can oxidize a large amount of ammonia into nitrite and nitrate, reduces the volatilization of ammonia gas, improves the content of nitrate in the compost, reduces environmental pollution, can form a large amount of nitrogen elements which can be utilized by the growth of crops, thereby improving the quality of the compost.
2. The composite microbial inoculum provided by the invention can be prepared from low-cost industrial and agricultural wastes such as bran, straw and cassava residues, is low in cost, has a good nitrogen preservation effect, can promote ammonia nitrogen to be converted into nitrate nitrogen in a composting process, reduces the generation of malodorous gases such as ammonia gas and the like, reduces environmental pollution and improves the quality of composting.
3. The composite microbial inoculum provided by the invention can be applied to the fields of composting, ammonia nitrogen oxidation and ammonia nitrogen sewage purification, and has the advantages of low application cost, less generated ammonia gas and good nitrogen preservation effect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a small indoor fermentation test apparatus in example 1 of the present invention;
FIG. 2 shows the form of microscopic cells in example 1 of the present invention;
FIG. 3 is a graph showing the growth of the strain in example 1 of the present invention;
FIG. 4 is a phylogenetic tree according to example 1 of the present invention;
FIG. 5 is a graph showing the temperature change during composting in example 3 of the present invention;
reference numerals:
1-constant temperature incubator; 2-a first boric acid absorption unit; 3-a second barium hydroxide absorption device.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The nitrogen-retaining strain provided by the invention is obtained by separating and screening compost and is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCC No. 61602.
The media used in the examples are as follows:
ammonia oxidation selective liquid medium: (NH)4)2SO4 2.0g，MgSO4·7H2O 0.25g，NaCl 0.5g， KH2PO4 1.0g，CaCl2 0.5g,FeSO4·7H2O 0.2g,H2O 1000mL，pH 7.0。
Ammonia oxidation selective solid medium 2% agar was added to the ammonia oxidation selective liquid medium.
The high-temperature bacterium liquid culture medium comprises macroelements and microelements, wherein the macroelements (unit: g/L): (NH)4)2SO40.50，KH2PO4 0.28，MgSO4·7H2O 0.25，CaCl20.01, Tryptone 2.00, Yeast extract 2.00; trace elements (mg/L): na (Na)2MoO4·2H2O 0.025，FeCl30.28，CuSO4 0.016，MnSO4·H2O 2.2，H3BO30.5，ZnSO4·7H2O 0.5，CoCl2·6H2O0.046; sterilizing at 121 deg.C for 20 min.
The high-temperature bacterium solid culture medium is added with 2 percent of agar on the basis of a high-temperature liquid culture medium.
Example 1
This example provides a nitrogen-conserving strain and a screening method thereof, comprising the following steps,
weighing 5g of pig manure compost sample in a high-temperature period, adding the pig manure compost sample into a triangular flask containing 45ml of sterile water, and oscillating the pig manure compost sample in a water bath shaking table at the temperature of 28 ℃ and at the speed of 150r/min for 30min to obtain a suspension of the compost sample;
inoculating 5ml of the suspension into 100ml of ammonia oxidation selective liquid culture medium, carrying out shake-flask culture in an incubator at 60 ℃ and 150r/min for 7 days, and obtaining the enrichment liquid after the culture medium is obviously turbid.
Inoculating the enrichment solution into a fresh ammonia oxidation selective liquid culture medium with the inoculation amount of 1%, culturing for 7 days, and continuously carrying out enrichment culture for 3 times according to the method to obtain the target flora.
Adding 1ml of final ammonia oxidation selective culture medium bacterial liquid into a sterile normal saline test tube containing 9ml, uniformly mixing by shaking, and then diluting to 10 times by weight-8Respectively taking 0.1ml 10-6、10-7、10-8Uniformly coating the diluent on an ammonia oxidation selective solid culture medium by using a plate coating method, then culturing for 24h in a constant-temperature incubator at 60 ℃, and selecting single colonies with different morphological characteristics for repeated streak culture to obtain a purified strain. Inoculating the purified strain into test tubes containing a high-temperature bacterium liquid culture medium, culturing at 60 ℃ for 24h at 120r/min, and dropwise adding a Grignard reagent into each test tube, wherein the red strain is a strain with nitrogen preservation;
performing an inter-strain antagonism experiment on a nitrogen-retaining strain with ammonia oxidation capacity and a decomposing bacterium (bacillus subtilis) commonly used for composting, and screening to obtain a strain which can exert a synergistic effect with the decomposing bacterium (bacillus subtilis) to obtain a target nitrogen-retaining strain; after the target policy-keeping strain is fermented, the nitrogen-keeping strain is obtained by detecting the nitrogen-keeping performance of the strain and screening again.
The method for testing the nitrogen retention performance of the strain comprises the following steps:
adding 100g of fresh pig manure biogas residues and 40g of wood chips into a 500mL triangular flask, respectively sucking 20mL of the preserved primary-screening strain fermentation liquid, adding the 20mL of the preserved primary-screening strain fermentation liquid into a biogas residue and wood chip mixture, and performing three parallels on each bacterial liquid by taking sterile distilled water as a blank control. Each treatment was carried out in a 60 ℃ constant temperature incubator, high temperature composting was simulated in accordance with the small indoor fermentation test apparatus shown in FIG. 1, air was introduced into the incubator by an air pump (air pump flow: 6L/min), and the produced ammonia gas and carbon dioxide gas were absorbed by the first boric acid absorption apparatus and the second barium hydroxide absorption apparatus. And judging the corrosion-promoting and nitrogen-preserving effect of the strain according to the amount of ammonia and carbon dioxide released by the simulated composting test.
1. Measuring NH3Amount of (2)
In absorbing NH3Dripping 2 drops of 0.1% methyl red ethanol solution and 1 drop of 0.1% methylene blue ethanol indicator into the boric acid solution, wherein the solution is green, titrating with 0.01mol/L hydrochloric acid until the solution becomes blue-purple, and does not fade within half a minute, reaching the titration end point, recording the volume of the used hydrochloric acid, and adding NH3HCl is used to calculate the molar quantity n of ammonia gas generated.
2. Measuring CO2Amount of (2)
(1) Get and absorb CO2Adding 1 drop of phenolphthalein indicator into the supernatant of the barium hydroxide solution, titrating the solution with 0.05mol/L hydrochloric acid until the solution is pink and does not fade within half a minute, and recording the volume of the hydrochloric acid used as the end point of titration2When the barium hydroxide is about 2HCl, the molar quantity n1 can be calculated. (2) The amount of the original barium hydroxide is N, the amount of the unconsumed barium hydroxide is N1, and then the carbon dioxide is reacted with CO2The amount of reacted barium hydroxide is N-N1 as CO2～Ba(OH)2The amount of carbon dioxide generated can be calculated as N-N1.
The identification method of the nitrogen-retaining strain comprises the following steps:
1. physiological and biochemical characterization
And (3) streaking and inoculating the nitrogen-retaining strain J3 obtained by re-screening into a high-temperature bacterium solid culture medium, culturing at 55 ℃ for 36h, observing colony morphology by naked eyes, and observing the bacterium morphology by a microscope, wherein the details are shown in Table 1 and figure 2.
TABLE 1 colony and thallus morphology characteristics
The physiological and biochemical indexes of the strain are identified, and specific results are shown in table 2.
TABLE 2 physiological and biochemical characteristics of the strains
| Substrate composition | 48h | Substrate composition | 48h | |
| Mannitol | + | Salicin | + | |
| Erythritol | + | D-Cellobiose | + | |
| D-arabinose | + | D-maltose | + | |
| L-arabinose | + | D-lactose | + | |
| D-ribose | + | D-melibiose | + | |
| D-xylose | + | D-sucrose | + | |
| L-xylose | + | D-trehalose | + | |
| D-adonitol | + | Inulin powder | + | |
| Methyl-beta D xylopyranosides | + | D-melezitose | + | |
| D-galactose | + | D-raffinose | + | |
| D-glucose | + | Starch | + | |
| D-fructose | + | Glycogen | + | |
| D-mannose | + | Xylitol, its preparation method and use | + | |
| L-sorbose | + | D-gentiobiose | + | |
| L-rhamnose | + | D-Talinum sugar | + | |
| Dulcitol | + | D-lyxose | + | |
| Inositol | + | D-tagatose | + | |
| Mannitol | + | D-fucose | + | |
| Sorbitol | + | L-fucose | + | |
| Methyl-alpha D-mannopyranoside | + | D-arabitol | + | |
| Methyl-alpha D-glucopyranoside | + | L-arabitol | + | |
| N-acetylglucosamine | + | Potassium gluconate | + | |
| Amygdalin | + | 2 Keto Potassium gluconate | - | |
| | + | 5 Keto-Glucose Potassium | + | |
| Esculin ferric citrate | + |
The growth characteristics of the strain and the growth curve under the optimal growth conditions were determined, and the specific results are shown in Table 3 and FIG. 3.
TABLE 3 growth characteristics of the strains
2. Molecular identification
Extraction of PCR template DNA
Inoculating the nitrogen-retaining strain into a 250mL conical flask containing 50mL of high-temperature bacteria liquid culture medium, culturing at the temperature of 55 ℃ at 150r/min for 36h to obtain thallus cells, and extracting the total genomic DNA by using the bacterial genomic DNA extraction kit.
② PCR amplification
Primer:
Primer A:5’-AGAGTTTGATCCTGGCTCAG-3’
Primer B:5’-TACGGCTACCTTGTTACGACTT-3’
the total volume of the PCR reaction system is 25 mu L, and the PCR amplification condition is 94 ℃ for 5 min; 94 ℃ for 45s, 56 ℃ for 45s, 72 ℃ for 45s, 30 cycles; 10min at 72 ℃.
Sequence determination
The PCR amplification product is detected by electrophoresis, purified and sequenced, and the sequence is shown as SEQ ID NO. 1. The obtained sequences were analyzed by comparison at NCBI and EzBioCloud websites, and the results are shown in FIG. 4, the 16S rRNA gene sequence of the strain J3 of the present invention and the model bacterium Aneurinibacillus thermoaerophilus DSM 10154TThe homology was highest and the similarity was 99.86%. Therefore, the strain J3 obtained by screening is a thermophilic aerophilis thiamine-decomposing bacillus Aneurinibacillus thermoaerophilus with ammonia oxidation capability.
Example 2
The embodiment provides a composite microbial inoculum comprising the nitrogen-preserving strain provided in the embodiment 1 and a preparation method thereof, and the preparation method comprises the following steps:
the composite microbial inoculum comprises a rotting strain and a nitrogen-preserving strain, wherein the rotting strain can degrade organic nitrogen into inorganic ammonia nitrogen in a mesophilic stage, the nitrogen-preserving strain further promotes ammonium nitrogen to be converted into nitrate nitrogen in a high-temperature stage, and the rotting-promoting and nitrogen-preserving effects are cooperatively exerted.
The decomposed bacteria are conventional compost bacteria bacillus subtilis and purchased from China center for culture Collection of industrial microorganisms (CICC); the nitrogen-retaining strain is the strain obtained by screening in example 1, i.e., Bacillus thermoaminolyticus (Aneurinibacillus thermoaerophilus J3).
(1) Preparation of decomposed fungus powder
Preparing a decomposed bacterium seed solution: the glycerol tube strain is stored in a refrigerator (-80 ℃), inoculated into 3mL LB culture medium (containing 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl) by aseptic technique, and cultured for 24h at 30 ℃ and 180r/min under shaking to prepare a fresh strain. Inoculating activated fresh strain 1mL in shaking flask LB liquid medium at sterile condition with a loading of 100mL/500mL, shake culturing at 30 deg.C and 220r/min for 24 hr until viable bacteria concentration is 1 × 109cfu/mL to obtain seed bacterial liquid.
Culturing in a fermentation tank: the culture medium in the fermentation tank is filled in an amount of 50% of the total volume, the seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 5% (volume percentage), the fermentation temperature is 30 ℃, the dissolved oxygen amount is controlled at 10%, the stirring speed is 180r/min, the tank pressure is kept at 0.03-0.05MPa, and the air displacement and the tank pressure are properly adjusted according to the foam condition after inoculation to prevent liquid escape. Sampling and performing microscopic examination once every 6 h; the fermentation end point is that the number of spores accounts for more than 90 percent of the total number of bacteria; the total number of bacteria was determined by plate counting. The bacteria content of the fermentation liquid is 9.5 × 109. Culture medium in fermenter: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl, pH 7.0.
Spray drying the obtained fermentation liquor: the inlet temperature is 190 deg.C, the outlet temperature is 80 deg.C, spray carrier soluble starch is added, the addition amount is 5%, and the effective viable count is about 3.8 × 109cfu/g of bacillus subtilis powder.
(2) Preparation of azotobacteria powder
Preparing a nitrogen-preserving strain seed solution: storing glycerol tube strain in refrigerator at (-80 deg.C), and performing aseptic techniqueInoculating into 3mL of fresh high-temperature bacteria liquid culture medium, and standing at constant temperature of 55 deg.C for 24h to obtain fresh strain. Inoculating 5mL of activated fresh strain into shake flask high temperature bacteria liquid culture medium with a loading of 100mL/500mL, standing at 55 deg.C for 24 hr until viable bacteria concentration is 1.0 × 108cfu/mL to obtain seed bacterial liquid.
Culturing nitrogen-retaining bacteria in a fermentation tank: the culture medium in the fermentation tank is filled by 50 percent of the total volume, the seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 5 percent (volume percentage), the fermentation temperature is 55 ℃, the dissolved oxygen is 1 to 5 percent, the tank pressure is kept at 0.03 to 0.05MPa, and the air displacement and the tank pressure are properly adjusted according to the foam condition after inoculation to prevent liquid escape. Sampling and performing microscopic examination once every 6 hours; the fermentation end point is that the number of spores accounts for more than 90 percent of the total number of bacteria. The total number of bacteria was determined by plate counting. The number of bacteria contained in the fermentation liquid is 1.5 × 109cfu/ml. Culture medium in fermenter: starch 10.0g/L, bean cake powder 10.0g/L, KH2PO4 1.0g/L，Na2HPO4 0.5g/L、 (NH4)2SO4 0.50g/L，MgSO4·7H2O 0.25g/L，CaCl2 0.01g/L，pH7.0。
Spray drying the obtained fermentation liquor: the inlet temperature is 190 deg.C, the outlet temperature is 80 deg.C, spray carrier soluble starch is added to obtain effective viable count of 6.0 × 108cfu/g of thermophilic aerophilic thiamine-decomposing bacillus powder.
(3) Preparation of complex microbial inoculum
Mixing azotobacteria powder and decomposed bacteria powder according to the mass ratio of 1:3 to obtain powder product with bacteria content of 2.8 × 109cfu/g or more. The microbial agent product is yellowish powder, has no odor, and can be stored in a cool and dry place with a validity period of 1 year.
Example 3
The embodiment provides the application effect of the complex microbial inoculum prepared in the embodiment 2 in pig manure biogas residue compost, and concretely comprises the following steps,
(1) the weight ratio of the materials is as follows: the amount of the microbial inoculum is 0.1 percent of the total mass of the biogas residues and the wood chips (the conditioner) (the composite microbial inoculum in the example 2), and the basic properties of the materials are shown in the following table 4.
TABLE 4 basic Properties of the compost Material
| Test materials | Water content (%) | pH | Organic C (g/kg) | All N (g/kg) | C/N |
| Wood chip | 59.94％ | -- | 478.79 | 1.91 | 250.67 |
| Biogas residue | 70.73％ | 7.92 | 211.10 | 23.34 | 9.04 |
(2) The activation process of the microbial inoculum: taking 50kg of mixed compost materials, dissolving the microbial inoculum in 5L of water, adding 1g of glucose,and mixing the solution with the mixed compost materials, and standing for 24 hours, wherein the number of the bacterial strains in the compost body is amplified.
(3) And (3) biogas residue composting process: biogas residues generated by fermentation of pig manure biogas provided by Jiangxi province ecological engineering company are used as compost raw materials, wood chips are used as a conditioner, C/N to 21 of the materials are regulated and controlled, activated microbial inoculum is mixed into a compost, and meanwhile compost treatment without the microbial inoculum is set as a control group. The composting season is 7 months in summer, the total composting time is 45 days, the pile is turned once every 2 days, the sampling time is 4 days, and the pile is turned after sampling each time. Two rectangular composting pools with the same size are constructed in a composting field, and the two rectangular composting pools are 300cm long, 180cm wide and 150cm high. Taking a solid sample once every four days, taking the solid sample from the upper, middle and lower parts of the stack body, and uniformly mixing the solid sample to be used as a sample for measuring total soluble nitrogen (DTN) and ammonia Nitrogen (NH)4 +) Nitrate radical (NO)3 -)。
(4) The test results are as follows:
influence of microbial inoculum on heap temperature
In aerobic composting, temperature is a key factor affecting microbial activity and the composting process. Pathogenic bacteria can be killed at high temperature, organic matters are degraded fastest in a proper temperature range, and the composting speed and the composting quality are determined by the temperature of a compost body. The high temperature period characteristics and the temperature trend of each treatment group are shown in table 5 and fig. 5. The temperature change of different treatment groups is roughly divided into 3 stages, namely a temperature rise stage, a high temperature stage and a temperature drop stage. The temperature change trends of the test groups are basically the same, but the holding time is different at the temperature of more than 50 ℃, the temperature of the inoculation treatment group can be continuously raised to the maximum temperature of 67.1 ℃, the high temperature duration is 37 days, and the temperature is far higher than that of the control group. In addition, the high-temperature initial period of the inoculation treatment group is 1 day, and the high-temperature initial period of the control group CK is 2 days, which shows that the inoculation of the compost nitrogen-retention microbial inoculum can quickly heat the compost and shorten the time for reaching the high-temperature period. As can be shown above, the addition of the microbial agent can accelerate the temperature rise of compost, increase the reaction temperature of the compost, prolong the duration time of high temperature, and effectively promote the decomposition of the compost, thereby accelerating the decomposition of organic matters in the compost.
TABLE 5 high temperature period (above 50 ℃) characteristics of the composting group
Influence of microbial inoculum on the number of microorganisms in the heap
The high-temperature stage is an important main stage of composting and is also the most main stage of massive volatilization of ammonia, and common ammonia oxidizing bacteria cannot survive in the center of the high-temperature compost, so that a large amount of nitrogen is volatilized in the form of ammonia. The microbial count of the microbial inoculum treated group at the high temperature stage of the compost is higher than that of the control group, wherein the microbial count is about 100 times of that of the control group, which shows that the thermophilic nitrogen-retaining microbial strain can effectively survive and reproduce in the compost, and the microbial inoculum treatment obviously increases the bacterial count at the high temperature stage, promotes the ammonia oxidation capability, reduces the loss of nitrogen, improves the total nitrogen content in the compost, and effectively promotes the deodorization and nitrogen retention of the compost.
TABLE 6 microbial counts in the high temperature period (above 60 ℃) of the composting group
| Treatment of | Number of bacteria (cfu/g) | Number of Actinomycetes (cfu/g) | Number of fungi (cfu/g) |
| Biogas residue, wood chips and microbial agent | 7.15×109 | 3.35×105 | 3.26×103 |
| Biogas residue and wood chips | 6.20×107 | 2.18×105 | 2.67×103 |
Influence of microbial inoculum on heap nitrogen change
As can be seen from Table 7, the increase rate of the total nitrogen content of the inoculation group at the end of composting is 18.54 percent respectively, and the increase rate of the total nitrogen content of the control group is 4.10 percent; inoculation group NH4+N loss as low as 22.00%; NO of inoculation group and control group3N has a growing trend, wherein the growth rate of the inoculation group is as high as 52.2 percent, and the growth rate of the CK group is 26.19 percent. From the data, the inoculated deodorization microbial agent can effectively reduce the loss of nitrogen, improve the total nitrogen content in the compost and effectively promote the deodorization and nitrogen retention of the compost.
TABLE 7 variation of nitrogenous substances in different treatments before and after composting
Example 4
The embodiment provides a nitrogen-retaining strain applied to ammonia nitrogen sewage purification.
The pig raising wastewater produced by the water-washed manure process of a certain pig raising plant in the west of the river is characterized by COD: 3000mg/L, ammonia nitrogen 1000mg/L, TP 40mg/L, TN 1500mg/L, pH 8.0. Inoculating nitrogen-retaining strain J3 into liquid culture medium of thermophilic bacteria, and culturing to logarithmAfter the end, the thalli are collected centrifugally, and a little sterile water is added into the thalli to prepare a bacterial suspension. Putting 100mL of pretreated and sterilized pig farm wastewater into a 500mL triangular flask, and adding 10g/L, KH g of glucose2PO42.0g/L and K2HPO42.0g/L, and adjusting the pH value to 7.5; adding the bacterial suspension to make the cell density be 1.0X 107CFU/mL, and meanwhile, the pig farm wastewater without bacteria is set as a blank control group CK. And (2) after inoculation, performing static culture in an incubator at 55 ℃, and detecting the ammonia nitrogen concentration in the wastewater every 1d (refer to 'national standard water ammonia nitrogen determination (GB7479-87) — Nashi reagent spectrophotometry'), the nitrite nitrogen concentration (refer to 'national standard water nitrite nitrogen determination (GB7493-87) — spectrophotometry'), and the nitrate nitrogen concentration determination refer to 'national standard water nitrate nitrogen determination (GB7480-87) — phenoldisulfonic acid spectrophotometry').
The experimental results show that the thallus cells grow rapidly and metabolize actively, and the total ammonium Nitrogen (NH) of the wastewater is compared with the non-inoculated control 4 days after the inoculation treatment4 +-N) concentration decrease of 92.5%, nitrite Nitrogen (NO)2 --N) concentration increased 1.36 times, nitrate Nitrogen (NO)3 -The concentration of-N) is increased by 2.9 times, which shows that the microbial inoculum has obvious ammonia oxidation effect and can efficiently convert ammonium nitrogen in wastewater into nitrite nitrogen and nitrate nitrogen.
TABLE 8 removal rate (%) of nitrogen-protecting bacteria to ammonia nitrogen in pig farm wastewater
| Treatment of | 1d | 2d | 3d | 4d |
| Treatment with microbial inoculum | 60.7％ | 64.2％ | 70.4％ | 92.5％ |
| Blank control | 5.5％ | 6.1％ | 8.6％ | 11.2％ |
TABLE 9 increase ratio (%)% of the nitrogen-conserving agent to nitrite nitrogen in wastewater from pig farm
| Treatment of | 1d | 2d | 3d | 4d |
| Treatment with microbial inoculum | 32.0％ | 46.9％ | 121.27％ | 136.16％ |
| Blank spaceControl | 2.7％ | 2.6％ | 4.0％ | 6.1％ |
TABLE 10 increase ratio (%)% of nitrogen-retaining agent to nitrate nitrogen in wastewater from pig farm
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
SEQUENCE LISTING
<110> institute of microbiology of academy of sciences of Jiangxi province
<120> nitrogen-retaining strain, complex microbial inoculum, preparation method and application of complex microbial inoculum
<130> NHA201900280
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1475
<212> DNA
<213> thermophilic aerophilic thiamine-decomposing bacillus
<400> 1
tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcgaaccg atggagtgct 60
tgcattcctg aggttagcgg cggacgggtg agtaacacgt aggcaacctg cctgtacgac 120
cgggataact ccgggaaacc ggagctaata ccggatagga tgccgaaccg catggttcgg 180
catggaaagg cctttgagcc gcgtacagat gggcctgcgg cgcattagct agttggtggg 240
gtaacggcct accaaggcga cgatgcgtag ccgacctgag agggtgaacg gccacactgg 300
gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360
gaaagtctga cggagcaacg ccgcgtgagt gaggaaggtc ttcggatcgt aaaactctgt 420
tgtcagggaa gaaccgccgg gatgacctcc cggtctgacg gtacctgacg agaaagcccc 480
ggctaactac gtgccagcag ccgcggtaat acgtaggggg caagcgttgt ccggaattat 540
tgggcgtaaa gcgcgcgcag gcggcttctt aagtcaggtg tgaaagccca cggctcaacc 600
gtggagggcc atctgaaact ggggagcttg agtgcaggag aggagagcgg aattccacgt 660
gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcgg ctctctggcc 720
tgtaactgac gctgaggcgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780
ccacgccgta aacgatgagt gctaggtgtt ggggagtcca cctcctcagt gccgcagcta 840
acgcaataag cactccgcct ggggagtacg gccgcaaggc tgaaactcaa aggaattgac 900
ggggacccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960
cagggcttga catcccgctg acccctccag agatggaggc ttccttcggg acagcggtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttgtcctttg ttgccagcat tcagttgggc actctaagga gactgccgtc 1140
gacaagacgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg tcctgggcta 1200
cacacgtgct acaatggacg gtacaacggg cgtgccaacc cgcgagggtg agccaatccc 1260
taaaaaccgt tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag ccggaatcgc 1320
tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggtcttgta cacaccgccc 1380
gtcacaccac gagagtttgc aacacccgaa gtcggtgagg taaccttctg gagccagccg 1440
ccgaaggtgg ggcagatgat tggggtgaag tcgta 1475
Claims (10)
1. The nitrogen-protecting strain is characterized by being thermophilic heterotrophic ammonia oxidizing bacteria, the preservation organization is Guangdong province microbial strain preservation center, the classification is named Aneurinibacillus thermoaerophilus J3, the preservation number is GDMCC No. 61602, and the preservation date is 2021, 4 and 12 days.
2. A complex microbial inoculant comprising the nitrogen-retaining strain according to claim 1.
3. The complex microbial inoculant according to claim 2, wherein the complex microbial inoculant comprises azotobacter strain powder and decomposed microbial inoculant powder;
the mass ratio of the nitrogen-retaining strain-containing bacterial powder to the decomposed bacterial powder is 1: (2.5-3.5);
the decomposed bacterium powder is bacillus subtilis and/or bacillus licheniformis;
the content of the decomposed bacteria powder is 2.6 multiplied by 109-5.0×109cfu/g；
The bacterium content of the bacterium powder containing the nitrogen-retaining strain is 4.5 multiplied by 108-7.5×108cfu/g。
4. The composite microbial inoculum of claim 3, wherein the raw materials of the powder containing the nitrogen-retaining strain comprise a culture medium substrate and growth factors for growth and fermentation of the nitrogen-retaining strain;
the culture medium matrix is at least one of starch, bean cake powder, bran, cassava residue and bean pulp;
the growth factor comprises at least one of a carbon source, a nitrogen source and an inorganic salt;
the mass ratio of the culture medium substrate to the growth factors is (20-35): (3-5).
5. The complex microbial inoculum according to claim 3 or 4, which has a bacterial content of 1.9 x 109-3.8×109cfu/g。
6. A method for preparing a composite bacterial agent according to any one of claims 2 to 5, which comprises fermenting the nitrogen-retaining strain according to claim 1.
7. The preparation method of the composite microbial inoculum according to claim 6, wherein the powder of the nitrogen-retaining strain is obtained by mixing a culture medium substrate, a growth factor and the nitrogen-retaining strain, fermenting and drying;
mixing the powder of the nitrogen-retaining strain and the powder of the decomposed bacteria to obtain the composite microbial inoculum;
optionally, the fermentation conditions are that the fermentation temperature is 50-55 ℃, the pressure is 0.03-0.05MPa, and the low dissolved oxygen is 1-5%.
8. The nitrogen-retaining strain of claim 1, the composite microbial inoculum of any one of claims 2 to 5 or the composite microbial inoculum prepared by the method of any one of claims 6 to 7 has the following application:
(1) application in preparing compost;
(2) the application in the field of ammonia nitrogen oxidation;
(3) the method is applied to ammonia nitrogen sewage purification.
9. A composting method, which is characterized in that composting raw materials are composted by using the nitrogen-retaining strain of claim 1, the complex microbial inoculum of any one of claims 2 to 5 or the complex microbial inoculum prepared by the method of any one of claims 6 to 7;
optionally, the amount of the composite microbial inoculum is 0.05-0.15% of the total mass of the raw materials and the conditioner;
optionally, the composting time is 40-50 days, and the pile is turned once every 2-3 days;
optionally, fresh pig manure biogas residues are used as a composting raw material, wood chips are used as a conditioner, and the C/N of the material is regulated to be 15-30.
10. A method for purifying ammonia nitrogen sewage is characterized in that the ammonia nitrogen sewage is inoculated with the nitrogen-protecting strain of claim 1, the composite microbial inoculum of any one of claims 2 to 5 or the composite microbial inoculum prepared by the method of any one of claims 6 to 7 for culture, and the ammonia nitrogen sewage is purified;
optionally, the temperature of the culture is 52-57 ℃.
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