CN113621546A - Aerobic denitrification strain and preparation and application of microbial inoculum thereof - Google Patents
Aerobic denitrification strain and preparation and application of microbial inoculum thereof Download PDFInfo
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Abstract
The aerobic denitrifying bacteria and the microbial inoculum thereof are prepared by screening new strains of acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38, respectively pre-culturing and propagating, then performing vacuum freeze drying to obtain corresponding single bacterium powder, mixing the single bacterium powder, and then mixing the mixed powder with an aerobic denitrifying bacteria extracellular polysaccharide crude product and auxiliary materials. The acinetobacter YTLJ-N-S10 and the bacillus YTLJ-N-B38 are preserved in Guangdong province microbial strain preservation center, and the preservation numbers are GDMCC No: 61690 and GDMCC No: 61774. the strain and the microbial inoculum thereof can enhance the heterotrophic nitrification and aerobic denitrification of water, have the effect of inhibiting pathogenic bacteria, and can be used for purifying various sewage and enhancing the removal of nitrogen pollutants.
Description
Technical Field
The invention relates to the technical field of environmental protection, in particular to an aerobic denitrification strain and preparation and application of a microbial inoculum thereof.
Background
Factory breeding is an important mode of modern fishery breeding. The influence of the large amount of the culture wastewater on the water quality of offshore culture water areas is great, and the problems of water environment pollution, frequent aquatic diseases and the like caused by large-scale industrial culture seriously trouble and obstruct the development of the aquaculture industry in China. The in-situ purification treatment technology of the aquaculture water has the advantages of little ecological environment damage, little tail water discharge and the like, can protect the offshore ecological environment and can obtain high-yield and high-quality aquatic products. Therefore, the in-situ purification treatment of the aquaculture water body is one of the key development directions in the aquaculture field. With the rapid development of economy and the enhancement of human activities, environmental pollution has become an important factor limiting the development of coastal ecologically vulnerable areas. The circulating water culture is the inevitable choice for sustainable development of land-based mariculture in China. Therefore, the development of the aquaculture water in-situ advanced treatment and purification technology with low cost and simple operation has important significance and application value.
At present, the technology for treating and recycling the water of the aquaculture system is less, the treatment cost is higher, and the popularization and the application of the recirculating aquaculture technology are greatly limited. In recent years, the biological floc technology has certain application in the aquaculture industry. Through literature retrieval, the situation that dane can be used in the 'formation condition and action effect of biological flocs in a closed litopenaeus vannamei culture experiment' is found, the biological floc technology is introduced into a closed litopenaeus vannamei culture system, the ammonia nitrogen and nitrite nitrogen concentration of a water body is kept at a lower level, and the survival rate of the litopenaeus vannamei is over 80% (Fishery science progress, 2012, 33(2): 69-75); plum bin and the like in the aspects of' regulation and control of water quality by biological flocs and apostichopus japonicusApostichopus japonicus) Influence of growth of young stichopus japonicus' it is mentioned that biological floccules formed by adding carbon sources can not only purify water quality, but also promote the growth of young stichopus japonicus (Fishery science progress 2014, 46(1): 197-205). However, through research summary, the above technologies all add a large amount of organic carbon sources, only promote the ammonia nitrogen in the water to generate organic nitrogen through assimilation, but not remove nitrogen pollutants from the water, and the accumulation of ammonia nitrogen and nitrite is easily caused when the ventilation is insufficient, which limits the wide application of the technology in the field of water treatment.
Disclosure of Invention
The invention aims to provide an aerobic denitrification strain, a microbial inoculum thereof and application of the aerobic denitrification strain and the microbial inoculum for purifying water, aiming at the defects of the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
an aerobic denitrifying strain is Acinetobacter YTLJ-N-S10 and/or Bacillus YTLJ-N-B38;
the acinetobacter YTLJ-N-S10 is preserved in Guangdong province microorganism strain preservation center, the preservation address is Guangzhou, China, the preservation date is 2021, 7 and 5 days, and the preservation number is GDMCC No: 61690, classification nameAcinetobactersp.。
The bacillus YTLJ-N-B38 is preserved in Guangdong province microorganism strain preservation center, the preservation address is Guangzhou, the preservation date is 2021, 7 and 5 days, and the preservation number is GDMCC No: 61774, classification nameBacillus sp.。
The Acinetobacter YTLJ-N-S10 and the bacillus YTLJ-N-B38 have the aerobic denitrification function. Different from common denitrifying bacteria (anaerobic bacteria) which can only carry out denitrification under strict anaerobic conditions, the two bacterial strains are aerobic bacteria, have facultative anaerobic survival characteristics, can generate nitrogen gas through denitrification under aerobic conditions (dissolved oxygen is more than 2mg/L) and microaerobic conditions (dissolved oxygen is more than 0.1 mg/L), and remove nitrogen in water in the form of nitrogen gas.
Wherein the denitrification efficiency of the bacillus YTLJ-N-B38 under microaerobic conditions is higher than that of the acinetobacter YTLJ-N-S10. The salinity of the strain suitable for growth is 0.01-4%. The strains have a heterotrophic nitrification function, can remove ammonia nitrogen and organic pollutants through nitrification, and is suitable for the condition that C/N is 0.5-50.
The application of aerobic denitrification strains, namely the application of the acinetobacter YTLJ-N-S10 and/or the bacillus YTLJ-N-B38 in purifying water bodies in a mode of aerobic denitrification and organic pollutant removal.
The microbial inoculum for purifying the water body contains the aerobic denitrification strain.
The microbial inoculum contains symbiotic microorganisms of acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38.
The aerobic denitrification microbial inoculum (Acinetobacter YTLJ-N-S10 and Bacillus YTLJ-N-B38) not only exerts the aerobic denitrification effect together, but also has complementary advantages to form an aerobic denitrification microbial symbiotic system. The acinetobacter YTLJ-N-S10 generates a zoogloea and promotes the formation of a biological film rich in the bacillus YTLJ-N-B38 so as to build a local micro-aerobic environment to strengthen the aerobic denitrification, particularly the micro-aerobic denitrification effect, of the bacillus YTLJ-N-B38 and also generate extracellular polysaccharide with disease-resistant bacteria (such as vibrio); the bacillus YTLJ-N-B38 can generate cyclopeptide quorum sensing signal molecules while performing aerobic denitrification, promote the colonizing and propagation of the added functional strains in a treatment system and strengthen the denitrification function, and in addition, the bacillus YTLJ-N-B38 has strong degradation capability on macromolecular organic matters and can generate small-molecular organic acids to supply acinetobacter YTLJ-N-S10 as a carbon source.
The microbial inoculum is prepared by mixing two symbiotic strains of acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38 in a ratio of 10-20: 1-10 (mass ratio), mixing the mixed bacteria with an aerobic denitrifying bacteria extracellular polysaccharide crude product and auxiliary materials, wherein the total effective viable count of the microbial inoculum is more than or equal to 1.0 multiplied by 109one/mL.
The aerobic denitrifying bacteria extracellular polysaccharide crude product is obtained by carrying out alcohol precipitation on an expanding propagation culture filtrate of acinetobacter YTLJ-N-S10 and then secreting the expanding propagation culture filtrate.
The method specifically comprises the following steps:
1) and (3) strain propagation: respectively inoculating acinetobacter YTLJ-N-S10 and Bacillus YTLJ-N-B38 into a proliferation culture medium containing antibiotics, culturing at 28 deg.C for 48-72 hr, separating and collecting precipitated bacteria;
2) preparing compound bacteria dry powder: freezing and drying the single acinetobacter YTLJ-N-S10 and the single bacillus YTLJ-N-B38 obtained by propagation, crushing to respectively obtain single strain dry powder, and mixing the acinetobacter YTLJ-N-B38 dry powder and the bacillus according to a mass ratio of 10-20: 1-10;
3) preparing a crude product of the aerobic denitrifying bacteria exopolysaccharide: adding 2-4 times volume of 95% ethanol into the filtrate obtained by separating the propagation culture solution of acinetobacter YTLJ-N-S10, standing to fully precipitate extracellular polysaccharide, washing to obtain crude product of aerobic denitrifying bacteria extracellular polysaccharide, and drying for later use;
4) preparing a complex microbial inoculum:
(1) mixing the prepared composite bacteria dry powder with the crude product of the aerobic denitrifying bacteria extracellular polysaccharide to obtain a mixture, wherein the mixing ratio is 100: 0.1-1 (mass ratio);
(2) 200-200 by mass ratio: 1-10: 1-10 mixing nitrogen-rich biochar (nitrogen content is more than 1%), brown sugar and peptone to obtain auxiliary materials; and (3) adding auxiliary materials into the mixture obtained in the step (1) according to the proportion of 1-10% to obtain the composite microbial inoculum.
The addition amount of the antibiotic in the propagation culture medium containing the antibiotic is 0.01-0.05% of the mass of the propagation culture medium;
the propagation culture medium is 3-5% K2HPO4·3H2O、1-3% NaCl、1-3% MgSO4·7H2O、0.05-0.5%NH4NO4、0.01-0.04% MnSO4·H2O、0.01-0.05% FeSO4·7H2O, 0.5-1.5% sodium succinate, 0.01-0.1% NaNO2、0.001-0.005% (NH4)2MoO4、0.001-0.005% CoCl2、0.001-0.005% Y(NO3)3。
The antibiotic is a mixture of tetracycline antibiotics, sulfonamide antibiotics, quinolone antibiotics and macrolide antibiotics, and the mass ratio of the antibiotics is 1: 1: 1: 1.
the nitrogen-rich biochar generally refers to various biochar with the nitrogen content higher than 1%.
The nitrogen-rich biochar generally refers to various biochar with nitrogen content higher than 1%, such as seaweed biochar, crab shell biochar, fecal residual bait biochar or high-nitrogen-content biochar prepared by doping nitrogen in a biomass carbonization process. Typically, the nitrogen content of biochar produced from terrestrial plant biomass is less than 1%.
The aerobic denitrifying bacteria exopolysaccharide is obtained by coarse purification of a large amount of exopolymers generated by a strain (Acinetobacter YTLJ-N-S10) in the propagation expanding culture process, has the effect of inhibiting various pathogenic bacteria, and can improve the immunity of aquatic animals after being ingested. 0.01-0.05% of antibiotic is added into a culture medium for strain propagation to stimulate the strain to generate aerobic denitrifying bacteria extracellular polysaccharide. After bacteria are harvested, fermentation liquor for propagation of the aerobic denitrification strains is mixed with 95% ethanol with 3 times of volume, standing is carried out to fully precipitate exopolysaccharide, the precipitate is washed with 95% ethanol and anhydrous acetone to remove impurities, a crude exopolysaccharide product is obtained after freeze drying, and the crude exopolysaccharide product (the content is more than 30%) of the aerobic denitrification strains is prepared by mixing and stored in a drying place for later use.
Antibiotic (0.01-0.05%) is added into the strain propagation culture medium to stimulate the strain to secrete aerobic denitrifying bacteria exopolysaccharide, and the survival capability of the strain in a sewage treatment system can be improved by antibiotic tolerance exercise. The antibiotic is a mixture of tetracycline antibiotics, sulfonamide antibiotics, quinolone antibiotics and macrolide antibiotics, and the mass ratio of the antibiotics is 1: 1: 1: 1; the tetracycline antibiotics refer to tetracycline, oxytetracycline, doxycycline and the like, the sulfonamide antibiotics refer to sulfadiazine, sulfamethoxazole, sulfamethoxydiazine and the like, the quinolone antibiotics refer to ofloxacin, ciprofloxacin, enrofloxacin and the like, and the macrolide antibiotics refer to erythromycin, clarithromycin, roxithromycin and the like.
The application of the microbial inoculum for purifying the water body is to remove nitrogen and organic pollutants in the water body.
The composite microbial inoculum is put into a sewage treatment system according to the mass ratio of 1-10% to remove organic pollutants, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and the like. The inoculation amount of the composite microbial inoculum is 1-10% (mass ratio), the composite microbial inoculum has good removal effect on nitrogen in various sewage under aerobic conditions, and the effective removal rate is more than 80%.
The invention has the advantages that:
the invention can thoroughly remove nitrogen from water to generate a special nitrogen metabolism function of nitrogen by virtue of aerobic denitrifying bacteria under an aerobic condition, realizes advantage complementation by mixing a plurality of newly discovered strains, develops an aerobic denitrifying microbial inoculum, is applied to the enhanced nitrogen removal of wastewater, particularly aquaculture water, promotes most nitrogen pollutants in sewage treatment, particularly aquaculture systems, to be converted into nitrogen for removal, and has important practical significance for enhancing the removal efficiency of nitrogen in wastewater.
1) The strains in the composite strain of the invention are Acinetobacter YTLJ-N-S10 (respectively)Acinetobactersp, YTLJ-N-S10) and Bacillus YTLJ-N-B38 (B)Bacillussp, YTLJ-N-B38). Acinetobacter YTLJ-N-S10 can produce extracellular polysaccharide, the yield of the extracellular polysaccharide can reach 0.5 g/g of cell dry matter, and the extracellular polysaccharide has the functions of aerobic denitrification and heterotrophic nitrification, and can remove macromolecular organic pollutants while removing nitrogen pollutants. The bacillus YTLJ-N-B38 has high denitrification efficiency under microaerobic conditions, and the cyclic peptide secreted by the bacillus YTLJ-N-B38 can promote the reproduction of symbiotic bacteria.
2) The compound bacterial agent obtained by the invention is prepared by mixing compound strains separated from a culture system, is easy to colonize and propagate in the culture system after being used, and ensures higher biomass in the environment. Has the economic and ecological benefits of less usage of microbial inoculum, wide strain source and no secondary pollution.
3) The microbial inoculum is a composite flora composed of multiple strains, each strain can form an aerobic denitrification microbial community with complementary advantages, the microbial inoculum has stable property, has no strict requirements on the pH, temperature, salinity and oxygen content of a feeding environment, can furthest retain the functional activity of the microorganisms in the in-situ pollution treatment process, realizes the simultaneous removal of organic pollutants, nitrogen and other nutrient elements, and belongs to a multipurpose environment-friendly microbial inoculum.
Drawings
FIG. 1 is FTIR spectrum of exopolysaccharide obtained from strain preparation provided by the embodiment of the invention.
Detailed Description
The present invention is further illustrated by the following examples, which, however, are not intended to limit the scope of the invention.
The invention utilizes the mutual cooperation of two specific strains, ammonia nitrogen or organic nitrogen can be firstly oxidized in the presence of oxygen to form nitrite nitrogen or nitrate nitrogen, and then nitrate or nitrite is converted into nitrogen gas through denitrification to be removed. Removing organic matters while removing nitrogen; the strain and the microbial inoculum thereof can enhance the heterotrophic nitrification and aerobic denitrification of water, have the effect of inhibiting pathogenic bacteria, and can be used for purifying various sewage and enhancing the removal of nitrogen pollutants.
Example 1
Acinetobacter YTLJ-N-S10 (Acinetobactersp, YTLJ-N-S10) and identification thereof
1. Isolation of the Strain
Taking tail water of a certain seawater fish culture system in coastal sea as stock solution, inoculating to sterilized propagation culture medium according to 3% inoculation amount, culturing at 30 ℃ for 3-7 days, performing dilution culture in LB culture medium, selecting single colony, performing streak separation on agar solid culture medium to obtain purified strain YTLJ-N-S10 (aAcinetobacter sp. YTLJ-N-S10）。
2. Identification of strains
2.1 morphological identification
The strain separated and purified in the step 1 is analyzed and compared with Acinetobacter.
2.2, 16S rDNA sequence homology analysis
And (2) amplifying the 16S rDNA fragment of the strain obtained in the step (1) by adopting a colony PCR method, amplifying the 16S rDNA gene fragment, and cloning and sequencing to show that the 16S rDNA of the strain has a nucleotide sequence of a sequence 1 in a sequence table. The similarity between the 16S rDNA of the strain and the 16S rRNA gene of Acinetobacter (Acinetobacter sp) reaches more than 90 percent.
The 16S rDNA gene sequence is:
CGAGCGGAGAGAGGTAGCTTGCTACTGATCTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTTAGTTAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTAGCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCAGCGAGTAATGTCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCTAGCGATAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGA
example 2
Bacillus YTLJ-N-B38 (Bacillussp, YTLJ-N-B38) and identification thereof
1. Isolation of the Strain
Inoculating the excrement residue of litopenaeus littoralis culture to sterilized propagation culture medium according to the inoculation amount of 1%, culturing for 5-7 days at 35 ℃, performing dilution culture in LB culture medium, selecting single colony, performing streak separation on agar solid culture medium to obtain purified strain YTLJ-N-B38 (aBacillus sp. YTLJ-N-B38）。
2. Identification of strains
2.1 morphological identification
And (3) analyzing and comparing the strain obtained by separation and purification in the step 1 with the bacillus.
2.2, 16S rDNA sequence homology analysis
And (3) amplifying the 16S rDNA fragment of the strain obtained in the step (1) by adopting a colony PCR method, amplifying the 16S rDNA gene fragment, and cloning and sequencing to show that the 16S rDNA of the strain has a nucleotide sequence of a sequence 2 in a sequence table. The similarity between the 16S rDNA of the strain and the 16S rRNA gene of Bacillus (Bacillus sp) reaches over 95 percent.
The 16S rDNA gene sequence is:
TCGAGCGGACAGAAGGGAGCTCGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAG
example 3
500 mL of simulated livestock and poultry breeding biogas slurry is placed in a conical flask, the ammonia nitrogen concentration of the simulated biogas slurry reaches 200 mg/L, the pH =7.06, the C/N ratio is 1, and the salinity is 0.2%. Adding pure Acinetobacter YTLJ-N-S10 bacterial liquid into the simulated culture biogas slurry according to the inoculation amount of 2%, and treating for 7 days at constant temperature of 30 ℃, wherein the ammonia nitrogen concentration is reduced to 36 mg/L, and the removal rate reaches 82%.
Adding the pure bacillus YTLJ-N-B38 bacterial liquid to the simulated food processing high-concentration organic wastewater according to the inoculation amount of 5%, wherein COD, ammonia nitrogen and nitrate nitrogen of the wastewater are 2000 mg/L, 40 mg/L and 5 mg/L respectively, dissolved oxygen is 0.5 mg/L, and the C/N ratio is 50. After the treatment for 14 days at the constant temperature of 28 ℃, the concentrations of COD, ammonia nitrogen and nitrate nitrogen are respectively reduced to 150 mg/L, 4mg/L and 0.5 mg/L, and the removal rates respectively reach 92.5 percent, 90.0 percent and 90.0 percent.
Then, the analysis of a titration method shows that volatile organic acid (micromolecule organic acid) (the concentration is 21 mg/L and is calculated by acetic acid) is generated in the wastewater after the treatment of the bacillus YTLJ-N-B38, the substances are not detected in the wastewater before the treatment, and the large molecular organic matters in the wastewater are degraded, and partial products are the micromolecule organic acid.
After further inoculating Acinetobacter YTLJ-N-S10 pure bacterial liquid (5%) to the treated wastewater containing the volatile organic acid, carrying out advanced treatment for 14 days, further reducing the concentrations of COD, ammonia nitrogen and nitrate nitrogen to 50, 1.0 and 0.2 mg/L, and not detecting the volatile organic acid (micromolecular organic acid), thereby confirming that the Acinetobacter YTLJ-N-S10 can utilize the micromolecular organic acid generated by the Bacillus YTLJ-N-B38 as a carbon source and carry out aerobic denitrification.
Adding 3% of pure bacillus YTLJ-N-B38 bacterial liquid into high-nitrogen wastewater according to the inoculation amount, wherein the nitrate concentration of the high-nitrogen wastewater reaches 300 mg/L, the pH value is =7.15, and the C/N ratio is 0.5. After the treatment for 5 days at 25 ℃, the concentration of the nitrate is reduced to 15 mg/L, and the removal rate reaches 95 percent; after 8 days of treatment, the nitrate concentration is reduced to 10 mg/L, the removal rate reaches 96.7 percent, the COD concentration is reduced to 18 mg/L from the initial 150 mg/L, and the removal rate reaches 88 percent.
Example 4
Preparing exopolysaccharide:
1) and (3) strain propagation: acinetobacter YTLJ-N-S10 was inoculated into a propagation medium containing antibiotics in an amount of 10%. Culturing at 28 deg.C for 48 hr, and separating and collecting the precipitated bacteria.
The addition amount of the antibiotic in the propagation culture medium containing the antibiotic is 0.01 percent of the mass of the culture medium.
The antibiotic is a mixture of tetracycline, sulfadiazine, ofloxacin and erythromycin, and the mixing mass ratio is 1: 1: 1: 1.
the propagation culture medium is 3 percent K2HPO4·3H2O、1% NaCl、1% MgSO4·7H2O、0.05%NH4NO4、0.01% MnSO4·H2O、0.01% FeSO4·7H2O, 0.5% sodium succinate, 0.01% NaNO2、0.001% (NH4)2MoO4、0.001% CoCl2、0.001% Y(NO3)3。
2) Preparing a crude product of the aerobic denitrifying bacteria exopolysaccharide: adding 95% ethanol with the volume 3 times that of the filtrate into the filtrate collected after the culture solution of the Acinetobacter YTLJ-N-S10 is separated after the propagation in the step 1), standing to ensure that extracellular polysaccharide in the filtrate is fully precipitated, washing the precipitate with 95% ethanol and anhydrous acetone to remove impurities, and freeze-drying to obtain an extracellular polysaccharide crude product (an FTIR spectrogram is shown in figure 1).
As can be seen from FIG. 1, the crude product thus prepared was found to be 1069 cm-1An absorption peak is nearby, the absorption peak is a characteristic absorption peak of the polysaccharide, and the crude extracellular polysaccharide is obtained.
Example 5
Preparing a complex microbial inoculum:
1) and (3) strain propagation: acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38 are respectively inoculated into a propagation medium containing antibiotics, and the inoculation amount is 10%. Culturing at 28 deg.C for 48 hr, separating, and collecting the precipitated bacteria.
The addition amount of the antibiotic in the propagation culture medium containing the antibiotic is 0.01 percent of the mass of the culture medium.
The antibiotic is a mixture of tetracycline, sulfadiazine, ofloxacin and erythromycin, and the mixing mass ratio is 1: 1: 1: 1.
the propagation culture medium is 5 percent K2HPO4·3H2O、3% NaCl、3% MgSO4·7H2O、0.5%NH4NO4、0.04% MnSO4·H2O、0.05% FeSO4·7H2O, 1.5% sodium succinate, 0.1% NaNO2、0.005% (NH4)2MoO4、0.005% CoCl2、0.005% Y(NO3)3。
2) Preparing compound bacteria dry powder: freezing and drying the single acinetobacter YTLJ-N-S10 and the single bacillus YTLJ-N-B38 obtained by propagation, grinding and crushing to obtain single strain dry powder, and mixing the single strain dry powder according to a certain ratio to obtain composite bacteria dry powder, wherein the ratio of the acinetobacter to the bacillus is 10: 1 (mass ratio).
3) Preparing a crude product of the aerobic denitrifying bacteria exopolysaccharide: adding 95% ethanol with the volume 3 times that of the filtrate into the filtrate collected after the culture solution of the Acinetobacter YTLJ-N-S10 is separated after the propagation in the step 1), standing to ensure that extracellular polysaccharide in the filtrate is fully precipitated, washing the precipitate with 95% ethanol and anhydrous acetone to remove impurities, freeze-drying to obtain an extracellular polysaccharide crude product (an FTIR spectrogram is shown in figure 1), mixing to obtain an aerobic denitrifying bacteria extracellular polysaccharide crude product (the content is more than 30%) and storing the aerobic denitrifying bacteria extracellular polysaccharide crude product in a drying place for later use.
4) Preparing a complex microbial inoculum:
(1) mixing the mixed bacteria dry powder prepared in the step 2) with the crude product of the aerobic denitrifying bacteria exopolysaccharide obtained in the step 3) to obtain the composite bacteria, wherein the ratio of the mixed bacteria dry powder to the aerobic denitrifying bacteria exopolysaccharide is 100: 0.1 (mass ratio).
(2) The auxiliary materials are 100 percent by mass: 1: 1 nitrogen-enriched biochar (nitrogen content > 1%), brown sugar and peptone.
(3) And adding the obtained auxiliary materials into the mixture of the mixed bacteria dry powder and the aerobic denitrifying bacteria extracellular polysaccharide according to the proportion of 1wt% to obtain the composite microbial inoculum. The nitrogen-rich biochar is seaweed biochar (the nitrogen content is 2 percent and is measured by an element analyzer), and the preparation method comprises the steps of placing seaweed biomass in a reaction kettle and carrying out hydrothermal carbonization for 3 hours at 180 ℃.
The complex microbial inoculum prepared in the embodiment is used for treating industrial aquaculture water:
the industrial aquaculture water to be treated is the tail water of a tilapia aquaculture system of an aquaculture plant, and the concentrations of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and COD respectively reach 0.6, 3, 0.4 and 10 mg/L.
The prepared microbial inoculum is put into an industrial circulating water culture system according to the mass ratio of 1 percent, the concentration of dissolved oxygen in water is 5 mg/L, after 10 days of treatment, the concentration of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and COD is respectively reduced to 0.11, 0.5, 0.04 and 1.5 mg/L, the removal rate is respectively 81.7 percent, 83.3 percent, 90 percent and 85 percent, the aerobic removal of nitrogen pollutants is realized, and organic pollutants are removed simultaneously.
Example 6
Preparing a complex microbial inoculum:
1) and (3) strain propagation: acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38 are respectively inoculated into a propagation medium containing antibiotics, and the amount of the seeds is 10 wt%. Culturing at 28 deg.C for 56 hr, separating, and collecting the precipitated bacteria.
The addition amount of the antibiotic in the propagation culture medium containing the antibiotic is 0.02 percent of the mass of the culture medium.
The antibiotic is a mixture of oxytetracycline, sulfamethoxazole, ciprofloxacin and clarithromycin, and the mixing mass ratio is 1: 1: 1: 1.
the propagation medium is the same as in the above example.
2) Preparing compound bacteria dry powder: freezing and drying the single acinetobacter YTLJ-N-S10 and the single bacillus YTLJ-N-B38 obtained by propagation, grinding and crushing to obtain single strain dry powder, and mixing the single strain dry powder according to a certain ratio to obtain composite bacteria dry powder, wherein the mixing ratio of the acinetobacter and the bacillus is 15: 5 (mass ratio).
3) Preparing a crude product of the aerobic denitrifying bacteria exopolysaccharide: adding 95% ethanol with the volume 3 times that of the filtrate into the filtrate collected after the culture solution of the Acinetobacter YTLJ-N-S10 is separated after the propagation in the step 1), standing to ensure that extracellular polysaccharide in the filtrate is fully precipitated, washing the precipitate with 95% ethanol and anhydrous acetone to remove impurities, freeze-drying to obtain an extracellular polysaccharide crude product, mixing to obtain an aerobic denitrifying bacteria extracellular polysaccharide crude product (the content is more than 30%) and storing the aerobic denitrifying bacteria extracellular polysaccharide crude product in a drying place for later use.
4) Preparing a complex microbial inoculum:
(1) mixing the mixed bacteria dry powder prepared in the step 2) with the crude product of the aerobic denitrifying bacteria exopolysaccharide obtained in the step 3) to obtain the composite bacteria, wherein the ratio of the mixed bacteria dry powder to the aerobic denitrifying bacteria exopolysaccharide is 100: 0.5 (mass ratio).
(2) The auxiliary materials are 150: 5: nitrogen-enriched biochar (nitrogen content > 1%), brown sugar and peptone.
(3) And adding the auxiliary materials into the mixture of the mixed bacteria dry powder and the aerobic denitrifying bacteria extracellular polysaccharide according to the proportion of 5wt% to obtain the composite microbial inoculum. The nitrogen-rich biochar is shrimp shell biochar (nitrogen content is 5%), and the preparation method comprises the step of carbonizing shrimp shell powder under the condition of 400 ℃ in an oxygen-deficient manner for 2 hours.
The shrimp culture wastewater is treated by the composite microbial inoculum prepared by the embodiment:
the prepared microbial inoculum is put into a sewage treatment system of a certain shrimp farm in east China nutrition according to the mass ratio of 5%, the dissolved oxygen in sewage is 3 mg/L, and the concentrations of sewage ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and COD are 0.2, 1.5, 0.2 and 2.1 mg/L respectively. After 2 weeks of treatment, the concentration of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and COD is reduced to 0.03, 0.2, 0.02 and 0.2 mg/L, the removal rate is respectively 85%, 86.7%, 90% and 90.5%, the aerobic removal of nitrogen pollutants is realized, and organic pollutants are removed at the same time.
Example 7
Preparing a complex microbial inoculum:
1) and (3) strain propagation: acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38 are respectively inoculated to a propagation medium containing antibiotics, and the amount of the seeds is 10 wt%. Culturing at 28 deg.C for 72 hr, separating, collecting precipitate, and collecting bacteria.
The addition amount of the antibiotic in the amplification culture medium containing the antibiotic is 0.05 percent of the mass of the culture medium.
The antibiotic is a mixture of doxycycline, sulfamethoxydiazine, enrofloxacin and roxithromycin, and the mixing mass ratio is 1: 1: 1: 1.
the propagation medium is as described in the examples above.
2) Preparing compound bacteria dry powder: freezing and drying the single acinetobacter YTLJ-N-S10 and the single bacillus YTLJ-N-B38 obtained by propagation, grinding and crushing to obtain single strain dry powder, and mixing the single strain dry powder according to a certain ratio to obtain composite bacteria dry powder, wherein the mixing ratio of the acinetobacter and the bacillus is 20:10 (mass ratio).
3) Preparing a crude product of the aerobic denitrifying bacteria exopolysaccharide: adding 95% ethanol with the volume 3 times that of the filtrate into the filtrate collected after the culture solution of the Acinetobacter YTLJ-N-S10 is separated after the propagation in the step 1), standing to ensure that extracellular polysaccharide in the filtrate is fully precipitated, washing the precipitate with 95% ethanol and anhydrous acetone to remove impurities, freeze-drying to obtain an extracellular polysaccharide crude product, mixing to obtain an aerobic denitrifying bacteria extracellular polysaccharide crude product (the content is more than 30%) and storing the aerobic denitrifying bacteria extracellular polysaccharide crude product in a drying place for later use.
4) Preparing a complex microbial inoculum:
(1) mixing the mixed bacteria dry powder prepared in the step 2) with the crude product of the aerobic denitrifying bacteria exopolysaccharide obtained in the step 3) to obtain the composite bacteria, wherein the ratio of the mixed bacteria dry powder to the aerobic denitrifying bacteria exopolysaccharide is 100: 1 (mass ratio).
(2) The auxiliary materials are 200 mass percent: 10: nitrogen-rich biochar (nitrogen content > 1%), brown sugar and peptone.
(3) And adding the obtained auxiliary materials into the mixture of the mixed bacteria dry powder and the aerobic denitrifying bacteria extracellular polysaccharide according to the proportion of 5wt% to obtain the composite microbial inoculum. The nitrogen-rich biochar is fecal residual bait biochar (nitrogen content is 3%), and the preparation method comprises placing fecal residual bait in a reaction kettle and performing hydrothermal carbonization for 1h at 200 ℃.
The complex microbial inoculum prepared by the embodiment is used for treating grouper culture wastewater:
the prepared microbial inoculum is put into a sewage treatment system of a rockfish farm according to the mass ratio of 10%, the dissolved oxygen in the sewage is 4mg/L, and the concentrations of sewage ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and COD are respectively 3, 5, 0.3 and 60 mg/L. After 20 days of treatment, the concentrations of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and COD are respectively reduced to 0.25, 0.4, 0.02 and 2mg/L, the removal rates are respectively 91.7%, 92%, 93.3% and 96.7%, the aerobic removal of nitrogen pollutants is realized, and organic pollutants are removed at the same time.
Sequence listing
<110> institute of tobacco pipe coastal zone of Chinese academy of sciences
<120> aerobic denitrification strain, preparation and application of microbial inoculum thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1367
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgagcggaga gaggtagctt gctactgatc ttagcggcgg acgggtgagt aatgcttagg 60
aatctgccta ttagtggggg acaacatttc gaaaggaatg ctaataccgc atacgtccta 120
cgggagaaag caggggatct tcggaccttg cgctaataga tgagcctaag tcggattagc 180
tagttggtgg ggtaaaggcc taccaaggcg acgatctgta gcgggtctga gaggatgatc 240
cgccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 300
ggacaatggg cgcaagcctg atccagccat gccgcgtgtg tgaagaaggc cttatggttg 360
taaagcactt taagcgagga ggaggctact ttagttaata cctagagata gtggacgtta 420
ctcgcagaat aagcaccggc taactctgtg ccagcagccg cggtaataca gagggtgcaa 480
gcgttaatcg gatttactgg gcgtaaagcg cgcgtaggcg gctaattaag tcaaatgtga 540
aatccccgag cttaacttgg gaattgcatt cgatactggt tagctagagt gtgggagagg 600
atggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accgatggcg 660
aaggcagcca tctggcctaa cactgacgct gaggtgcgaa agcatgggga gcaaacagga 720
ttagataccc tggtagtcca tgccgtaaac gatgtctact agccgttggg gcctttgagg 780
ctttagtggc gcagctaacg cgataagtag accgcctggg gagtacggtc gcaagactaa 840
aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 900
aacgcgaaga accttacctg gccttgacat agtaagaact ttccagagat ggattggtgc 960
cttcgggaac ttacatacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1020
ggttaagtcc cgcaacgagc gcaacccttt tccttatttg ccagcgagta atgtcgggaa 1080
ctttaaggat actgccagtg acaaactgga ggaaggcggg gacgacgtca agtcatcatg 1140
gcccttacgg ccagggctac acacgtgcta caatggtcgg tacaaagggt tgctacctag 1200
cgataggatg ctaatctcaa aaagccgatc gtagtccgga ttggagtctg caactcgact 1260
ccatgaagtc ggaatcgcta gtaatcgcgg atcagaatgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccatgg gagtttgttg caccaga 1367
<210> 2
<211> 1380
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcgagcggac agaagggagc tcgctcccgg atgttagcgg cggacgggtg agtaacacgt 60
gggtaacctg cctgtaagac tgggataact ccgggaaacc ggagctaata ccggatagtt 120
ccttgaaccg catggttcaa ggatgaaaga cggtttcggc tgtcacttac agatggaccc 180
gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc 240
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 300
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 360
ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtgcaagagt aactgcttgc 420
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 480
cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag ggctcgcagg cggtttctta 540
agtctgatgt gaaagccccc ggctcaaccg gggagggtca ttggaaactg ggaaacttga 600
gtgcagaaga ggagagtgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 660
acaccagtgg cgaaggcgac tctctggtct gtaactgacg ctgaggagcg aaagcgtggg 720
gagcgaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 780
gggggtttcc gccccttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 840
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 900
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caaccctaga 960
gatagggctt tcccttcggg gacagagtga caggtggtgc atggttgtcg tcagctcgtg 1020
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatcttag ttgccagcat 1080
tcagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1140
aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggaca gaacaaaggg 1200
ctgcgagacc gcaaggttta gccaatccca caaatctgtt ctcagttcgg atcgcagtct 1260
gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1320
tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgca acacccgaag 1380
Claims (10)
1. An aerobic denitrification strain is characterized in that: the strain is Acinetobacter YTLJ-N-S10 and/or Bacillus YTLJ-N-B38;
the acinetobacter YTLJ-N-S10 is preserved in Guangdong province microorganism strain preservation center, the preservation address is Guangzhou, China, the preservation date is 2021, 7 and 5 days, and the preservation number is GDMCC No: 61690, classification nameAcinetobacter sp. ；
The bacillus YTLJ-N-B38 is preserved in Guangdong province microbial strainsThe collection center has a preservation address of Guangzhou China, a preservation date of 2021, 7 months and 5 days, and a preservation number of GDMCC No: 61774, classification nameBacillus sp. 。
2. The use of aerobic denitrifying bacteria as defined in claim 1, wherein: the Acinetobacter YTLJ-N-S10 and/or the Bacillus YTLJ-N-B38 are applied to water purification in a mode of aerobic denitrification and organic pollutant removal.
3. A microbial inoculum for purifying water bodies is characterized in that: the bacterial agent contains the aerobic denitrification strain of claim 1.
4. The microbial inoculum for purifying a water body according to claim 3, wherein: the microbial agent comprises symbiotic microorganisms of Acinetobacter YTLJ-N-S10 and Bacillus YTLJ-N-B38 as defined in claim 1.
5. The microbial inoculum for purifying a water body according to claim 4, wherein: the microbial inoculum is prepared by mixing two symbiotic strains of acinetobacter YTLJ-N-S10 and bacillus YTLJ-N-B38 according to the mass ratio of 10-20: 1-10, and mixing the mixed bacteria with an aerobic denitrifying bacteria extracellular polysaccharide crude product and auxiliary materials, wherein the total effective viable count of the microbial inoculum is more than or equal to 1.0 multiplied by 109one/mL.
6. The microbial inoculum for purifying a water body according to claim 5, wherein: the aerobic denitrifying bacteria extracellular polysaccharide crude product is obtained by carrying out alcohol precipitation on an expanding propagation culture filtrate of acinetobacter YTLJ-N-S10 and then secreting the expanding propagation culture filtrate.
7. The microbial inoculum for purifying a water body according to any one of claims 4 to 6, which is characterized in that:
1) and (3) strain propagation: respectively inoculating acinetobacter YTLJ-N-S10 and Bacillus YTLJ-N-B38 into a proliferation culture medium containing antibiotics, culturing at 28 deg.C for 48-72 hr, separating and collecting precipitated bacteria;
2) preparing compound bacteria dry powder: freezing and drying the single acinetobacter YTLJ-N-S10 and the single bacillus YTLJ-N-B38 obtained by propagation, crushing to respectively obtain single strain dry powder, and mixing the acinetobacter dry powder and the bacillus according to the mass ratio of 10-20: 1-10;
3) preparing a crude product of the aerobic denitrifying bacteria exopolysaccharide: adding 2-4 times volume of 95% ethanol into the filtrate obtained by separating the propagation culture solution of acinetobacter YTLJ-N-S10, standing to fully precipitate extracellular polysaccharide, washing to obtain crude product of aerobic denitrifying bacteria extracellular polysaccharide, and drying for later use;
4) preparing a complex microbial inoculum:
(1) mixing the prepared composite bacteria dry powder with the crude product of the aerobic denitrifying bacteria extracellular polysaccharide to obtain a mixture, wherein the mixing ratio is 100: 0.1-1 mass ratio;
(2) 200-200 by mass ratio: 1-10: 1-10 mixing nitrogen-rich biochar, brown sugar and peptone to obtain auxiliary materials; and (3) adding auxiliary materials into the mixture obtained in the step (1) according to the proportion of 1-10% to obtain the composite microbial inoculum.
8. The microbial inoculum for purifying a water body according to claim 7, wherein:
the addition amount of the antibiotic in the propagation culture medium containing the antibiotic is 0.01-0.05% of the mass of the propagation culture medium;
the propagation culture medium is 3-5% K2HPO4·3H2O、1-3% NaCl、1-3% MgSO4·7H2O、0.05-0.5%NH4NO4、0.01-0.04% MnSO4·H2O、0.01-0.05% FeSO4·7H2O, 0.5-1.5% sodium succinate, 0.01-0.1% NaNO2、0.001-0.005% (NH4)2MoO4、0.001-0.005% CoCl2、0.001-0.005% Y(NO3)3；
The antibiotic is a mixture of tetracycline antibiotics, sulfonamide antibiotics, quinolone antibiotics and macrolide antibiotics, and the mass ratio of the antibiotics is 1: 1: 1: 1.
9. the microbial inoculum for purifying a water body according to claim 7, wherein: the nitrogen-rich biochar generally refers to various biochar with the nitrogen content higher than 1%.
10. The application of the microbial inoculum for purifying water body in claim 3, which is characterized in that: the microbial inoculum is applied to water purification in a mode of removing nitrogen and organic pollutants in water.
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