CN116555069A - Alkana diesel oil bacterium with salt-tolerant aerobic denitrification characteristic - Google Patents
Alkana diesel oil bacterium with salt-tolerant aerobic denitrification characteristic Download PDFInfo
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- denitrification
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- 241000894006 Bacteria Species 0.000 title claims abstract description 33
- 239000002283 diesel fuel Substances 0.000 title abstract description 13
- 239000002351 wastewater Substances 0.000 claims abstract description 29
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- 241000611272 Alcanivorax Species 0.000 claims abstract description 12
- 239000010865 sewage Substances 0.000 claims abstract description 12
- 241000814601 Alcanivorax dieselolei Species 0.000 claims abstract description 11
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- 238000004321 preservation Methods 0.000 claims abstract description 6
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- 239000008103 glucose Substances 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 229940074404 sodium succinate Drugs 0.000 claims description 5
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
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- 150000001335 aliphatic alkanes Chemical class 0.000 abstract description 5
- 230000000593 degrading effect Effects 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 20
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 11
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- 229910052760 oxygen Inorganic materials 0.000 description 11
- 229910002651 NO3 Inorganic materials 0.000 description 10
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 10
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- 230000009471 action Effects 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
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- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- QALQXPDXOWOWLD-UHFFFAOYSA-N [N][N+]([O-])=O Chemical compound [N][N+]([O-])=O QALQXPDXOWOWLD-UHFFFAOYSA-N 0.000 description 2
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
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- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
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- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 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
- C12N1/205—Bacterial isolates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/22—Nature of the water, waste water, sewage or sludge to be treated from the processing of animals, e.g. poultry, fish, or parts thereof
- C02F2103/24—Nature of the water, waste water, sewage or sludge to be treated from the processing of animals, e.g. poultry, fish, or parts thereof from tanneries
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2209/00—Controlling or monitoring parameters in water treatment
- C02F2209/05—Conductivity or salinity
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention provides a strain of diesel oil alcanivorax with salt-tolerant aerobic denitrification characteristic, which is diesel oil alcanivorax (Alcanivorax dieselolei) DFC1 with a preservation number of CGMCC No.25879, and also provides an application of the diesel oil alcanivorax DFC1 in biological denitrification of high-salt sewage/wastewater. The novel strain DFC1 of the Alkania dieselvora provided by the disclosure can perform denitrification in a high-salt environment. The diesel oil edible alkane bacteria are widely found in the marine environment and are the most important special alkane degrading bacteria in the marine environment, but the diesel oil edible alkane bacteria DFC1 is found to have a new denitrification function for the first time, and can synchronously play the denitrification function in other application scenes, such as mariculture industry, so that the problem of nitrogen pollution of mariculture tail water is solved.
Description
Technical Field
The present disclosure belongs to the technical field of environmental microorganisms, and in particular relates to an alkane-eating diesel bacterium with salt-tolerant aerobic denitrification characteristics.
Background
In recent years, wild fishery resources have been drastically reduced due to artificial overdrawing and serious environmental damages. However, the demand for seafood is increasing, so that the mariculture industry is rapidly developing. However, a great amount of nitrogen-containing pollutants are generated in the mariculture process and released into the surrounding environment, and if untreated water is directly discharged into the sea, the content of nutrient components in the received water body can be increased, so that the water body is eutrophicated, and the coastal ecosystem is seriously threatened. Thus, proper treatment of mariculture wastewater to remove nitrogen-containing compounds has been urgent.
Various researches have been conducted on the treatment of mariculture wastewater so far, and physical, chemical and biological methods are three common methods for treating mariculture wastewater. Compared with physical and chemical methods, biological methods for treating mariculture wastewater have received great attention in terms of high denitrification efficiency, low energy consumption, low running cost and the like. Autotrophic aerobic nitrification and heterotrophic anaerobic denitrification are two steps in a typical biological denitrification process. However, these nitrifying and denitrifying bacteria have certain disadvantages in the process of removing nitrogen from seawater wastewater: (1) Autotrophic nitrifying bacteria grow slowly, are difficult to maintain high biological concentrations, are easily eliminated in wastewater treatment, and cannot compete with heterotrophic nitrifying bacteria for nutrients. (2) In the conventional biological denitrification system, autotrophic nitrifying bacteria are grown under aerobic conditions, and denitrification is performed under anoxic conditions, so that a nitrification tank and a denitrification tank are necessarily constructed separately. The fractional nitrification and denitrification process is long, and the capital cost and the running cost are easy to increase.
In recent decades, research reports have demonstrated that some strains are also capable of denitrification under aerobic conditions. Aerobic denitrifying bacteria under aerobic condition, NO is utilized 3 - As an electron acceptor reductive denitrification, the possibility of simultaneous nitrification and denitrification in one reactor is provided. However, the existing aerobic denitrifying bacteria are almost screened in fresh water or soil environment, and can not effectively treat high salinity wastewater such as mariculture wastewater, seafood processing, tanning oil and the like. In high salinity environments, an increase in osmotic pressure inhibits cellular metabolism and enzymatic activity. Therefore, the separation of salt-tolerant aerobic denitrifying bacteria and the research of the aerobic denitrifying performance under the high salinity condition have important scientific and practical significance. Meanwhile, the method is also helpful for us to review and solve the problem of nitrogen pollution in the mariculture wastewater.
The inventor newly screens out a salt-tolerant aerobic denitrification strain DFC1 which is identified as Alkania dieselvora (Alcanivorax dieselolei) and has not been found to have the salt-tolerant aerobic denitrification characteristic before. The strain can realize the efficient denitrification treatment of the salt-containing wastewater, and provides a new idea for realizing the synchronous nitrification and denitrification process of the sewage plant.
Disclosure of Invention
The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. It should be understood that this summary is not an exhaustive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.
In order to solve the technical problems, the technical scheme provided by the present disclosure is as follows:
in a first aspect, the disclosure provides an alcanivorax diesel with salt-tolerant aerobic denitrification characteristics, which is alcanivorax diesel (Alcanivorax dieselolei) DFC1 with a preservation number of CGMCC No.25879.
The disclosed diesel oil edible fungi DFC1 is separated from a water sample of an anoxic section of the culture tail water of Bo Feng aquatic products (eastern) limited company, pure bacteria are separated through enrichment and domestication, gradient dilution and plate streaking, and the identification shows that the strain has higher homology with various bacteria of the genus Alcanivora (Alcanivora) and is the diesel oil edible fungi (Alcanivorax dieselolei). Experiments show that the strain can realize the high-efficiency denitrification treatment of salt-containing wastewater such as cultivation wastewater, industrial wastewater and the like.
In a second aspect, the present disclosure also provides the use of Alkania diesel DFC1 in biological denitrification of high salt wastewater.
Preferably, the salinity of the high-salt sewage/wastewater is 1-6% (such as 1.5%, 2%,3%,4%,5%, 5.5%, etc.); more preferably, the salinity of the high-salt sewage/wastewater is 1-4%.
Preferably, in the biological denitrification, the carbon source used can be any one or more selected from ethanol, sodium acetate, glucose, sodium citrate and sodium succinate; more preferably ethanol or sodium acetate.
Preferably, in the biological denitrification, the treatment time is 12-48h (such as 15h, 18h, 20h, 24h, 28h, 32h, 35h, 40h, 45h, etc.); more preferably 16 to 32 hours, still more preferably 20 to 32 hours.
Preferably, in the biological denitrification, the volume ratio of the logarithmic phase bacterial liquid of the Alkania dieselvorans (Alcanivorax dieselolei) DFC1 to the salinity of the high-salt sewage/wastewater is 1:10.
Preferably, in the biological denitrification, a dry powder microbial inoculant of the DFC1 of the alkane-eating bacteria (Alcanivorax dieselolei) is used for the nitrogen removal in the sewage/wastewater; more preferably, the dry powder microbial inoculum is obtained by fermenting and culturing the diesel oil edible alkane fungus DFC1 to a logarithmic phase, mixing with a certain amount of rice bran and turfy soil, and drying.
In a third aspect, the present disclosure also provides a dry powder microbial inoculant for biological denitrification of high-salinity wastewater/wastewater, the preparation method thereof comprising: fermenting and culturing the alkane-eating bacteria DFC1 to bacterial liquid in the logarithmic phase, mixing the bacterial liquid with a certain amount of turfy soil and rice bran, centrifuging to remove supernatant, and drying to obtain the alkane-eating bacteria.
Preferably, in the preparation method, 100ml of rice bran and 50ml of turfy soil are added into each 1L of bacterial liquid.
Compared to the prior art, the benefits of the present disclosure include, but are not limited to:
first, the novel strain DFC1 of alcanivorax diesel provided by the present disclosure is capable of denitrification in a high salt environment. In recent years, the mariculture industry in China rapidly develops, and a large amount of mariculture tail water needs to be discharged, so that proper treatment of the mariculture tail water is particularly important. However, the denitrification treatment process in the wastewater is greatly affected due to the existence of salt. Therefore, it is important to screen out aerobic denitrifying bacteria capable of resisting salt. The novel strain DFC1 of the Alkania dieselvora provided by the disclosure provides a good foundation for solving the denitrification problem of high-salt wastewater.
Secondly, the alkane-eating bacteria of diesel oil have been found to widely exist in the marine environment and are the most important special alkane degrading bacteria in the marine environment, but the alkane-eating bacteria DFC1 of diesel oil provided by the disclosure also has a new denitrification function, and can synchronously play the denitrification function in other application scenes, such as mariculture industry, so that the problem of nitrogen pollution of tail water of mariculture is alleviated.
The new strain of the Alkania dieselvorax disclosed by the disclosure has a preservation date of 2022, 10 months and 08 days, a preservation number of CGMCC No.25879 and a classification name of: alkanvorax diesel (Alcanivorax dieselolei) DFC1, accession number: china general microbiological culture Collection center (CGMCC) with the address: the number 3 of North Chen West Lu 1 in the Chaoyang area of Beijing city is 100101.
Drawings
FIG. 1 is a phylogenetic tree of Alkania diesel DFC1 in example 1;
FIG. 2 is a denitrification curve of Alkania diesel DFC1 under high salt (salinity 3%) condition in example 2;
FIG. 3 is a denitrification curve of Alkanolaminetobacter DFC1 of example 3 under different carbon source conditions;
FIG. 4 is a denitrification profile of Alkania diesel DFC1 of example 4 under different salinity conditions;
FIG. 5 is a denitrification curve of Alkanolaminetobacter DFC1 of example 5 under different dissolved oxygen conditions;
FIG. 6 is a bar graph of the nitrogen removal after 24 hours for the different strains isolated and purified in example 1;
FIG. 7 is a denitrification curve within 32 hours of the dry microbial agent prepared in application example 1.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions of the methods, steps or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Technical aspects of the present disclosure will be described hereinafter with reference to exemplary embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: screening, identifying and preserving the salt-tolerant aerobic denitrifying bacteria.
(1) Strain and culture Medium preparation
The strain is derived from a water sample of an anoxic section of the culture tail water of Bo Feng aquatic (eastern) limited company.
The liquid salt tolerant denitrification medium (nitrogen concentration of about 100 mg/L) comprises the following chemicals: CH (CH) 3 COONa 1.71g/L，NaNO 3 0.6g/L，K 2 HPO 4 1.6g/L，MgSO 4 ·7H 2 O 0.1g/L，CaCl 2 0.02g/L，FeSO 4 ·7H 2 0.005g/L of O, 30g/L of NaCl and 0.1mL of trace element solution. Wherein the trace element solution comprises MgSO 4 ·H 2 O 0.0344g/L、H 3 BO 3 0.05g/L、ZnCl 2 0.07g/L、Na 2 MoO 4 ·2H 2 O 0.0726g/L、CaCl 2 ·2H 2 O 0.02g/L、NiCl 2 ·6H 2 O 0.024g/L、CoCl 2 ·6H 2 O 0.08g/L、FeSO 4 ·7H 2 O 1.0g/L。
The agar plate culture medium is prepared by adding 1mL of BTB solution (dissolved in 1% absolute alcohol) and 20g of agar per liter of the liquid salt-tolerant denitrification culture medium, and other components are unchanged.
All media were sterilized at 120 ℃ for 90 minutes before use to ensure no microbial contamination.
(2) Enriching and domesticating: 10mL of the cultivation tail water was added to a conical flask containing 90mL of sterile liquid salt tolerant denitrification medium. The inoculated medium was incubated at 30℃and 120rpm until liquid turbidity was achieved. Then, 10ml of the cell suspension was transferred to a new 90ml of sterile liquid salt-tolerant medium and acclimatized under the same conditions. And finally, finishing the third enrichment domestication under the same condition.
(3) And (3) separating and purifying: and (3) carrying out gradient dilution on the enriched and domesticated bacterial suspension obtained in the step (2). The diluted cell suspension is evenly coated on an agar plate culture medium and is placed in a biochemical incubator with constant temperature of 30 ℃ for culture. At any time, when a small bacterial colony grows on the flat plate, different bacterial colonies are picked and respectively subjected to flat plate streaking and separation for a plurality of times, after the last step of flat plate streaking is carried out, bacterial colonies with different forms are selected, 30ml of the liquid salt-tolerant denitrification medium in the step (1) is inoculated for culture and activation for 14 hours until the bacterial strain grows to the logarithmic phase, and different pure bacterial strain bacterial solutions are obtained, and the numbers are A1, A2, B1, B2, C1 and C2 respectively.
The six bacteria are respectively subjected to an initial removal experiment with the nitrate nitrogen of 100mg/L, and the steps are as follows: respectively taking 3ml of each of the 6 pure strain bacterial liquids obtained after separation and purification, respectively adding the bacterial liquids into 30ml of liquid salt-tolerant denitrification culture medium, culturing at 30 ℃, and calculating the nitrate nitrogen removal rate after 24 hours, wherein the result is shown in figure 6. As is clear, the highest nitrate removal rate after 24 hours was 99.32% for strain C1, and therefore strain C1 was selected as a subject for subsequent study and identified.
(4) Identification of strains: the strain C1 genomic DNA was extracted using the soil FastDNA SPIN kit (MPbio, america) and the 16S rDNA gene was amplified by Polymerase Chain Reaction (PCR). PCR amplified products were sequenced by Magi biomedica (Shanghai). The obtained sequence was compared with other related bacteria in the GenBank database using BLAST tool, and BLAST comparison result shows that the strain has higher homology with various bacteria of genus Alcanivorax (Alcanivorax) and identified as Alcanivorax diesel (Alcanivorax dieselolei). A phylogenetic tree of the strain was constructed using MEGA software (version 6.0) using neighborhood ligation, as shown in FIG. 1.
The inventor names the strain as Alkania dieselvorans (Alcanivorax dieselolei) DFC1, and submits the strain to be preserved on the 10 th month 08 day 2022, with the preservation number of CGMCC No.25879, and the classification names are: alkanvorax diesel (Alcanivorax dieselolei) DFC1, accession number: china general microbiological culture Collection center (CGMCC) with the address: the number 3 of North Chen West Lu 1 in the Chaoyang area of Beijing city is 100101.
Example 2: denitrification experiment of Strain under high salt (salinity 3%) condition
The alkane-eating diesel bacteria DFC1 obtained by the primary screening in the example 1 is activated according to the method in the example 1 to obtain bacterial liquid, 3ml of the bacterial liquid is inoculated into 30ml of liquid salt-tolerant denitrification culture medium (salinity is 3%), shake culture is carried out for 48 hours at 30 ℃, the content of nitrate nitrogen, nitrite nitrogen and ammonia nitrogen in the culture liquid is measured at intervals, and the degradation rate of the nitrate nitrogen is calculated, so that the salt-tolerant aerobic denitrification bacteria obtained by enrichment, domestication, separation and purification are subjected to performance test.
The absorbance of nitronitrogen was measured by ultraviolet spectrophotometry at wavelengths 275nm and 220nm, the absorbance of nitronitrogen was measured by N- (1-naphthalene) -ethylenediamine spectrophotometry at 540nm, and the absorbance of ammonia nitrogen was measured by Nami reagent spectrophotometry at 420 nm. And calculating the calculated corrected absorbance, and calculating the corresponding nitrate nitrogen, nitrite nitrogen and ammonia nitrogen concentrations according to a standard curve, wherein the obtained data is multiplied by the corresponding dilution times.
The results of the removal of the nitrate nitrogen by the strain are shown in FIG. 2. The result shows that the bacterial strain has better effect of removing the nitrate nitrogen under the condition of high salt (3%). After the strain is cultured for 32 hours, the removal rate of the paranitronitrogen reaches 100% even under the salinity of 3%, so that the strain effectively realizes the denitrification process under the high-salt condition.
Example 3: denitrification experiment of bacterial strain under different carbon sources
The Alkania diesel edible fungi DFC1 screened in example 1 was inoculated into a liquid salt-tolerant denitrification medium containing sodium acetate as a carbon source and activated in a shaker at 30℃and 120r/min (same as in example 1). After the strain grows to the logarithmic growth phase, 3mL of bacterial liquid is taken and is inoculated into fresh 30mL of denitrification culture medium with carbon sources of methanol, sodium citrate, glucose, sodium acetate, ethanol and sodium succinate respectively, and shake culture is carried out in a shaking table at 30 ℃ and 120 r/min. A small amount of bacterial liquid is taken every 2 hours, the bacterial liquid is centrifuged at 12000rpm for 2 minutes, the supernatant is taken to respectively measure the concentration of nitrate nitrogen and nitrite nitrogen, and the result is shown in figure 3.
As can be seen from FIG. 3, when methanol is used as the only carbon source, the growth rate of the strain is slow, and NO is produced after 32 hours 3 - The N removal was only 13.24%. When glucose is the sole carbon source, NO in the medium 3 - The N concentration drops rapidly between 18h and 32h from 73.79mg/l for 18 hours to 21.05mg/l for 32 hours, NO 3 - The removal rate of N is improved from 9.69% to 74.2%. In a culture medium with sodium citrate and sodium succinate as carbon sources, NO after 18h 3 - The removal rate of N is about 70% and 50%, respectively. In addition, when ethanol is used as the sole carbon source, NO is obtained after 20 hours 3 - The removal rate of N can reach 100 percent. When sodium acetate is used as the sole carbon source, NO 3 - The removal rate of N is between 95% and 100%, and is less than 100%. From the analysis of the above data, ethanol is the most effective carbon source for NO 3 - The removal rate of the-N is highest, and the second is sodium acetate, glucose, sodium citrate and sodium succinate, when methanol is used as a substrate, NO 3 - The N removal rate is the worst.
Example 4: denitrification experiment of bacterial strain under different salinity conditions
The Alkania diesel oil bacteria DFC1 screened in example 1 was activated in advance (same as in example 1), after the strain had grown to the logarithmic growth phase, 3mL of the bacterial liquid was inoculated into 30mL of denitrification medium (formulation reference example 1, only salinity was adjusted) with fresh salinity adjusted to 0%,1%,2%,3%,4%,5% and 6%, and shaking culture was carried out in a shaker at 30℃and 120 r/min. Then, a small amount of bacterial liquid was taken every 2 hours, centrifuged at 12000rpm for 2 minutes, and the supernatant was taken to determine the nitrosamine concentration, and the result is shown in FIG. 4.
FIG. 4 shows denitrification rates of strains at different salinity. In the absence of sodium chloride added to the medium, there was essentially no change in nitrate concentration over 32 hours. When the salinity is 1%, the nitrate concentration is reduced to 0mg/L at 18h, and the removal rate is 100%. As salinity increases, the time required for nitrate concentration to reach a minimum increases. Nitrate was completely removed over 22 hours and 24 hours at salinity of 2% and 3%, respectively. And at a salinity of 4%, the strain has a 32h denitrification rate of 94.06%, and at a salinity of 5%, the strain has a 32h denitrification rate reduced to 73.70%. When the salinity is continuously increased to 6%, the denitrification rate of the strain is obviously reduced, the nitrate concentration is reduced from 97.63mg/l to 50mg/l after the strain is cultured for 32 hours, and the removal rate is 51.50%. These data indicate that the Alkanka DFC1 can achieve denitrification in a medium with a salinity of 1-6%, but can still maintain a high nitrate removal efficiency only with a salinity of 1-3%. Thus, the strain is classified as a salt tolerant strain.
Example 5: denitrification experiment of bacterial strain under different dissolved oxygen conditions
The headspace of the serum bottle was emptied by pure helium, then a needle tube filled with oxygen was inserted into the rubber plug of the serum bottle, and different volumes of oxygen were supplemented into the serum bottle so that the volume of Dissolved Oxygen (DO) was 0%, 10%, 20%, 30%, 50% and 100% of the headspace volume of the serum bottle, respectively, thereby examining the effect of dissolved oxygen on denitrification of the novel strain Alkana dieselvorax DFC1 obtained in example 1, and other experimental conditions were the same as in example 1. Similarly, prior to the experiment, the strain needs to be activated in advance, and the activation step is the same as in example 1. The change in nitrate nitrogen and nitrite nitrogen was monitored every 2 hours, and the results are shown in fig. 5.
As can be seen from FIG. 5, when the oxygen volume ratio is 0%, i.e. under anaerobic conditions, the nitrate removal rate is relatively slow, and the nitrate concentration is reduced from the initial 90.46mg/L to 30.38mg/L at 32h, NO 3 - The final removal of N reaches 66.42%. Furthermore, as can be seen from FIG. 5, microbial denitrification is essentially enhanced with increasing DO concentration. The increase in dissolved oxygen concentration does not inhibit the denitrification of the strain, but rather has a promoting effect. The presence of oxygen does not inhibit the activity of the nitrate reductase, denitrificationEnzymes may coexist with the aerobic respiratory system. Thus, the strain can be identified as aerobic denitrifying bacteria. When the dissolved oxygen concentration was further increased, the nitrate removal rate was slightly decreased at an oxygen volume ratio of 100% because the strain activity was suppressed.
Application example 1:
the immobilized microbial inoculum is prepared by an adsorption method, and turfy soil and rice bran are selected as carrier materials. Culturing the bacterial liquid to logarithmic growth phase, adding 100ml rice bran and 50ml turfy soil (all weighed by measuring cylinders, and the turfy soil is sieved by a 10-mesh sieve in advance) into each 1L bacterial liquid, and centrifuging for 5min by a high-speed centrifuge at 12000 rpm/min. After centrifugation, removing the supernatant, retaining the precipitate, and placing into a sterile container, drying at low temperature in a constant temperature drying oven at 35 ℃, and preserving for later use.
The microbial inoculum obtained above was added to laboratory simulated saline wastewater (formulation as in example 1) at intervals of a small amount of water sample, centrifuged at 12000rpm for 2min, and the supernatant was taken to determine the nitrate nitrogen concentration, respectively, as shown in fig. 7. The result shows that the microbial inoculum prepared by the method has a certain denitrification effect when being added into the saline wastewater, and the nitrate nitrogen removal rate reaches 70.56% after 32 hours.
Finally, it is further noted that in this disclosure, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
While the disclosure has been disclosed by the foregoing description of specific embodiments thereof, it will be understood that various modifications, improvements, or equivalents may be devised by those skilled in the art that will fall within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this disclosure.
Claims (10)
1. A strain of Alkania dieselvorax with salt-tolerant aerobic denitrification characteristic is Alkania dieselvorax (Alcanivorax dieselolei) DFC1, and the preservation number is CGMCC No.25879.
2. Use of the alcanivorax diesel DFC1 according to claim 1 in biological denitrification of high salt sewage/wastewater.
3. Use according to claim 2, wherein the salinity of the high-salt sewage/wastewater is 1-6%; preferably the salinity of the high salt sewage/wastewater is 1-4%.
4. The use according to claim 2 or 3, wherein the carbon source used in the biological denitrification is selected from any one or more of ethanol, sodium acetate, glucose, sodium citrate and sodium succinate; ethanol or sodium acetate is preferred.
5. The use according to any one of claims 2-4, wherein the treatment time in the biological denitrification is 12-48 hours; preferably 16 to 32 hours, and more preferably 20 to 32 hours.
6. The use according to any one of claims 2-5, wherein in the biological denitrification, the volume ratio of the log phase bacterial liquid of the alcanivorax diesel edible fungi DFC1 to the salinity of the high-salt sewage/wastewater is 1:10.
7. The use according to any one of claims 2-4, wherein the biological denitrification is performed using a dry microbial agent comprising alcanivorax diesel DFC1 to remove nitrogen from sewage/wastewater.
8. The use according to claim 7, wherein the dry powder microbial inoculum is obtained by fermenting and culturing the alkane-eating bacteria DFC1 until the alkane-eating bacteria reach a logarithmic phase, mixing the alkane-eating bacteria with a certain amount of rice bran and turfy soil, and drying the mixture.
9. The dry powder microbial inoculum for biological denitrification of high-salt sewage/wastewater is characterized in that the preparation method of the dry powder microbial inoculum comprises the following steps: fermenting and culturing the Alkanka DFC1 as defined in claim 1 to bacterial liquid in a logarithmic phase, mixing the bacterial liquid with a certain amount of turfy soil and rice bran, centrifuging to remove supernatant, and drying to obtain the Alkanka DFC 1.
10. The dry microbial inoculant according to claim 9, wherein 100ml of rice bran and 50ml of turfy soil are added to 1L of microbial inoculum.
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