CN114703093B - Facultative anaerobic complete denitrification gas producing bacterium Y23 and application thereof - Google Patents
Facultative anaerobic complete denitrification gas producing bacterium Y23 and application thereof Download PDFInfo
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- C09K8/58—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids
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Abstract
The invention discloses a Pseudomonas stutzeri capable of completely denitrifying and denitrifying under higher oxygen concentrationPseudomonas stutzeriY23 and application thereof. The completely denitrified Pseudomonas stutzeri Y23 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.24240 in the year 1 and 5 of 2022. The pseudomonas stutzeri Y23 has a complete denitrification path, can convert nitrate into nitrogen under the aerobic and anaerobic conditions, and is suitable for the fields of nitrogen-containing sewage denitrification treatment, oil field microorganism enhanced crude oil recovery, oil reservoir acidification and pipeline corrosion prevention and control caused by sulfate reducing bacteria, and the like.
Description
Technical Field
The invention belongs to the fields of microbial technology and environmental biotechnology, and relates to pseudomonas stutzeri capable of completely denitrifying and denitrifying under higher oxygen concentrationPseudomonas stutzeriY23 and application thereof.
Background
Nitrate-reducing bacteria are a type of bacteria capable of reducing nitrate or nitrite to produce N 2 O and N 2 Is also known as bacteria. Nitrate-reducing bacteria are mostly anaerobic bacteria, and a small part of them are facultative anaerobic bacteria. The facultative anaerobic nitrate reducing bacteria preferentially perform aerobic respiration to grow fast bacteria in an aerobic environment, and under an anoxic condition, the nitrate reducing bacteria perform anaerobic respiration by taking nitrate or nitrite as an electron acceptor to generate N 2 O and N 2 . At present, nitrate-reducing bacteria are widely applied to the fields of sewage denitrification treatment and microbial oil recovery.
The sewage denitrification by using nitrate-reducing bacteria is one of the main means of sewage denitrification at present. Microbial denitrification is usually performed under anoxic conditions, and oxygen affects the microbial denitrification effect. Thus, NO can be released at a higher oxygen concentration 3 Sequential conversion of N to NO 2 - 、NO、N 2 O and N 2 The complete denitrification microorganism has higher application value in the aspect of strengthening sewage denitrification treatment. In addition, the nitrate reducing bacteria with complete denitrification capability can also reduce the greenhouse gas N to a certain extent 2 O is discharged.
In microbial oil recovery processes, carbohydrates and nitrates are often injected into the reservoir to activate the subterranean microorganisms to achieve microbial enhanced oil recovery. On the other hand, the nitrate reducing bacteria can obtain electron donors (organic carbon sources) more preferentially than the sulfate reducing bacteria, so that the nitrate reducing bacteria can inhibit the growth of the sulfate reducing bacteria through biological competition, and the problems of oil reservoir acidification, pipeline corrosion, personal safety and the like caused by hydrogen sulfide generated by metabolism of the sulfate reducing bacteria are solved. Nitrous oxide (N) produced by denitrifying bacteria 2 O) gas and CO 2 Has a similar molecular structure and higher solubility in nonpolar liquids than CO 2 Can function like CO 2 Oil displacement (dissolution in crude oil to expand the crude oil to reduce the viscosity of the crude oil); n produced by denitrifying bacteria 2 Crude oil recovery may be enhanced by increasing reservoir pressure.
The complete denitrifying bacteria are separated from oil reservoirs subjected to microbial oil recovery, nitrate solution is injected into the oil reservoir for a long time to perform microbial oil recovery, and nitrate-reducing bacteria are dominant microbial groups of the oil reservoir blocks. The strain can realize complete denitrification under higher oxygen concentration, and has higher application value in the fields of enhanced sewage denitrification treatment and enhanced microbial oil recovery.
Disclosure of Invention
An object of the present invention is to provide a Pseudomonas stutzeri capable of complete denitrification at a relatively high oxygen concentrationPseudomonas stutzeriY23 has a preservation number of CGMCC No.24240 in China general microbiological culture Collection center.
The invention also aims to provide a bacterial suspension containing the microorganism strain, a fermentation broth or a bacterial agent containing the microorganism strain and a new application.
The invention provides the Pseudomonas stutzeri for completely denitrifying gas productionPseudomonas stutzeriY23, bacterial suspension thereof, culture solution thereof, fermentation product thereof and application of microbial agent containing the same in microbial oil recovery.
The invention also provides the Pseudomonas stutzeri for completely denitrifying gas productionPseudomonas stutzeriY23, bacterial suspension thereof, culture solution thereof, fermentation product thereof and microbial inoculum containing the same are applied to the preparation of microbial oil extraction products.
The invention provides the Pseudomonas stutzeri for completely denitrifying gas productionPseudomonas stutzeriY23, bacterial suspension thereof, culture solution thereof, fermentation product thereof and application of bacterial agent containing the same in sewage treatment.
The invention also provides the Pseudomonas stutzeri for completely denitrifying gas productionPseudomonas stutzeriY23, bacterial suspension thereof, culture solution thereof, fermentation product thereof and bacterial agent containing the same are applied to the preparation of sewage treatment products or equipment.
It is a further object of the present invention to provide a product comprising the above Pseudomonas stutzeri as an active ingredientPseudomonas stutzeriY23, or a bacterial suspension thereof, or a culture solution thereof, or a fermentation product thereof, or a microbial inoculum containing the sameThe product has any one of functions of microbial oil extraction and sewage treatment.
It is still another object of the present invention to provide a method for microbial oil recovery comprising the following steps: during oil extraction, the pseudomonas stutzeri is preparedPseudomonas stutzeriInjecting Y23 fermentation liquor and nutrient into stratum from oil sleeve annulus of oil well, utilizing anaerobic metabolism of said bacterium to produce gas to raise single well yield, at the same time preventing and controlling oil reservoir acidification resulted from sulfate reducing bacterium; the Pseudomonas stutzeri is preparedPseudomonas stutzeriThe Y23 fermentation liquid and the nutrient are injected into the ground by a water injection well, the bacteria are utilized to metabolize gas in the ground to improve the recovery ratio of crude oil, and simultaneously, the oil reservoir acidification caused by sulfate reducing bacteria is prevented and controlled.
The final object of the invention is to provide a sewage treatment method, comprising the following steps: during the sewage treatment, the pseudomonas stutzeri is addedPseudomonas stutzeriY23 bacterial suspension, bacterial agent or Pseudomonas stutzeri which has been film-coated on fillerPseudomonas stutzeri Y23。
Drawings
FIG. 1 is a phylogenetic tree of strain Y23 constructed based on the 16S rRNA gene and its homologous sequences.
FIG. 2 shows denitrification effect of strain Y23 in a sequencing batch bioreactor.
FIG. 3 shows the gas production of strain Y23 at different carbon sources.
FIG. 4 shows the gas components produced by strain Y23 and their relative amounts.
Detailed Description
Example 1: isolation and identification of Strain Y23
Strain Y23 was isolated from oilfield produced fluid. The oil field injects nitrate solution into an oil reservoir for a long time to develop microbial oil recovery, and nitrate-reducing bacteria are one of dominant microorganism groups of the oil reservoir. The specific steps of strain isolation are as follows: 10ml of oilfield produced fluid is inoculated into a 500ml sterile plastic bottle containing 200mL sterile denitrifying gas-producing bacteria enrichment medium, redundant air is removed, and the bottle cap is screwed down and is cultured in an incubator until gas is produced. The denitrification gas producing bacteria enrichment culture medium comprises the following components: disodium hydrogen phosphate 0-1 g, potassium dihydrogen phosphate 0-1 g, sodium nitrate 0-10 g, ferrous sulfate 0-1 g, magnesium sulfate hexahydrate 0-1 g, calcium chloride 0.01 g, yeast extract powder 0.01 g, glucose 0-10 g, distilled water, pH 6-8, and sterilizing with high pressure steam at 121deg.C for 20min. Taking 5mL of enrichment culture solution, inoculating the enrichment culture system of the denitrifying gas-producing bacteria again, and culturing in a constant temperature box until no gas is produced. The single bacteria were isolated by dilution coating. And verifying the gas production of the single strain, and selecting the strain with the largest gas yield, which is named as Y23.
The genome of the strain is extracted by using a genome extraction kit, the strain 16S rRNA gene is obtained by PCR amplification by using primers 27f (5 '-AGA GTT TGA TCC TGG CTC AG-3') and 1492r (5 '-GGT TAC CTT GTTACG ACT T-3'), and the obtained PCR product is sequenced to obtain the 16S rRNA sequence information. The nucleotide sequence of the 16S rRNA of the Y23 strain is submitted to GenBank, and the results of BLAST search and homology analysis comparison show that the 16S rDNA sequence of the Y23 strain is compared with the known strainPseudomonas stutzeriThe similarity of the strain is 99%, which indicates that the strain is a Pseudomonas stutzeri strain, and the strain is named as Pseudomonas stutzeri by combining the identification resultsPseudomonas stutzeriY23. Based on the nucleotide sequence of the 16S rRNA of Y23 strain, a phylogenetic tree was constructed using software MEGA as shown in FIG. 1.
The strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.24240 in 1 month and 5 days of 2022, wherein the address is 1 st part 3 rd institute of microbiological study, china academy of sciences, beijing, and the preservation number is CGMCC No.24240.
Example 2: strain Y23 denitrification gene detection
The strain genome is used as a template, and the strain denitrification gene sequence fragment is obtained by PCR amplification using specific primers of the denitrification genes and amplification conditions disclosed in academic papers. The PCR product was checked by 1% agarose gel electrophoresis and sent to Beijing ao Dingsheng biotechnology Co. And submitting the sequenced sequence to NCBI database for BLAST analysis to determine the most similar functional genes.
Sequencing analysis of denitrifying gene PCR amplicon shows that the strainY23 has genes encoding Membrane-bound nitrate reductase (Membrane-bound nitrate reductase, nar), periplasmic nitrate reductase (Periplasmic-bound nitrate reductase, nap), nitrite reductase (Nitrite reductase, nir), nitric oxide reductase (Nitric oxide reductase, norB) and nitrous oxide reductase (Nitrous oxide reductase, nos) involved in the denitrification process, capable of converting NO 3 Sequential conversion of N to NO 2 - 、NO、N 2 O and N 2 . Thus, strain Y23 is a denitrifying bacterium having complete denitrification. The membrane-bound nitrate reductase Nar is sensitive to oxygen molecules and plays a main role in denitrification of anaerobic denitrifying organisms. Periplasmic nitrate reductase Nap is located in the periplasm of the cell and is insensitive to oxygen molecules. The presence of the periplasmic nitrate reductase-encoding gene means that strain Y23 is capable of denitrification under aerobic conditions, i.e.simultaneously utilizing oxygen and nitrate as electron acceptors for energy-producing metabolism.
Example 3: denitrification effect of Strain Y23 in sequencing batch bioreactor
Inoculating the Y23 strain into 50 ml of LB liquid medium for overnight culture; centrifuging the culture solution at 4000r/min for 5min, and pouring out the supernatant; suspending thalli sunk to the bottom of the centrifuge tube by using a sterile denitrification culture medium, centrifuging for 5min at 4000r/min, pouring out supernatant, and adding 50 mL sterile denitrification culture medium to suspend thalli; strain Y23 was inoculated in an inoculum size of 1% to a sequencing batch bioreactor, which was used to discharge the treated wastewater every 12 hours while supplementing the same amount of wastewater (hydraulic retention time of 12 hours). Simulated wastewater C 4 H 4 Na 2 O 4 ·6H 2 O、NaNO 3 、KH 2 PO 4 、MnSO 4 ·7H 2 O and FeSO 4 ·7H 2 O. And (3) sampling and detecting the content of nitrate nitrogen and nitrite nitrogen in the treatment liquid every 30 minutes on the 24 th day after the reactor runs stably, and measuring the change of pH value.
As shown in fig. 2, the strain Y23 has good denitrification effect in the sequencing batch bioreactor, the degradation of the strain on nitrate nitrogen is mainly concentrated in the first 4 hours, the denitrification efficiency is close to 100%, nitrite accumulation is basically avoided in the process, and the pH value of treated wastewater is raised to a certain extent.
Example 4: gas production effect of strain Y23 under different carbon sources
In the carbon source experiment, the gas production of the Y23 strain under different carbon sources is explored by taking carbon source saccharides (glucose, sucrose, molasses), organic acid, small molecular alcohol (glycerol), petroleum hydrocarbon and the like which are commonly used in industry as unique carbon sources. The dosage of various carbon sources is glucose 0-5 g/L, glycerol 0-5 g/L, sodium succinate 0-5 g/L, sodium acetate 0-5 g/L, sucrose 0-5 g/L, toluene 0-5 ml/L.
As shown in FIG. 3, the strain can produce gas in a liquid culture medium with glucose, sodium succinate, glycerol, sodium acetate, molasses and sucrose as the only carbon sources, but the gas production is greatly different under different carbon source substrates. In a culture solution taking sugar (molasses, glucose and sucrose) as a carbon source, the gas yield of the strain is highest; in the culture solution with glycerol, sodium succinate and sodium acetate as carbon sources, the gas production effect is reduced. Therefore, from the application point of view, molasses can be used as the optimal carbon source for the gas production of strain Y23.
Example 5: analysis of gas Components produced by Denitrification of Strain Y23
Inoculating the seed liquid into a 1L feed supplement bottle containing 1L denitrification gas production culture medium according to the inoculation amount of 2%, screwing up a bottle cap, and reserving two-way connectors on the bottle cap, wherein each connector is connected with a gas collecting bag (Bikerman organism). In order to eliminate the influence of air on the analysis of gas components generated by the bacterial strain, in the early culture stage, an air inlet valve of one air collecting bag is opened, the air inlet valve of the air collecting bag is closed when the gas collection amount reaches about 200ml, and the air inlet valve of the other air collecting bag is opened for gas collection. The gas analysis used an Agilent 7890A gas chromatograph with detectors of Thermal Conductivity (TCD) and hydrogen Flame Ionization (FID) at a detection limit of 1ppm.
As shown in FIG. 4, the gas generated by the denitrifying gas-producing bacteria Y23 mainly comprises nitrogen, nitrous oxide and carbon dioxide. N (N) 2 The content reaches 56%, and the generated N 2 Crude oil recovery may be enhanced by increasing reservoir pressure. N (N) 2 O content reaches 32%, and the gas and CO 2 Has a similar molecular structure and higher solubility in nonpolar liquids than CO 2 Can function like CO 2 Oil displacement. CO 2 The content reaches 9 percent, which is far higher than the concentration (0.03 to 0.04 percent) of carbon dioxide in the air, is mainly generated by the respiration of microorganisms, can be dissolved in the crude oil, can reduce the viscosity of the crude oil, and has the oil displacement effect.
Example 6: preparation of Strain Y23 fermentation broth and bacterial suspension
The preparation method of the fermentation liquor comprises the following steps: inoculating strain Y23 into LB liquid culture medium, standing or shake culturing on shaking table of 120 rpm, culturing at 16-45deg.C for 12-36 hr until the bacterial concentration reaches 10 9 -10 10 individual/mL fermentation broth.
And a second fermentation liquid preparation method: inoculating the strain Y23 into LB liquid culture medium, shake culturing on a shaking table of 120 rpm to logarithmic phase; transferring the strain to a sterile fermentation culture medium (comprising disodium hydrogen phosphate 0-1 g, potassium dihydrogen phosphate 0-1 g, sodium nitrate 0-10 g, ferrous sulfate 0-1 g, magnesium sulfate hexahydrate 0-1 g, calcium chloride 0.01 g, yeast extract 0.01 g and molasses 0-10 g) according to an inoculum size of 5%, culturing at 16-45deg.C for 12-36h, and dissolving oxygen amount of 0-10mg/L; after the cultivation is finished, the bacterial concentration reaches 10 9 individual/mL fermentation broth.
Preparation of bacterial strain Y23 suspension: diluting the bacterial liquid prepared by the first fermentation liquid preparation method and the second fermentation liquid preparation method according to a certain proportion to obtain bacterial suspension with corresponding concentration; or the bacterial liquid prepared by the first fermentation liquid preparation method and the second fermentation liquid preparation method is filtered or centrifuged to collect bacterial bodies, and then the bacterial bodies are added into the corresponding solution according to a certain proportion to obtain bacterial suspension with corresponding concentration.
Claims (7)
1. Pseudomonas stutzeriPseudomonas stutzeriY23, its characterized in that its microorganism preservation number is CGMCC No.24240.
2. Pseudomonas stutzeri according to claim 1Pseudomonas stutzeriY23The application of the bacterial suspension or the bacterial agent thereof in microbial oil extraction or in the preparation of microbial oil extraction products.
3. Pseudomonas stutzeri according to claim 1Pseudomonas stutzeriThe application of Y23, bacterial suspension thereof or bacterial agent thereof in sewage treatment or in preparing products or equipment for sewage treatment, wherein the sewage treatment refers to degradation of nitrate nitrogen in wastewater.
4. A product for microbial oil recovery or sewage treatment comprising the Pseudomonas stutzeri according to claim 1 as an active ingredientPseudomonas stutzeriY23 or a bacterial suspension thereof.
5. A product for microbial oil recovery or sewage treatment comprising the Pseudomonas stutzeri according to claim 1 as an active ingredientPseudomonas stutzeriMicrobial inoculum of Y23.
6. A method for microbial enhanced oil recovery comprising the steps of: during oil recovery, pseudomonas stutzeri according to claim 1Pseudomonas stutzeriThe fermentation liquor and the nutrient of Y23 are injected into the stratum from the oil sleeve annulus of the oil well, the anaerobic metabolism of the strain is utilized to produce gas to improve the yield of a single well, and simultaneously, the acidification of the oil reservoir caused by sulfate reducing bacteria is prevented and controlled; or Pseudomonas stutzeri according to claim 1Pseudomonas stutzeriThe fermentation liquor and nutrient of Y23 are injected into the ground by a water injection well, and the bacterium is utilized to metabolize gas in the ground to improve the recovery ratio of crude oil, and simultaneously prevent and control the acidification of oil reservoirs caused by sulfate reducing bacteria.
7. The sewage treatment method is characterized by comprising the following steps of: during sewage treatment, adding the pseudomonas stutzeri according to claim 1Pseudomonas stutzeriBacterial suspension, bacterial agent of Y23 or Pseudomonas stutzeri which is coated on fillerPseudomonas stutzeriY23, the sewage treatment refers to degradation of nitrate nitrogen in the wastewater.
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| WO2012061350A2 (en) * | 2010-11-01 | 2012-05-10 | E. I. Du Pont De Nemours And Company | Methods, strains, and compositions useful for microbially enhanced oil recovery: pseudomonas stutzeri |
| CN103497908A (en) * | 2013-08-26 | 2014-01-08 | 温州大学 | Pseudomonas stutzeri and its culture, immobilization and use |
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| CN110157639A (en) * | 2019-05-14 | 2019-08-23 | 清华大学 | A kind of preparation method and application being resistant to denitrifying bacterium and its microbial inoculum with high salt |
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| WO2012061350A2 (en) * | 2010-11-01 | 2012-05-10 | E. I. Du Pont De Nemours And Company | Methods, strains, and compositions useful for microbially enhanced oil recovery: pseudomonas stutzeri |
| CN103497908A (en) * | 2013-08-26 | 2014-01-08 | 温州大学 | Pseudomonas stutzeri and its culture, immobilization and use |
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| CN110157639A (en) * | 2019-05-14 | 2019-08-23 | 清华大学 | A kind of preparation method and application being resistant to denitrifying bacterium and its microbial inoculum with high salt |
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