CN117586922A - Application of aerobic denitrification synergistic flora in petroleum pollution system - Google Patents
Application of aerobic denitrification synergistic flora in petroleum pollution system Download PDFInfo
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- CN117586922A CN117586922A CN202311711487.XA CN202311711487A CN117586922A CN 117586922 A CN117586922 A CN 117586922A CN 202311711487 A CN202311711487 A CN 202311711487A CN 117586922 A CN117586922 A CN 117586922A
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Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
-
- 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
- C02F3/02—Aerobic processes
-
- 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
-
- 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
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
-
- C—CHEMISTRY; METALLURGY
- 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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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Biotechnology (AREA)
- Water Supply & Treatment (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to an application of aerobic denitrification synergistic bacteria in petroleum pollution systems, wherein the bacteria comprise 2 strains of aerobic denitrification petroleum degradation synergistic bacteria: fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 with a strain preservation number of CCTCC NO: m20231378; microbacterium phyllosum (Microbacterium foliorum) CRH with a strain preservation number of CCTCC NO: m20231379, both of which are mixed in equal proportion. The bacterial colony is a bacterial strain separated and screened from aged oil-contaminated soil for many years, can secrete denitrification reductase and dioxygenase simultaneously under aerobic conditions, and can synchronously carry out oxidation and reduction, so that the degradation rate of nondegradable components in petroleum can be remarkably improved, and a foundation is laid for pollution control of oil-containing sludge, petroleum-contaminated soil, petroleum wastewater and petrochemical products.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and relates to application of an aerobic denitrification synergistic flora in petroleum pollution systems.
Background
As the global industry enters the petroleum era, petroleum becomes a well-known and real industrial blood, and is a catalyst for the modern industry. Petroleum has penetrated into each industry and the massive demand for petroleum and its products has also led to the entry of large amounts of petroleum and its processed products into soil, water. The method brings a serious disaster to the earth animals, plants and human beings. Benzene series and polycyclic aromatic hydrocarbon in petroleum have carcinogenic, teratogenic and mutagenic effects, and threaten food safety, drinking water safety and human health. Leading to ecological system imbalance and species diversity crisis. In particular, the oily sludge of the oil field contains a large amount of toxic substances with malodor such as benzene series, phenols, anthracene, pyrene and the like, if the oily sludge is directly discharged, a large amount of cultivated land is occupied, and serious pollution is caused to soil, water and air, and the oily sludge also contains a large amount of pathogenic bacteria, parasites (eggs), heavy metals such as copper, zinc, chromium, mercury and the like, and durable carcinogens such as salts, polycyclic aromatic hydrocarbons and the like. The nation has classified oily sludge as a hazardous solid waste requiring forced treatment. The treatment of oily sludge has been a major concern for each field and is a first problem that plagues the oil industry for environmental safety.
With the economic development, petroleum products are used in a large amount in industry and agriculture, petroleum and products thereof enter the environment in a large amount, the environmental quality is unprecedented to deteriorate, the human survival is threatened, and the treatment is urgently needed. The microorganism has the characteristics of strong vitality, multiple metabolic types, large specific surface area and rapid propagation, thereby playing an increasingly important leading role in the repair of the polluted environment worldwide. The natural homing of petroleum in natural environment is degradation by microorganisms, complete mineralization of petroleum hydrocarbon and conversion into CO 2 And H 2 O, etc., which is the result of natural evolution of billions of years of manufacturers, the use of microbiological techniques to treat petroleum pollution is the most scientific choice. Microbial remediation technology has been honored by the world academy as a green treatment technology for petroleum contaminated soil, and the U.S. national Environmental Protection Agency (EPA) publication shows that: more than 48% of the soil remediation programs supported by the U.S. super-fund program after 2000 use microbial remediation technology. Microbial remediation in the european union soil remediation program accounts for 35% of the market share, with in situ biological treatment accounting for 18.33% and ectopic biological treatment accounting for 16.67%. As the main body of the environment restoration technology, the microbial treatment technology plays a role as a killer in soil restoration by the advantages of high efficiency, low cost, safe process, simple and convenient operation, no secondary pollution to the environment and the like. However, the microbial remediation technology of China has the bottleneck problems of long remediation period, low degradation efficiency, low tolerance to toxic substances and the like all the time because of wide soil types, complex structure, large climate difference, a large amount of biomass inhibition and other limiting factors in China. Therefore, by utilizing the advantage of rich microbial resources in China, a set of high-efficiency engineering bacteria agent for treating the hazardous waste of soil, water and oil sludge is developed, and the improvement of the biodegradation efficiency is the key for solving the treatment of petroleum and petrochemical organic pollution.
At present, biotechnology is the most widely used method for treating petroleum pollution. The bioremediation technology achieves the effect of thoroughly mineralizing pollutants by utilizing biological enzymes of microorganisms, does not produce secondary pollution, and achieves good application effect in some foreign petroleum pollution accidents in recent years. However, for the macromolecular normal paraffins (paraffin), heterocycles, aromatic hydrocarbons and other nondegradable organic components in petroleum, the traditional biological aerobic treatment effect is not obvious, and particularly for the petroleum components such as halohydrocarbons, asphaltenes, colloid and the like, the existing aerobic technology is not ideal. Therefore, it is necessary to design a bacterial population which can be degraded efficiently under aerobic conditions.
Disclosure of Invention
The first object of the invention is to provide an aerobic denitrification synergistic flora, which overcomes the problem of pain points with unsatisfactory petroleum biodegradation effect.
A second object of the present invention is to provide the use of the above-mentioned flora in the biological treatment of petroleum pollution.
It is a third object of the present invention to provide a method for the biological remediation of petroleum pollution by the above-mentioned flora.
The invention is realized by the following technical scheme:
1. an aerobic denitrification synergistic flora, the flora comprising 2 strains of aerobic denitrification petroleum degrading bacteria: fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 with a strain preservation number of CCTCC NO: m20231378; microbacterium phyllosum (Microbacterium foliorum) CRH with a strain preservation number of CCTCC NO: m20231379, both of which are mixed in equal proportion.
2. According to the application of the flora, the flora is applied to the biological treatment of petroleum pollution.
Further, the petroleum pollution biological treatment comprises oil-containing sludge treatment, petroleum pollution soil treatment and petroleum wastewater treatment. Wherein the oily sludge comprises floor oil sludge, tank bottom sludge and oil-based drilling mud; petroleum contaminated soil includes petroleum and petroleum-based organic contaminated arable land, grassland, woodland, mines (coal mine and petroleum mining area restoration, in particular oil well site in situ restoration); the petroleum wastewater comprises petroleum industrial wastewater, petroleum collecting water, fracturing fluid, tank washing water and mineral oil polluted surface water.
3. According to the application, the method for treating the petroleum pollution organisms by the flora comprises the following steps: adding flora fermentation liquor into petroleum pollutant in the proportion of 0.01-10% by weight, and mixing with phthalic acid: methanol=1:8 is a co-metabolism substrate, the addition amount is 0.45-45 g/T, nitrate nitrogen is added as an activator, and C: n=4:1-12:1, the temperature is kept at 30-35 ℃, and the repair is completed within 4-30 days.
Further, the flora fermentation liquid is obtained by inoculating equal amounts of Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 and Microbacterium phyllosum (Microbacterium foliorum) CRH,180r/min on a culture medium by an inoculating needle, culturing for 48 hours at 30 ℃, pouring into a fermentation tank, fermenting at 30 ℃, performing aerobic aeration to keep dissolved oxygen at about 8mg/L, stirring at a rotation speed of 120r/min, and fermenting for 48 hours.
The technical scheme has the advantages that: the Rhodococcus erythropolis (rhodococcus_qingshengii) PW03 and the microbacterium phyllosum (Microbacterium foliorum) CRH are bacterial strains separated and screened from aged greasy soil for many years, can secrete denitrification reductase and dioxygenase simultaneously under aerobic conditions, synchronously carry out oxidation and reduction, can obviously improve the degradation rate of refractory components in petroleum, and lays a foundation for pollution control of oily sludge, petroleum polluted soil, petroleum waste water and petrochemical products; the synergistic flora and the metabolic regulation method thereof provide an innovative green clean treatment technology for innocent treatment of petroleum pollution soil, oil-containing sludge dangerous waste and petroleum petrochemical pollution in China.
The important innovation point 1 of the invention is as follows: the flora successfully solves the pain point of high-concentration oxygen electron acceptor stress in the aerobic denitrification process, and can start the metabolic pathway of degrading petroleum hydrocarbon by aerobic denitrification under the condition of saturated dissolved oxygen;
the important innovation point 2 of the invention is as follows: the aerobic denitrification process can produce intermediate metabolite nitrite nitrogen accumulation, and the high-concentration nitrite nitrogen can exert physiological stress on microbial cells to terminate the denitrification process. And the microbacterium phyllosum Microbacterium foliorum CRH can take 2g/L nitrous acid nitrogen as the only nitrogen source, and degrade petroleum substances through aerobic denitrification. Therefore, the complex flora constructed by CRH and PW03 can completely and efficiently denitrify and degrade petroleum under the condition of saturated dissolved oxygen, and the reaction is not stressed by nitrite nitrogen and dissolved oxygen;
the important innovation point 3 of the invention is as follows: the aerobic denitrification coupling co-metabolism petroleum degradation technology starts an aerobic denitrification metabolism path through nutrition regulation and control, and synchronously starts biological enzyme oxidation, biological enzyme reduction and co-metabolism, wherein the petroleum removal rate is up to more than 99% in 4-30 days. The composite flora provided by the invention is widely applied, and can be used in the fields of harmless treatment of dangerous wastes such as oil-containing sludge, bioremediation of organic contaminated soil such as petroleum, advanced treatment of oil-contaminated water such as petroleum water collection and the like, and environmental remediation and treatment.
Drawings
FIG. 1 is a streak culture plot of Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 at Fan Qingsheng;
FIG. 2 is a pure culture diagram of Rhodococcus rhodochrous (Rhodococcus_qingshengii) PW03 of Fan Qingsheng;
FIG. 3 is a streak culture plot of micro-bacillus phyllorum (Microbacterium foliorum) CRH;
FIG. 4 is a pure culture diagram of Microbacterium phyllosum (Microbacterium foliorum) CRH;
FIG. 5 is a 16S rRNA-based evolutionary branch tree of Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 of Fan Qingsheng;
FIG. 6 is a 16S rRNA based evolutionary branch tree of Microbacterium phyllosum (Microbacterium foliorum) CRH;
FIG. 7 is a graph showing Total Nitrogen (TN) removal rate;
FIG. 8 is a schematic of petroleum hydrocarbon removal rate.
The invention relates to Fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 which is subjected to patent program preservation in China patent office or International patent organization (International) acknowledged preservation center in 2023, namely China Center for Type Culture Collection (CCTCC) for short, wherein the preservation unit address is: chinese, university of armed chinese, accession number: cctccc NO: m20231378.
The invention relates to a microbacterium phyllorum (Microbacterium foliorum) CRH which is subjected to patent program preservation in China general microbiological culture Collection center (CCTCC) at 25 th of 2023 in the following month 07 and is admitted by China general purpose culture Collection center (China center for type culture collection), wherein the preservation unit is called as CCTCC for short, and the address of the preservation unit is: chinese, university of armed chinese, accession number: cctccc NO: m20231379.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but should not be construed as limiting the present invention:
example 1
This example illustrates the screening of bacterial species.
Strain screening culture medium:
NO 3 - -BTB modified basal medium: KNO (KNO) 3 20.0g/L, 0.5g/L, CH 3 OH 4.0g/L，MgS0 4 ·7H 2 0 1.0g/L，FeCl 3 ·6； 2 0 0.05g/L，KH 2 PO 4 1.0g/L，CaCl 2 ·2H 2 0.2g/L, 1mL of 1% BTB alcohol solution, 0.5mL of trace elements, 20g of agar and 1000mL of deionized water.
The trace element solution comprises EDTA 50.0g/L and ZnS0 4 2.2g/L，CaCl 2 5.5g/L，MnCl 2 ·4H 2 0 5.0g/L，FeS0 4 ·7H 2 0 5.0，CuS0 4 ·5H 2 0.57 g/L, 1000mL deionized water, and adjusting the pH to 7.0.
Screening strains:
(1) Enrichment of petroleum degrading bacteria: taking 50g of perennial aged oil sludge of Daqing oilfield, and inoculating with petroleum as unique carbon source, NO 3 - The BTB modified culture medium is subjected to liquid culture, 180rpm and 30 ℃ for 3d, 1mL of culture solution is taken and is connected with the fresh enrichment culture medium to continue culture, and the culture is repeated for a plurality of times until the batch change of petroleum emulsification efficiency is small (visual), and enrichment is stopped.
(2) Separation of petroleum degrading bacteria: adopting a plate scribing separation method, dipping the enriched liquid in NO in an ultra-clean workbench by using an inoculating loop 3 - Streaking on the BTB complete medium plate, resting at 30℃for 5d, further picking single colonies, repeatedly streaking until the end single colonies are completely isolated. The single colony was transferred to a test tube and kept at 5℃until use.
(3) Screening petroleum degrading bacteria: the isolated strains were inoculated into liquid medium (NO 3 - -replacing carbon source with petroleum based on BTB modified culture medium without adding agar powder), culturing with initial petroleum hydrocarbon content of 10g/L,30 ℃,180rpm, shake culturing with shaking table, measuring petroleum hydrocarbon content of 7d culture medium, calculating petroleum hydrocarbon removal rate, and measuring petroleum in parallel of each experimental group, wherein petroleum is measured by infrared spectrophotometry for soil petroleum determination of national environmental protection standard of the people's republic of China (HJ 1051-2019). Finally, two high-efficiency degrading bacteria PW03 and CRH are obtained, and the strain is sent to Shanghai worker for strain species identification, the plate culture of Rhodococcus erythropolis (Rhodococcus qingshengii) PW03 of Fan Qingsheng is shown in figures 1 and 2, and the plate culture of Microbacterium phyllosum (Microbacterium foliorum) CRH is shown in figures 3 and 4.
(4) Sequencing bacterial PW03 genome (with sequence shown as SEQ ID No. 1) by using a Shanghai worker second generation whole genome sequencing technology method, and further carrying out species identification and obtaining identification results of species level.
16S rRNA-based phylogenetic tree: the predicted 16s rRNA sequence of the gene was aligned with the 16s database of NCBI using blast+ and parameter identification >95 was set. Then, the first 30 16s rRNA sequences with highest idetify (full extraction if not enough) are selected, and after multiple comparison and cutting of sequences are carried out by using the mafft software, a phylogenetic tree is constructed by using FastTree/iqtree/raxml software, as shown in figure 5.
And (3) carrying out inference by combining the results of the second-generation sequencing genome comparison, the 16SrRNA comparison information, ANI analysis and the like: the best matching strains are searched in the NT library: rhodococcus_qingshengii_PM1GCA_ 025370975.1,ANI index was 98.9001%, which exceeds 95%, and seed levels could be identified. The species to which the sample belongs is presumed to be: rhodococcus qingshengii is designated Fan Qingsheng Rhodococcus erythropolis (Rhodococcus qingshengii) PW03.
(5) The bacterial CRH genome is sequenced (the sequence is shown as SEQ ID No. 2) by adopting a mature second-generation whole genome sequencing technical method of Shanghai, so that species identification is carried out, and an identification result of the species level is obtained.
16S rRNA-based phylogenetic tree: the predicted 16s rRNA sequence of the gene was aligned with the 16s database of NCBI using blast+ and parameter identification >95 was set. Then, the first 30 16s rRNA sequences with highest idetify (full extraction if not enough) are selected, and after multiple comparison and cutting of sequences are carried out by using the mafft software, a phylogenetic tree is constructed by using FastTree/iqtree/raxml software, as shown in figure 6.
And (3) carrying out inference by combining the results of the second-generation sequencing genome comparison, the 16SrRNA comparison information, ANI analysis and the like: the best matching strains are searched in the NT library: microbacterium_foolium_ 122GCA_002024885.1,ANI index is 98.8393%, which exceeds 95%, and can be identified at a seed level. The species to which the sample belongs is presumed to be: microbacterium_folium was designated as Microbacterium phyllum (Microbacterium foliorum) CRH.
(6) Determination of total nitrogen removal rate: initial petroleum hydrocarbon concentration 20g/L, initial nitrogen source: 2.6g/L of nitrate nitrogen, the other components are unchanged (refer to NO 3 - BTB medium), 200mL of bacterial liquid per experimental assembly, 3 in parallel. Wherein the experimental group comprises PW03 group, CRH group and PW03+CRH group, the same batch of culture medium is used as blank reference liquid, PW03 and CRH bacterial liquid fermented for 48h are taken during measurement, and diluted with sterilized deionized water and OD is obtained 600 The values are adjusted to be the same, and the standard error is less than 0.1%, wherein the PW03+CRH group bacterial liquid is formed by mixing 100mL of PW03 and 100mL of CRH bacterial liquid respectively. The contents of 0d, 3d, 5d, 7d and 9d Total Nitrogen (TN) are respectively measured, and the Total Nitrogen (TN) is measured by adopting the method of ultraviolet spectrophotometry for digestion of alkaline potassium persulfate for the determination of total nitrogen in water quality, HJ636-2012 of national environmental protection standard of the people's republic of China, wherein TN removal rate= [ initial total nitrogen concentration (0 dTN content) -measured total nitrogen concentration]Total nitrogen initial concentration (0 dTN content) ×100% and the experimental results are shown in fig. 7.
(7) Determination of Petroleum Hydrocarbon removal Rate: initial petroleum hydrocarbon concentration 20g/L, initial nitrogen source: 2.6g/L of nitrate nitrogen, the other components are unchanged (refer to NO 3 - -BTB medium), 200mL of each experimental assembly liquid, wherein the experimental group comprises PW03 group, CRH group, PW03+CRH group and medium blank control group, PW03 and CRH bacterial liquid fermented for 48h are taken during measurement, and then the experimental group is prepared byDilution with sterile deionized water and OD 600 The values are adjusted to be the same, and the standard error is less than 0.1%, wherein the PW03+CRH group bacterial liquid is formed by mixing 100mL of PW03 and 100mL of CRH bacterial liquid respectively. Respectively measuring 0d, 3d, 5d, 7d and 9d, wherein the residual petroleum hydrocarbon content of the soil is 15 in parallel, randomly taking 3 parallel experiment groups each time, drying at 45 ℃ to remove water, respectively measuring the petroleum hydrocarbon content of the dry soil, wherein the petroleum content of the soil is measured, and the method adopts the infrared spectrophotometry for measuring the petroleum in the national environmental protection standard of the people's republic of China, HJ1051-2019, wherein the petroleum hydrocarbon removal rate= [ initial concentration-measured concentration ]]Initial concentration 100%, and the experimental results are shown in fig. 8.
As can be seen from the figure, fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 and Microbacterium phyllosum (Microbacterium foliorum) CRH form a synergistic interaction relationship, and under the combined action of the Rhodococcus erythropolis PW03 and the Microbacterium phyllosum, the petroleum degradation efficiency is greatly improved, and the removal rate of the petroleum hydrocarbon of the compound flora of the CRH and PW03 reaches 97.1 percent in 9 days.
Example 2
This example illustrates the preparation of a bacterial flora broth.
1. Preparation of the culture medium: KNO (KNO) 3 8.0g/L, sodium citrate 4g/L, phthalic acid 0.5g/L, CH 3 OH 4.0g/L，MgS0 4 ·7H 2 0 1.0g/L，FeCl 3 ·6； 2 0 0.05g/L，KH 2 PO 4 1.0g/L，CaCl 2 ·2H 2 0.2g/L, 1mL of 1% BTB alcohol solution, 0.5mL of trace elements, and 1000mL of deionized water. The trace element solution comprises EDTA 50.0g/L and ZnS0 4 2.2g/L，CaCl 2 5.5g/L，MnCl 2 ·4H 2 0 5.0g/L，FeS0 4 ·7H 2 0 5.0，CuS0 4 ·5H 2 0.57 g/L, 1000mL deionized water, and adjusting the pH to 7.0.
2. Preparation of the parent: and (3) subpackaging 150mL of the prepared culture medium into 250mL conical flasks, covering tightly by using a silica gel plug or a cotton sieve, and sealing by using a sealing film. Sterilizing at 121deg.C for 20min with high pressure steam. After the medium had cooled to room temperature, the culture was aseptically handled on an ultra clean bench, 1 Erlenmeyer flask was dipped with the ring of inoculating needle in each of the Fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 and Microbacterium phylloides (Microbacterium foliorum) CRH of claim 1, and rinsed into the Erlenmeyer flask medium, incubated at 180r/min at 30℃for 48 hours, and the culture was observed and if the clear medium indicator showed blue color, the strain was proved to be usable.
3. Liquid submerged fermentation: transferring the mother strain into a fermentation tank for liquid submerged fermentation, wherein the fermentation parameter temperature is 30 ℃, the dissolved oxygen is 8mg/L or so, the floating up and down is preferably not more than 1mg/L, the initial pH7.5 of the culture medium is achieved, the stirring speed is 120r/min, the fermentation is carried out for 48 hours, and the pH is controlled to be between 7.5 and 8.5 by automatically regulating the acid and the alkali. As the fermentation proceeds, the pH will rise and the fermentation broth will change from green to blue and the fermentation broth will be clear and normal.
Example 3
This example illustrates a method for innocent treatment of oily sludge using a bacterial population.
(1) Fine picking up device
Mechanical fine picking is adopted to remove impurities, and stone, brick blocks and plastic products are removed by using a sieve with multiple layers of apertures, so that the grain size of the oil sludge is smaller than 2mm.
(2) Hot washing
The first step is water washing desorption: the initial concentration of the petroleum hydrocarbon of the oil sludge is 20-35%, the water-soil ratio is 1:3-1:5, the water temperature is 50-60 ℃, the pH value is 7.0-9.5, the mechanical stirring is carried out, the rotating speed is determined according to the actual materials, the materials are ensured to be fully and uniformly mixed, and the time is controlled to be 1-5 min; standing for 10-15 min to thoroughly layer the oil-water mud.
And step two, air floatation demulsification: the pressurized dissolved air floatation technology is adopted to release air bubbles with the size of 20-40 microns at the lower layer of the water phase, and the characteristics of the floating air bubbles carrying charges to absorb organic matters are utilized to physically demulsify. Finally, scraping crude oil components on the water phase surface by overflow or a mechanical scraper, repeating the process (when the thickness of an oil layer on the water phase surface is less than or equal to 0.1 mm), stopping the operation procedure, and at the moment, the petroleum hydrocarbon content of the oil sludge is about 15%, and the thermal washing removal rate of the petroleum hydrocarbon is 34-57%. The lower layer material is transferred into a slurry reactor for biological treatment.
(3) Biological treatment
The sludge after the crude oil is recovered by hot water washing is transferred into a slurry reactor through a slurry pump, and the bacterial community is added for biological treatment. Modulating sludge and water 1: 3-1: 4, adding the flora fermentation liquor according to the proportion of 10 percent by weight at 35 ℃, stirring at 59rpm, and adjusting pH 7.0-9.5 to obtain phthalic acid: methanol=1:8 as co-metabolizing substrate, added in an amount of 4.5g/T, and nitrate nitrogen as activator to let C: n=4:1. According to different sources and characteristics of the oil sludge, oil scraping is carried out for 1 time in 2-12 hours, the whole biological treatment is completed within 4 days, the treated oil-containing sludge needs to be subjected to filter pressing or centrifugal dehydration, the concentration of petroleum hydrocarbon in the dehydrated sludge is less than or equal to 3 per mill, the oil-containing sludge comprehensive utilization pollution control standard of oil fields in Heilongjiang province is reached, the requirements related to the solid waste pollution control method of the people's republic of China are met, and the petroleum hydrocarbon removal rate of a biological treatment program can reach 98 percent at most.
Example 4
This example illustrates the method of the flora to treat soil in petrochemical high-concentration organic contaminated sites.
Step 1: hot washing, namely, preparing the stripped surface layer polluted soil by hot water at 50-75 ℃, wherein the mud-water ratio is 1: 3-1: 5, according to the nature of the polluted chemical, preparing a proper pH value, mechanically stirring and uniformly mixing for 1-3min, standing for 10-15 min, and removing the upper organic matters for 30 min/batch. The pressurized dissolved air floatation technology is adopted to release air bubbles with the size of 20-40 microns at the lower layer of the water phase, and the characteristics of the floating air bubbles carrying charges to absorb organic matters are utilized to physically demulsify. Finally, the organic phase component on the water phase surface is scraped by overflow or a mechanical scraping plate, and the process is repeated (the thickness of the oil layer on the water phase surface is less than or equal to 0.1 mm). And stopping the operation procedure, wherein the removal rate of the nonpolar organic petrochemical pollutants in the process is about 30-60% on average.
Step 2: chemical preoxidation, transferring the lower layer mud water into a stainless steel oxidation tank, regulating pH value to 7.0-9.5, adding H 2 O 2 Adding O 3 ，O 3 :H 2 O 2 1 g, residence time of 5-30 min (determined by subsequent biochemistry), stirring at 59rpm for 5-10 min, standing for 30min, air-floating scraping upper layer foam, press-filtering and dewatering the material for later use, chemical preoxidation efficiency is low, but 2.8ev hydroxyl free radical is generated, the hydroxyl free radical has strong oxidizing and electrophilic properties, O 3 And H is 2 O 2 Bonding ofMake up for O 3 The defect of low gas dissolution efficiency can open the conjugated double bonds and large pi bonds of macromolecular structures such as organic chlorine, organic phosphorus, polycyclic aromatic hydrocarbon and the like which are difficult to degrade, thereby creating conditions for subsequent biochemical treatment.
Step 3: biological cleaning and deep degradation of sludge, adding flora fermentation liquor according to the proportion of 1% by weight, and reacting under the system condition: 59rpm, controlling the temperature at 33 ℃, and controlling the pH value at 7.0-9.5, wherein the reaction product is prepared by the following steps of: methanol=1:8 as co-metabolizing substrate, added in an amount of 4.5g/T, and nitrate nitrogen as activator to let C: n=4:1, pressurized dissolved gas micron air flotation demulsification is carried out in the period, mud water is subjected to filter pressing and dehydration after repair is completed for 15 days, straw leavening agent is added into dehydrated sludge, humic acid is 0.01%, stirring is carried out uniformly, water body recycling is carried out, and petrochemical organic pollutant removal rate reaches 60-99%.
Example 5
This example illustrates the method of treating petroleum contaminated water by the flora.
Step 1, biological flocculation; regulating pH of water body to 7.0-9.5 with lime and waste hydrochloric acid, controlling temperature at 33 deg.c, aeration stirring for 30min, and flocculating polyacrylamide polymer with charge and large floc inclined plate settlement. The upper liquid is discharged into a pressurized dissolved air floatation unit, the air floatation is carried out for removing impurities, a scraping plate pushes and flows to remove micro-flocs and oil foam, the process is repeated for 1 to 3 times, and the supernatant fluid is discharged into the next procedure.
Step 2, O 3 +H 2 O 2 Preoxidation, O 3 :H 2 O 2 =3:10, high concentration O 3 Oxidizing the residual polyacrylamide for 30min, and consuming the residual O still 3 。
Step 3, biological advanced treatment, wherein the flora fermentation liquor is added according to the weight percentage of 0.01%, and phthalic acid is used for: methanol=1:8 is a co-metabolizing substrate, added in an amount of 0.45g/T, and nitrate nitrogen is added as activator to let C: n=12:1, and the temperature is controlled to be about 33 ℃. After aerobic culture for 4-8 h, residual pollutants are synchronously degraded by aerobic denitrification of the flora, pressurized dissolved air flotation demulsification is carried out, upper foam is removed by scraping plate plug flow, the supernatant is discharged into a sand filter, a carbon filter is used for filtration and recycling, the whole bioremediation time is about 7 days, the petroleum hydrocarbon content of petroleum pollution water is lower than 5mg/L, and the suspended matters meet the standard of reuse water.
Example 6
This example illustrates a method for in situ treatment of petroleum contaminated soil by a bacterial population.
And 1, stripping high-concentration greasy dirt soil, leveling low-concentration greasy dirt soil, ploughing and leveling the greasy dirt soil to be repaired, and removing sundries such as plastic products, large stones and the like for later use.
Step 2, adding a treatment repair microbial inoculum, and adding a flora fermentation liquor according to the weight percentage of 0.1%, wherein the fermentation liquor comprises the following components in percentage by weight: methanol=1:8 is a co-metabolizing substrate, added in an amount of 45g/T, and nitrate nitrogen is added as activator to let C: and N=8:1, mechanically rotary tillage and uniformly stirring, wherein the rotary tillage depth is controlled within 35 cm.
And step 3, watering, and keeping the humidity of the soil to be repaired at 15-35%.
And step 4, controlling the temperature, and keeping the optimal soil temperature at 30 ℃. The bioremediation process is completed every 3-7 d by turning the soil for 1 time and 30 days, the field test is carried out near two mine lifting 2935 and 39 inclined 33 well sites in eight factories of Daqing oil extraction, the field test is carried out for 30d, and the ecological function of the soil can be recovered in the current year. The concentration of petroleum hydrocarbon in the restored soil is reduced from 1-2% to 1-2%.
According to the invention, a special microorganism Fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 and a microbacterium phyllosum (Microbacterium foliorum) CRH innovatively metabolize and regulate means, and a nitrate reductase system and a aerobic oxidase system are synchronously started on the premise of saturated dissolved oxygen stress. The aerobically degraded dioxygenase system and the reductively degraded nitrate reductase system are allowed to function simultaneously. The nitrite reductase has remarkable effect on the decyclization of electron-withdrawing R functional groups, so that the strain has an important effect on the dehalogenation of halogenated hydrocarbons, the ring opening of heterocycle and the electron-withdrawing functional groups of benzene rings. By adopting the aerobic denitrification synergistic flora and the metabolic petroleum regulation method, petroleum and petrochemical products can be rapidly degraded by the flora. The bacterial colony is a bacterial strain separated and screened from aged greasy soil for many years, can secrete denitrification reductase and dioxygenase simultaneously under aerobic conditions, and synchronously performs oxidation and reduction, so that the degradation rate of nondegradable components in petroleum can be remarkably improved, and a foundation is laid for oil-containing sludge, petroleum polluted soil, petroleum wastewater and petrochemical pollution treatment; the symbiotic flora and the metabolic regulation method thereof provide an innovative green clean treatment technology for innocent treatment of petroleum pollution soil, oil-containing sludge dangerous waste and petroleum petrochemical pollution in China.
Claims (5)
1. An aerobic denitrification synergistic flora, which is characterized in that: the flora comprises 2 strains of aerobic denitrification petroleum degradation synergistic bacteria: fan Qingsheng Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 with a strain preservation number of CCTCC NO: m20231378; microbacterium phyllosum (Microbacterium foliorum) CRH with a strain preservation number of CCTCC NO: m20231379, both of which are mixed in equal proportion.
2. The use of a flora according to claim 1, wherein: the application of the flora in biological treatment of petroleum pollution.
3. The use according to claim 2, characterized in that: the petroleum pollution biological treatment comprises oil-containing sludge treatment, petroleum pollution soil treatment and petroleum wastewater treatment.
4. Use according to claim 2, characterized in that: the method for treating the petroleum pollution organisms by the flora comprises the following steps: adding flora fermentation liquor into petroleum pollutant in the proportion of 0.01-10% by weight, and mixing with phthalic acid: methanol=1:8 is a co-metabolism substrate, the addition amount is 0.45-45 g/T, nitrate nitrogen is added as an activator, and C: n=4:1-12:1, the temperature is kept at 30-35 ℃, and the repair is completed within 4-30 days.
5. The use according to claim 4, characterized in that: the flora fermentation liquor is obtained by inoculating equal amounts of Rhodococcus erythropolis (Rhodococcus_qingshengii) PW03 and Microbacterium phyllosum (Microbacterium foliorum) CRH with an inoculating needle on a culture medium, culturing at 30 ℃ for 48 hours, pouring into a fermentation tank, fermenting at 30 ℃, performing aerobic aeration to keep dissolved oxygen at about 8mg/L, stirring at a rotation speed of 120r/min, and fermenting for 48 hours.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342392A (en) * | 2014-10-30 | 2015-02-11 | 中国石油天然气股份有限公司 | Microbacterium oxydans for degrading polycyclic aromatic hydrocarbon and application thereof |
| CN105018390A (en) * | 2015-08-05 | 2015-11-04 | 天津翌石开元科技发展有限公司 | Rhodococcus erythropolis and application thereof |
| CN113957018A (en) * | 2021-11-30 | 2022-01-21 | 自然资源部第一海洋研究所 | Flora with petroleum degrading function under low temperature condition and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342392A (en) * | 2014-10-30 | 2015-02-11 | 中国石油天然气股份有限公司 | Microbacterium oxydans for degrading polycyclic aromatic hydrocarbon and application thereof |
| CN105018390A (en) * | 2015-08-05 | 2015-11-04 | 天津翌石开元科技发展有限公司 | Rhodococcus erythropolis and application thereof |
| CN113957018A (en) * | 2021-11-30 | 2022-01-21 | 自然资源部第一海洋研究所 | Flora with petroleum degrading function under low temperature condition and application thereof |
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