CN117586922B - Application of aerobic denitrification synergistic flora in petroleum pollution system - Google Patents

Application of aerobic denitrification synergistic flora in petroleum pollution system Download PDF

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CN117586922B
CN117586922B CN202311711487.XA CN202311711487A CN117586922B CN 117586922 B CN117586922 B CN 117586922B CN 202311711487 A CN202311711487 A CN 202311711487A CN 117586922 B CN117586922 B CN 117586922B
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慕庆峰
沈斯
刘聪
徐晶
宋有春
李万海
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Daqing Jindi Environmental Remediation Co ltd
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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 (rhodococcus_ qingshengii) PW03, the strain preservation number of which is CCTCC NO: m20231378; microbacterium phyllosum (Microbacterium foliorum) CRH with a strain preservation number of CCTCC NO: m20231379, and mixing the two 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

Application of aerobic denitrification synergistic flora in petroleum pollution system
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, conversion into CO 2, H 2 O, etc., which are the result of natural evolution of hundreds of millions of years of manufacturers, so that the treatment of petroleum pollution by microbial technology 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 colony 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 (rhodococcus_ qingshengii) PW03, the strain preservation number of which is CCTCC NO: m20231378; microbacterium phyllosum (Microbacterium foliorum) CRH with a strain preservation number of CCTCC NO: m20231379, and mixing the two 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 pollutants according to the weight percentage of 0.01-10%, and using 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, maintaining the temperature at 30-35 ℃ and finishing the repair 4-30 days.
Further, the flora fermentation liquid is obtained by inoculating equal amounts of Fan Qingsheng Rhodococcus rhodochrous (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.
The technical scheme has the advantages that: the Rhodococcus Fan Qingsheng (rhodococcus_ qingshengii) PW03 and the bacillus phyllorum (Microbacterium foliorum) CRH are strains separated and screened from aged oil-contaminated 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 components difficult to degrade in petroleum, and lay 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 phyllobacterium 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 Fan Qingsheng (Rhodococcus_ qingshengii) PW 03;
FIG. 2 is a pure culture diagram of Rhodococcus Fan Qingsheng (Rhodococcus_ qingshengii) PW 03;
FIG. 3 is a streak culture plot of a colony of Microbacterium phyllosum (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 Fan Qingsheng (Rhodococcus_ qingshengii) PW 03;
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 Rhodococcus Fan Qingsheng PW03 (rhodococcus_ qingshengii) of the present invention is preserved in the collection center of China patent office or International patent organization at 25 th year of 2023, and the collection unit is called China center for type culture Collection, abbreviated as CCTCC, and the address of the collection unit is: chinese, university of armed chinese, accession number: cctccc NO: m20231378.
The invention relates to a Microbacterium phyllosum (Microbacterium foliorum) CRH which is subjected to patent program preservation in China center for patent office or International patent organization (International) at 25 th of 2023, wherein the preservation unit is called China Center for Type Culture Collection (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 3 20.0.0 g/L, phthalic acid 0.5 g/L,CH3OH 4.0 g/L,MgS04·7H20 1.0 g/L,FeCl3·6;20 0.05 g/L,KH2PO4 1.0 g/L,CaCl2·2H20 0.2 g/L,1%BTB alcohol solution 1mL, trace elements 0.5mL, agar 20 g, deionized water 1000 mL.
The trace element solution component :EDTA 50.0 g/L,ZnS04 2.2 g/L,CaCl2 5.5 g/L,MnCl2·4H20 5.0g/L,FeS04·7H20 5.0,CuS04·5H20 1.57 g/L, is deionized water 1000 mL, and the pH value is adjusted to 7.0.
Screening strains:
(1) Enrichment of petroleum degrading bacteria: 50g of aged oil sludge of Daqing oilfield for many years is taken, petroleum is taken as a unique carbon source, NO 3 - -BTB modified culture medium, liquid culture is carried out, 180rpm and 30 ℃ culture are carried out for 3d, 1mL culture solution is taken, fresh enrichment culture medium is taken, culture is continued, and the process is repeated for many times until the batch change of petroleum emulsification efficiency is small (visual) and enrichment is stopped.
(2) Separation of petroleum degrading bacteria: and (3) adopting a plate streak separation method, dipping the enrichment solution in an ultra-clean workbench by using an inoculating loop, streaking on a NO 3 - -BTB complete culture medium plate, standing at 30 ℃ for 5 days, and further picking single colony for repeated streaking until the single colony at the tail end is completely separated. The single colony was transferred to a test tube and kept at 5℃until use.
(3) Screening petroleum degrading bacteria: and (3) respectively inoculating the separated strains into a liquid culture medium with petroleum as a unique carbon source (the petroleum is used for replacing the carbon source on the basis of an NO 3 - -BTB modified culture medium, agar powder is not added), culturing, wherein the initial content of petroleum hydrocarbon is 10g/L, the temperature is 30 ℃, the speed is 180rpm, shake culturing is carried out by a shaking table, the petroleum hydrocarbon content of a 7d culture medium is measured, the petroleum hydrocarbon removal rate is calculated, 3 experiments are parallel, wherein the petroleum is measured, and the infrared spectrophotometry for measuring the petroleum is adopted in HJ1051-2019 of the national environmental protection standard of the people's republic of China. Finally, two high-efficiency degrading bacteria PW03 and CRH are obtained, and are sent to Shanghai engineering for strain species identification, the plate culture of Fan Qingsheng Rhodococcus (Rhodococcus_ qingshengii) PW03 is shown in figure 1 and figure 2, and the plate culture of Microbacterium phyllosum (Microbacterium foliorum) CRH is shown in figure 3 and figure 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 the mafft software is used for carrying out multiple sequence comparison and cutting, and FastTree/iqtree/raxml software is used for constructing a phylogenetic tree, 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 of 98.9001%, which exceeds 95%, can be identified at seed level. The species to which the sample belongs is presumed to be: rhodococcus_ qingshengii was designated Fan Qingsheng Rhodococcus (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 the mafft software is used for carrying out multiple sequence comparison and cutting, and then FastTree/iqtree/raxml software is used for constructing a phylogenetic tree, 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 foliorum-122 GCA_002024885.1,ANI index was 98.8393%, which exceeded 95%, and seed levels could be identified. The species to which the sample belongs is presumed to be: microbacterium_ foliorum 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), 200 mL bacteria solutions are used in each experimental assembly liquid, and 3 bacteria solutions are parallel. The experimental group comprises a PW03 group, a CRH group and a PW03+CRH group, wherein the same batch of culture medium is used as a blank reference solution, PW03 and CRH bacterial solutions fermented for 48 hours are taken for measurement, the PW03 and CRH bacterial solutions are diluted by sterilized deionized water and are adjusted to be the same by OD 600, the standard error is considered to be the same below 0.1%, and the PW03+CRH bacterial solutions are obtained by mixing each 100mL of PW03 and CRH bacterial solutions. The Total Nitrogen (TN) content was measured for 0d, 3d, 5d, 7d, and 9d, respectively, and the Total Nitrogen (TN) content was measured using the basic potassium persulfate digestion uv spectrophotometry for water quality total nitrogen, HJ636-2012, wherein the TN removal rate= [ total nitrogen initial concentration (0 dTN content) -total nitrogen measurement concentration ]/total nitrogen initial concentration (0 dTN content) ×100%, and the experimental result is 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, and other components are unchanged (refer to NO 3 - -BTB medium), wherein each experimental assembly liquid is 200 mL, the experimental group comprises a PW03 group, a CRH group, a PW03+CRH group and a medium blank control group, PW03 and CRH bacterial solutions fermented for 48 hours are taken during measurement, diluted by sterilized deionized water and OD 600 values are adjusted to be the same, and standard errors are considered to be the same below 0.1%, wherein the PW03+CRH bacterial solutions are obtained by mixing PW03 and CRH bacterial solutions respectively by 100 mL. And respectively measuring the residual petroleum hydrocarbon content of the soil by 0d, 3d, 5d, 7d and 9d, taking 15 parallel groups of 3 parallel experiments at random each time, drying at 45 ℃ to remove water, and respectively measuring the petroleum hydrocarbon content of the dry soil, wherein the measurement of the petroleum in the soil is carried out by adopting the infrared spectrophotometry for measuring the petroleum in the soil of the national environmental protection standard of the people's republic of China, wherein the petroleum hydrocarbon removal rate= [ initial concentration-measured concentration ]/initial concentration is 100%, and the experimental result is shown in figure 8.
As can be seen from the figure, fan Qingsheng Rhodococcus (Rhodococcus_ qingshengii) PW03 and Microbacterium phyllosum (Microbacterium foliorum) CRH form a synergistic interaction relationship, under the combined action of the two, the petroleum degradation efficiency is greatly improved, and the removal rate of the petroleum hydrocarbon of the compound bacteria of the CRH and PW03 reaches 97.1 percent in 9 days.
Example two
This example illustrates the preparation of a bacterial flora broth.
1. Preparation of the culture medium: KNO 3 8.0.0 g/L, sodium citrate 4 g/L, phthalic acid 0.5 g/L,CH3OH 4.0 g/L,MgS04·7H20 1.0 g/L,FeCl3·6;20 0.05 g/L,KH2PO4 1.0 g/L,CaCl2·2H20 0.2 g/L,1%BTB alcohol solution 1mL, trace elements 0.5mL, deionized water 1000 mL. The trace element solution component :EDTA 50.0 g/L,ZnS04 2.2 g/L,CaCl2 5.5 g/L,MnCl2·4H20 5.0g/L,FeS04·7H20 5.0,CuS04·5H20 1.57 g/L, is deionized water 1000 mL, and the pH value is adjusted to 7.0.
2. Preparation of the parent: and subpackaging the prepared culture medium 150 mL into 250mL conical flasks, covering with a silica gel plug or cotton sieve, and sealing with a sealing film. Sterilizing at 121deg.C for 20min with high pressure steam. After the medium was cooled to room temperature, the culture was aseptically handled on an ultra clean bench, 1 Erlenmeyer flask was dipped with Fan Qingsheng Rhodococcus (Rhodococcus: qingshengii) PW03 and Microbacterium phyllum (Microbacterium foliorum) CRH, respectively, with an inoculating needle loop, and rinsed into Erlenmeyer flask medium, incubated at 180r/min at 30℃for 48h, and the culture was observed if the clear medium indicator showed blue color, demonstrating that the strain was 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 III
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 is 7.0-9.5, the mechanical stirring is carried out, the rotating speed is determined according to actual materials, the materials are ensured to be fully and uniformly mixed, and the time is controlled to be 1-5 minutes; standing for 10-15 min to thoroughly layer the oil-water mud.
And step two, air floatation demulsification: and releasing 20-40 micron-sized bubbles at the lower layer of the water phase by adopting a pressurized dissolved air floatation technology, and performing physical demulsification by utilizing the characteristic that floating bubbles carry charges to adsorb organic matters. Finally, scraping crude oil components on the water phase surface through 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-off 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 4.5 g/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 met, the related requirements of 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 IV
This example illustrates the method of the flora to treat soil in petrochemical high-concentration organic contaminated sites.
Step 1: heat 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, preparing a proper pH value according to the properties of the polluted chemicals, mechanically stirring and uniformly mixing for 1-3min, standing for 10-15 min, and removing the upper organic matters for 30 min/batch. And releasing 20-40 micron-sized bubbles at the lower layer of the water phase by adopting a pressurized dissolved air floatation technology, and performing physical demulsification by utilizing the characteristic that floating bubbles carry charges to adsorb organic matters. 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 muddy water into a stainless steel oxidation tank, adjusting the pH value to 7.0-9.5, adding H 2O2, adding O 3,O3: H2O2 =0.3-0.5 (g) 1 (g), keeping for 5-30 min (determined by subsequent biochemistry), stirring at 59rpm for 5-10 min, standing for 30min, air floatation scraping off upper foam, press-filtering and dewatering materials for later use, wherein the chemical preoxidation efficiency is not high, but 2.8ev hydroxyl free radicals are generated, the hydroxyl free radicals have strong oxidizing property and electrophilicity, O 3 is combined with H 2O2, the defect of low gas dissolving efficiency of O 3 is overcome, and the conditions for subsequent biochemistry treatment are created by opening nondegradable organic matters such as conjugated double bonds and large pi bonds of macromolecular structures such as organic chlorine, organic phosphorus and polycyclic aromatic hydrocarbon.
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 using phthalic acid: methanol=1:8 as co-metabolizing substrate, added in an amount of 4.5 g/T, and nitrate nitrogen as activator to let C: n=4:1, pressurized dissolved gas micron air flotation demulsification is performed during the period, mud water is subjected to filter pressing dehydration after repair is completed for 15 days, straw leavening agent is added into dehydrated sludge, humic acid is 0.01%, stirring is uniform, water body recycling is performed, and petrochemical organic pollutant removal rate reaches 60-99%.
Example no
This example illustrates the method of treating petroleum contaminated water by the flora.
Step 1, biological flocculation; adjusting pH 7.0-9.5 of the water body by using quicklime and waste hydrochloric acid, controlling the temperature at 33 ℃, aerating and stirring for 30min, and flocculating polyacrylamide polymer by charging metabolites such as biosurfactant, extracellular proteins, polysaccharides and the like generated by flora and settling large floc sloping plates. And discharging the upper liquid into a pressurized dissolved air flotation unit, removing impurities by air flotation, removing micro-flocs and oil foam by scraping plate pushing, repeating for 1-3 times, and discharging the supernatant into the next procedure.
Step 2, pre-oxidizing O 3+H2O2, wherein O 3:H2O2 =3:10, oxidizing residual polyacrylamide with high concentration O 3 for 30min, and standing to consume residual O 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 0.45 g/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 hours, 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 six
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 45 g/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 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 nitrate reductase system and a aerobic oxidase system are synchronously started on the premise of saturated dissolved oxygen stress according to a novel metabolic regulation means of special microorganisms Fan Qingsheng Rhodococcus rhodochrous (Rhodococcus_ qingshengii) PW03 and Microbacterium phyllosum (Microbacterium foliorum) CRH. 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 (rhodococcus_ qingshengii) PW03, the strain preservation number of which is CCTCC NO: m20231378; microbacterium phyllosum (Microbacterium foliorum) CRH with a strain preservation number of CCTCC NO: m20231379, and mixing the two 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 pollutants according to the weight percentage of 0.01-10%, and using 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, maintaining the temperature at 30-35 ℃ and finishing the repair 4-30 days.
5. The use according to claim 4, characterized in that: the flora fermentation liquor is obtained by inoculating equal amounts of Fan Qingsheng Rhodococcus rhodochrous (Rhodococcus_ qingshengii) PW03 and Microbacterium phyllorum (Microbacterium foliorum) CRH on a culture medium by an inoculating needle, culturing at 180r/min for 48h 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 h.
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