CN109097309B - Petroleum hydrocarbon degrading bacteria and fermentation method thereof - Google Patents
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Abstract
The invention discloses a petroleum hydrocarbon degrading bacterium and a fermentation method thereof, wherein the petroleum hydrocarbon degrading bacterium is a brevibacillus brevis, and the strain name is as follows: GWN-2, preserved in China center for culture Collection of microorganisms with the preservation number: CGMCC No. 13903. It is characterized in that the gene sequence is shown in a sequence table. The petroleum hydrocarbon degrading bacteria are inoculated, proliferated and cultured to obtain a petroleum hydrocarbon degrading bacteria product capable of degrading oily sludge, and the oil content of the treated oily sludge is reduced by about 80% in 20 days, so that a new way is provided for the treatment of the oily sludge of the oil field.
Description
Technical Field
The invention relates to a fermentation production method of bacillus for removing oil from sludge, which is particularly suitable for treating oily sludge generated in the fields of exploitation and treatment of crude oil in oil fields, oily rock debris generated in the drilling process and oil falling to the ground, and belongs to the technical field of biology.
Background
The oily sludge is a solid waste which is generated in the processes of petroleum exploitation, oil and gas gathering and transportation, refining and processing and oily sewage treatment and consists of crude oil, water, heavy metals and sludge. The sludge contains a large amount of residual oil, toxic substances such as benzene series, anthracene, phenols, pyrene and the like, and toxic and harmful substances such as heavy metal, polychlorinated biphenyl, kakakaempferia, radioactive elements and the like. On one hand, the petroleum enters the soil to change the composition and structure of soil organic matters, so that the carbon-nitrogen ratio and the carbon-phosphorus ratio are changed, and a soil microorganism area system is changed, and meanwhile, various petroleum hydrocarbons have certain toxicity to microorganisms and have certain inhibition effects on various soil microorganisms and soil enzyme activity. On the other hand, the petroleum-contaminated soil changes the soil characteristics to block the respiration and absorption of plant roots, and meanwhile, high-concentration petroleum contaminants have acute and chronic toxicity to plant growth, affect the metabolism and physiological functions of plants, and directly cause plant death or affect the plant growth.
The biological treatment technology is an effective oily sludge treatment technology. The degradation of petroleum hydrocarbons by natural microbial species present in the soil environment is one of the basic pathways for the elimination of petroleum hydrocarbons and other hydrocarbon contaminants from soil. The microorganisms utilize a biological self-regulation mechanism and a comprehensive purification function of pollutants to treat petroleum hydrocarbon pollutants in soil, so that the petroleum hydrocarbon pollutants are converted and degraded more thoroughly in the biological metabolism process, and CO is finally generated2And water. Therefore, the biological treatment technology is a development direction for treating the oily sludge in the oil field in the future.
The sludge degrading bacteria in the petroleum polluted environment can stimulate proliferation, but the rapid degradation of petroleum hydrocarbon containing oil sludge is difficult to realize depending on the efficiency of proliferation and degradation of endogenous indigenous bacteria. The method is characterized in that sludge degrading bacteria are produced in batches through separation and screening of high-activity petroleum hydrocarbon and industrial fermentation, the content of the sludge degrading bacteria in the oil-containing sludge is increased through external source addition, and the improvement of the degradation efficiency is the key for realizing the oil sludge indigenous biological strengthening technology.
The defects of the prior art are as follows: the biochemical degradation speed of the existing oily sewage or sludge is slow, the degradation efficiency is low, and the culture process of bacteria is complex.
Disclosure of Invention
The invention aims to provide a petroleum hydrocarbon degrading bacterium aiming at the defects in the current oil-containing sludge biodegradation technology, so that the degrading bacterium has the advantages of high efficiency, high speed, less equipment investment, simple flow, easiness in operation, less power consumption and low raw material cost on oil in oil sludge.
Therefore, the invention provides a petroleum hydrocarbon degrading bacterium, which is a Brevibacillus brevis (the Latin literature name is Brevibacillus parbrevibacterium), and the strain name is as follows: GWN-2, deposited in the China Committee for culture Collection of microorganisms, with the deposit number: CGMCC No. 13903.
The gene sequence is shown in the sequence table after sequencing.
The brevibacillus brevis is obtained from an oil sludge pool of an oil well of an oil field of Jiangsu, is identified as brevibacillus brevis by separating and screening sludge degrading bacteria, and has obvious degradation and metabolism effects on oil sludge and petroleum hydrocarbon. The oil sludge biodegradation microbial inoculum developed by promoting the activity of oil sludge degrading bacteria through the biological reinforcing agent can realize the rapid degradation of the oil-containing sludge, the oil content of the treated oil sludge is reduced by about 80 percent in 20 days, and a new way is provided for the treatment of the oil-containing sludge of oil fields.
The invention also provides a fermentation method of the petroleum hydrocarbon degrading bacteria, which comprises the following steps:
1) inoculating the brevibacillus brevis of claim 1 or 2 on a sterile solid culture medium in a sterile streak way, and performing static culture at 33-35 ℃ for 2-3 days to obtain a single colony of activated brevibacillus brevis;
2) inoculating the activated single colony on a solid culture medium inclined plane in a sterile square bottle with the capacity of 250mL, and performing static culture at 33-35 ℃ for 4-6 days to form bacterial spores; washing spores on the surface of a solid culture medium in 3-4 square bottles with sterile water to obtain bacterial thallus or spore suspension of sludge degrading bacteria as seed liquid, and counting the content of spores by microscopic examination by a microscope to be more than 1 multiplied by 1010cfu/mL；
3) Will be step 2)Inoculating the obtained seed liquid into a sterile liquid culture medium in a fermentation tank; the fermentation conditions were temperature: the inoculation temperature is 35 ℃; inoculating the spore for 1-8 h in a germination period, and controlling the temperature to be 34 ℃; inoculating the strain for 8-15 h in logarithmic growth phase, controlling the temperature to be 33 ℃, inoculating the strain for 16-35 h in lag phase, and controlling the temperature to be 32 ℃; ventilation volume: the fermentation ventilation volume is 0.1 +/-0.02 m after 1-8 h of inoculation3Min, the pressure of the fermentation tank is kept at 0.2 +/-0.02 MPa, and the air flow for inoculation for 9-35 h is 3 +/-0.02 m3The tank pressure is kept at 0.3 +/-0.02 MPa; constant stirring is adopted in the fermentation process, and the rotating speed is 230 rpm;
4) culturing in the liquid culture medium of the step 3) for 35 hours, observing by a microscope to obtain oval spores with higher density and consistent shape, and collecting fermentation culture solution to obtain the petroleum hydrocarbon degrading bacteria product when the spore rate is more than 90%.
The solid culture medium comprises the following components in percentage by mass: 0.4-0.6% of yeast extract, 0.8-1.2% of peptone, 1.5-2.0% of agar powder and the balance of water; the components are integrally mixed and stirred into a solidified state, the pH value is 7.0-7.2, and the sterilization conditions of the culture medium are as follows: 120 ℃, 0.11 +/-0.02 Mpa, and the time lasts for 25-35 min.
The method is further improved in that the spore suspension is subjected to water bath at 70-75 ℃ for 10-15 min to obtain the spore suspension with strong germination potential and uniform development as a seed solution.
The liquid culture medium in the step 3) comprises the following components in percentage by mass: 1.8-2.5% of bean cake powder, 1.0-1.5% of starch, 0.4-0.6% of corn steep liquor, 0.05-0.1% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.05-0.1% of dipotassium hydrogen phosphate, 0.01-0.015% of zinc sulfate, 0.02-0.03% of natural enemy, the balance of water and the pH value of 7.0-7.2.
The volume of the fermentation tank in the step 3) is 10000L, and the filling amount of the liquid culture medium is 5000L.
The brevibacillus brevis utilizes an industrial fermentation tank to carry out primary spore inoculation and an intermittent liquid submerged fermentation process, has the characteristics of less equipment investment, simple production flow, easy operation and control, short fermentation period, low power energy consumption and low raw material cost, and simultaneously the temperature and pH parameters of the fermentation process can be used as monitoring and control indexes of the fermentation process to indicate the fermentation process, thereby realizing the industrial production of sludge degrading bacteria and accelerating the field application of the oil sludge biodegradation microbial inoculum.
The fermentation method has the beneficial effects that: the culture medium has easily obtained components, low cost, simple process parameters, short fermentation period, high count of the obtained Brevibacillus brevis, and count of a blood count plate of 1 × 109More than cfu/ml.
Detailed Description
Example 1
Brevibacillus brevis (bravibacillus parbrevius) strain name: GWN-2, deposited in the China Committee for culture Collection of microorganisms, with the deposit number: CGMCC No. 13903.
The gene sequence is shown in a sequence table.
The brevibacillus brevis is used for fermentation to obtain a petroleum hydrocarbon degrading bacteria product, and the specific fermentation method is carried out according to the following steps:
1) brevibacillus brevis stored in glycerol at-70 ℃ was streaked on a solid medium in a pre-sterilized petri dish in a clean bench. The culture medium comprises the following components in percentage by mass: 0.4% of yeast extract, 0.8% of peptone, 1.5% of agar powder and the balance of water, and continuously sterilizing for 30min at the temperature of 120 ℃ and 0.11Mpa at the pH of 7.0. The culture dish is placed in an incubator at 33 ℃ for 3d in an inverted way to obtain the activated brevibacillus brevis single colony.
2) Single colonies were picked by aseptic manipulation and suspended in 20ml of sterile distilled water. A sterilized pipette sucks 1mL of the culture medium to inoculate the culture medium on a solid culture medium inclined plane in a sterile square bottle with the capacity of 250mL (the culture medium comprises, by mass, 0.4% of yeast extract, 0.8% of peptone, 1.5% of agar powder and the balance of water, and the pH value is 7.0), and the square bottle is slightly shaken to uniformly spread the single colony suspension on the surface of the solid culture medium. Placing the square bottle in an incubator to keep the surface of the solid medium horizontal, and performing static culture at 33 ℃ for 5d to form bacterial spores. Taking 4 square bottles for 5d culture, adding 100ml of sterile distilled water into each bottle to elute bacterial spores on the surface of the solid culture medium to obtain 200ml of sporesThe bacterial content of the suspension is 1.38 multiplied by 10 according to the observation of a microscope blood counting chamber10cfu/ml, and the spore suspension is used as a fermentation inoculation seed solution through a water bath at 70 ℃ for 10 min.
3) The prepared seed solution in 2 was inoculated into a 10000L fermentor loaded with 5000L sterile liquid medium, and the fermentation conditions were controlled. Temperature: the inoculation temperature is 35 ℃; inoculating for 1-8 h, and controlling the temperature to be 34 ℃; controlling the temperature to be 33 ℃ for 8-15 h and 32 ℃ for 16-35 h; ventilation volume: initial ventilation after inoculation 0.1 + -0.02M3Min, the tank pressure is 0.2 +/-0.02 MPa; the ventilation volume in the middle and later stages of fermentation (9-35 h) is 3 +/-0.02 m3The/min, the tank pressure is kept at 0.3 plus or minus 0.02 MPa. The fermentation process adopts constant stirring, and the rotating speed is 230 rpm.
The culture medium comprises the following components in percentage by mass: 1.8% of bean cake powder, 1.0% of starch, 0.4% of corn steep liquor, 0.05% of magnesium sulfate, 0.1% of sodium chloride, 0.05% of dipotassium hydrogen phosphate, 0.01% of zinc sulfate, 0.02% of sodium nitrite and the balance of water, wherein the pH value is 7.0.
4) Culturing and proliferating the brevibacillus brevis in a fermentation tank culture medium for 35h, taking a sample, observing by a microscope to obtain elliptic spores with high density and consistent shape, wherein the spore rate is more than 90 percent, the pH of a culture solution is 7.8, and counting the bacterium content of the brevibacillus brevis in the culture solution by a blood counting chamber is 1.2 multiplied by 109cfu/ml. Collecting the fermentation culture solution to obtain the petroleum hydrocarbon degrading bacteria product.
Example 2
The brevibacillus brevis of example 1 is used for fermentation to obtain a petroleum hydrocarbon degrading bacteria product, and the specific fermentation method is carried out according to the following steps:
1) the brevibacillus brevis preserved in glycerol at-70 ℃ is streaked and inoculated on a solid culture medium in a pre-sterilized culture dish in a super-clean workbench (the culture medium consists of the following components in percentage by mass: 0.5% of yeast extract, 1% of peptone, 1.8% of agar powder and the balance of water, wherein the yeast extract, the peptone and the agar powder are sterilized continuously for 30min at the pH of 7.2 and the temperature of 120 ℃ under 0.11 +/-0.02 Mpa. The culture dish is placed in an incubator at 33 ℃ for 3d in an inverted way to obtain the activated brevibacillus brevis single colony.
2) Single colonies were picked and suspended in 20ml sterile distilled waterIn (1). 1mL of the culture medium is absorbed by a sterilized pipette and inoculated on a solid culture medium inclined plane in a sterile square bottle with the capacity of 250mL (the culture medium comprises, by mass, 0.5% of yeast extract, 1% of peptone, 1.8% of agar powder and the balance of water, and the pH value is 7.2), and the square bottle is slightly shaken to ensure that the single colony suspension is uniformly spread on the surface of the solid culture medium. Placing the square bottle in a constant temperature incubator in an inclined manner to keep the surface of the solid medium horizontal, and performing static culture at 35 ℃ for 4 d to form bacterial spores. Taking 4 square bottles cultured for 5 days, adding 100ml of sterile distilled water into each bottle to elute bacterial spores on the surface of the solid culture medium to obtain 200ml of spore suspension, and observing and calculating the bacterial content by using a microscope blood counting chamber to be 2.51 multiplied by 1010cfu/ml, and the spore suspension is used as fermentation inoculation seed liquid through water bath at 75 ℃ for 10 min.
3) Inoculating the prepared seed solution in the step 2 into a 10000L fermentation tank loaded with 5000L of sterile liquid culture medium, wherein the culture medium comprises the following components in percentage by mass: 2% of bean cake powder, 1.2% of starch, 0.5% of corn steep liquor, 0.08% of magnesium sulfate, 0.15% of sodium chloride, 0.07% of dipotassium hydrogen phosphate, 0.0125% of zinc sulfate, 0.025% of sodium silicate and the balance of water, wherein the pH value is 7.1.
The fermentation control conditions are temperature: the inoculation temperature is 35 ℃; inoculating for 1-8 h, and controlling the temperature to be 34 ℃; controlling the temperature to be 33 ℃ for 8-15 h and 32 ℃ for 16-35 h; ventilation volume: initial aeration 0.01M after inoculation3Min, the tank pressure is 0.2 Mpa; the ventilation volume in the middle and later stages of fermentation (9-35 h) is 3m3The/min, the pot pressure is kept at 0.3 MPa. The fermentation process adopts constant stirring, and the rotating speed is 230 rpm.
4) Culturing and proliferating for 35h, sampling, observing with microscope, with spore rate of above 90%, pH of culture solution of 7.8, and counting with blood counting plate to obtain culture solution containing Bacillus brevis of 1.71 × 109cfu/ml. Collecting the fermentation culture solution to obtain the petroleum hydrocarbon degrading bacteria product.
Example 3
The brevibacillus brevis of example 1 is used for fermentation to obtain a petroleum hydrocarbon degrading bacteria product, and the specific fermentation method is carried out according to the following steps:
1) inoculating the brevibacillus brevis stored in glycerol at the temperature of 70 ℃ below zero on a solid culture medium in a pre-sterilized culture dish in a streak manner, wherein the culture medium comprises the following components in percentage by mass: 0.6% of yeast extract, 1.2% of peptone, 2% of agar powder and the balance of water, wherein the pH value is 7.2. The culture dish is placed in an incubator at 33 ℃ for 2d in an inverted way to obtain activated brevibacillus brevis single colony.
2. Single colonies were picked and aseptically suspended in 20ml of sterile distilled water. The sterilized pipette sucks 1mL of solid medium slant inoculated in a sterile square bottle with the capacity of 250mL, and the square bottle is slightly shaken to ensure that the single colony suspension is uniformly spread on the surface of the solid medium. The culture was allowed to stand at 35 ℃ to form bacterial spores. Taking 4 square bottles for 6d culture, adding 100ml sterile distilled water into each bottle to elute bacterial spores on the surface of the solid culture medium to obtain 200ml spore suspension, and observing and calculating the bacterial content by using a microscope blood counting chamber to be 4.1 multiplied by 1010cfu/ml, and the spore suspension is used as fermentation inoculation seed liquid through water bath at 75 ℃ for 10 min.
3) The prepared seed solution in 2 was inoculated into a 10000L fermentor loaded with 5000L of sterile liquid medium. The inoculation temperature is 35 ℃; inoculating for 1-8 h, and controlling the temperature to be 34 ℃; controlling the temperature to be 33 ℃ for 8-15 h and 32 ℃ for 16-35 h. Initial aeration 0.1 + -0.02M after inoculation3Min, the tank pressure is 0.2 +/-0.02 MPa; the ventilation volume in the middle and later stages of fermentation (9-35 h) is 3 +/-0.02 m3The/min, the tank pressure is kept at 0.3 plus or minus 0.02 MPa. The constant stirring speed was 230 rpm. The fermentation tank liquid culture medium comprises, by mass, 2.5% of bean cake powder, 1.5% of starch, 0.6% of corn steep liquor, 0.1% of magnesium sulfate, 0.2% of sodium chloride, 0.1% of dipotassium phosphate, 0.015% of zinc sulfate, 0.03% of sodium hydrogen chloride, and the balance of water, wherein the pH value of the fermentation tank liquid culture medium is 7.2.
4) Culturing and proliferating for 35h, sampling, observing with microscope, with spore rate of above 90%, pH of culture solution of 8.0, and counting with blood counting plate to obtain Bacillus brevis content of 6.1 × 109cfu/ml. Collecting the fermentation culture solution to obtain the petroleum hydrocarbon degrading bacteria product.
Example 4
The brevibacillus brevis of example 1 is used for fermentation to obtain a petroleum hydrocarbon degrading bacteria product, and the specific fermentation method is carried out according to the following steps:
1) the brevibacillus brevis preserved in glycerol at-70 ℃ is streaked and inoculated on a solid culture medium in a pre-sterilized culture dish in a super-clean workbench (the culture medium consists of the following components in percentage by mass: 0.45 percent of yeast extract, 1.1 percent of peptone, 1.8 percent of agar powder and the balance of water, and the pH value is 7.2). The culture dish is placed in an incubator at 33 ℃ for 3d in an inverted way to obtain the activated brevibacillus brevis single colony.
2) Single colonies were picked and aseptically suspended in 20ml of sterile distilled water. The sterilized pipette sucks 1mL of solid medium slant inoculated in a sterile square bottle with the capacity of 250mL, and the square bottle is slightly shaken to ensure that the single colony suspension is uniformly spread on the surface of the solid medium. Placing the square bottle in a constant temperature incubator in an inclined manner to keep the surface of the solid medium horizontal, and performing static culture at 35 ℃ for 4 d to form bacterial spores. Taking 4 square bottles cultured for 5 days, adding 100ml of sterile distilled water into each bottle to elute bacterial spores on the surface of the solid culture medium to obtain 200ml of spore suspension, and observing and calculating the bacterial content by using a microscope blood counting chamber to be 1.97 multiplied by 1010cfu/ml, and the spore suspension is used as fermentation inoculation seed liquid through water bath at 75 ℃ for 10 min.
The solid culture medium comprises the following components in percentage by mass: 0.45 percent of yeast extract, 1.1 percent of peptone, 1.8 percent of agar powder and the balance of water, and the pH value is 7.2
3) Inoculating the prepared seed solution in the step 2) into a 10000L fermentation tank loaded with 5000L of sterile liquid culture medium, wherein the liquid culture medium of the fermentation tank comprises the following components, by mass, 1.8% of bean cake powder, 1.5% of starch, 0.5% of corn steep liquor, 0.08% of magnesium sulfate, 0.15% of sodium chloride, 0.08% of dipotassium hydrogen phosphate, 0.01% of zinc sulfate, 0.025% of natural enemy, and the balance of water, and the pH value is 7.0.
The fermentation control conditions are temperature: the inoculation temperature is 35 ℃; inoculating for 1-8 h, and controlling the temperature to be 34 ℃; controlling the temperature to be 33 ℃ for 8-15 h and 32 ℃ for 16-35 h; ventilation volume: initial aeration 0.1 + -0.02 m after inoculation3Min, the tank pressure is 0.2 +/-0.02 MPa; the ventilation volume is 3 +/-0.02 m within 9-35 h3The/min, the tank pressure is kept at 0.3 plus or minus 0.02 MPa. The fermentation process adopts constant stirring, and the rotating speed is 230 rpm.
4) Culturing and proliferating for 35h, sampling, observing with microscope, with spore rate of above 90%, pH of culture solution of 7.5, and counting with blood counting plate to obtain Bacillus brevis content of 1.33 × 109cfu/ml. Collecting the fermentation culture solution to obtain the petroleum hydrocarbon degrading bacteria product.
The petroleum hydrocarbon degrading bacteria obtained in the above embodiments 1 to 4 can be used for degrading oily sludge, and the treatment method comprises the following steps:
1) adding oily sludge into a square or round open sludge tank; the oily sludge is raw sludge containing 40% of oil by weight; sludge tank capacity 100m3；
2) Wheat bran or attapulgite is used as a biological carrier, a biological enhancer and a petroleum hydrocarbon degrading bacterium product are added to obtain a carrier-carrying bacterium agent, and the weight ratio of the components is as follows: petroleum hydrocarbon degrading bacteria product: bio-enhancer = 100: 0.1: 0.04; the biological enhancer is potassium phosphate or potassium nitrate;
3) adding the carrier-carrying microbial inoculum obtained in the last step into a sludge pool, wherein the weight ratio of the carrier-carrying microbial inoculum to the oily sludge = 1: 10;
4) and the aeration treatment is carried out on the sludge tank for 20 days by adopting a lower aeration mode, so that the oil in the sludge is fully degraded.
The oil content of the treated sludge is less than or equal to 0.3 percent, and the oil removal efficiency reaches more than 80 percent.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> China petrochemical Co., Ltd
Sinopec Jiangsu Oilfield Co.
<120> petroleum hydrocarbon degrading bacteria and fermentation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1394
<212> DNA
<213> Brevibacillus brevis (Brevibacillus parbreborvis)
<400> 2
gtcgagcgag ggtcttcgga ccctagcggc ggacgggtga gtaacacgta ggcaacctgc 60
ctctcagacc gggataacat agggaaactt atgctaatac cggataggtt tttggattgc 120
atgatccgaa aagaaaagat ggcttcggct atcactggga gatgggcctg cggcgcatta 180
gctagttggt ggggtaacgg cctaccaagg cgacgatgcg tagccgacct gagagggtga 240
ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatt 300
ttccacaatg gacgaaagtc tgatggagca acgccgcgtg aacgatgaag gtcttcggat 360
tgtaaagttc tgttgtcagg gacgaacacg tgccgttcga atagggcggt accttgacgg 420
tacctgacga gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 480
aagcgttgtc cggatttatt gggcgtaaag cgcgcgcagg cggctatgta agtctggtgt 540
taaagcccgg agctcaactc cggttcgcat cggaaactgt gtagcttgag tgcagaagag 600
gaaagcggta ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc 660
gaaggcggct ttctggtctg taactgacgc tgaggcgcga aagcgtgggg agcaaacagg 720
attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taggtgttgg gggtttcaat 780
accctcagtg ccgcagctaa cgcaataagc actccgcctg gggagtacgc tcgcaagagt 840
gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgaa gaaccttacc aggtcttgac atcccgctga ccgctctgga gacagagctt 960
cccttcgggg cagcggtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tatctttagt tgccagcatt cagttgggca 1080
ctctagagag actgccgtcg acaagacgga ggaaggcggg gatgacgtca aatcatcatg 1140
ccccttatga cctgggctac acacgtgcta caatggttgg tacaacggga tgctacctcg 1200
cgaggggacg ccaatctctg aaaaccaatc tcagttcgga ttgtaggctg caactcgcct 1260
acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccacgg gagtttgcaa cacccgaagt cggtgaggta 1380
accgcaagga gcca 1394
Claims (6)
1. A petroleum hydrocarbon degrading bacterium, characterized in that the petroleum hydrocarbon degrading bacterium is a brevibacillus brevis (Brevibacillus brevis)Brevibacillus parabrevis) The strain name: GWN-2, deposited in the China Committee for culture Collection of microorganisms, with the deposit number: CGMCC No. 13903.
2. The method of claim 1, comprising the steps of:
1) streaking and inoculating the brevibacillus brevis of claim 1 in an aseptic environment on an aseptic solid culture medium, and performing static culture at 33-35 ℃ for 2-3 days to obtain activated single colonies of the brevibacillus brevis;
2) inoculating the activated single colony on a solid culture medium slope in a sterile square bottle with the capacity of 250mL, and performing static culture at 33-35 ℃ for 4-6 days to form bacterial spores; washing spores on the surface of a solid culture medium in 3-4 square bottles with sterile water to obtain bacterial thalli or spore suspension as seed liquid;
3) inoculating the seed liquid obtained in the step 2) into a sterile liquid culture medium in a fermentation tank; the fermentation conditions are warmDegree: the inoculation temperature is 35 ℃; inoculating the spore for 1-8 h in a germination period, and controlling the temperature to be 34 ℃; inoculating the strain for 8-15 h in logarithmic growth phase, controlling the temperature to be 33 ℃, inoculating the strain for 16-35 h in lag phase, and controlling the temperature to be 32 ℃; ventilation volume: the fermentation ventilation volume is 0.1 +/-0.02 m after 1-8 h of inoculation3Min, the pressure of the fermentation tank is kept at 0.2 +/-0.02 MPa, and the air flow for inoculation for 9-35 h is 3 +/-0.02 m3The tank pressure is kept at 0.3 +/-0.02 MPa; stirring continuously in the fermentation process; constant stirring is adopted in the fermentation process;
4) culturing in the liquid culture medium obtained in the step 3) for 30-35 h, and collecting the fermentation culture solution to obtain the petroleum hydrocarbon degrading bacteria product when the spores are observed to have higher density and consistent shape through microscope dyeing and the spore rate is more than 90%.
3. The method of claim 2, wherein the solid medium comprises the following components in percentage by mass: 0.4-0.6% of yeast extract, 0.8-1.2% of peptone, 1.5-2.0% of agar powder and the balance of water; the components are integrally mixed and stirred, the pH value is 7.0-7.2, and the sterilization conditions of the culture medium are as follows: 120 ℃, 0.11 +/-0.02 Mpa, and the time lasts for 25-35 min.
4. The fermentation method of petroleum hydrocarbon degrading bacteria as claimed in claim 2, wherein the spore suspension obtained in step 2) is subjected to water bath at 70-75 ℃ for 10-15 min to obtain a spore suspension with strong germination potential and uniform development as a seed solution.
5. The fermentation method of petroleum hydrocarbon degrading bacteria of claim 2, wherein the liquid medium in step 3) comprises the following components by weight percent: 1.8-2.5% of bean cake powder, 1.0-1.5% of starch, 0.4-0.6% of corn steep liquor, 0.05-0.1% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.05-0.1% of dipotassium hydrogen phosphate, 0.01-0.015% of zinc sulfate, 0.02-0.03% of natural enemy, the balance of water and the pH value of 7.0-7.2.
6. A fermentation method of petroleum hydrocarbon degrading bacteria as claimed in claim 2, wherein the volume of the fermentation tank in step 3) is 10000L, and the liquid medium loading is 5000L.
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