CN109097309A - A kind of petroleum hydrocarbon degradation bacterium and its fermentation process - Google Patents
A kind of petroleum hydrocarbon degradation bacterium and its fermentation process Download PDFInfo
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Abstract
The invention discloses a kind of petroleum hydrocarbon degradation bacterium and its fermentation process, which is a type Brevibacillus brevis, and strain name: GWN-2 is preserved in Chinese microorganism strain collection, deposit number: CGMCC No.13903.It is characterized in that its gene order is as shown in sequence table.The petroleum hydrocarbon degradation bacterium obtains the petroleum hydrocarbon degradation bacterium product of degradable oily sludge by inoculation, Multiplying culture, and the oil content decline 80% or so in 20 days of processing greasy filth provides new approach for the processing of Sludge in Oilfields.
Description
Technical field
The present invention relates to a kind of deoiling of sludge fermentation of bacillus production methods, are particularly suitable for crude oil exploitation and place
The oil patch of the oily sludge, drilling process generation that generate in the fields such as reason, landing oil processing, belong to field of biotechnology.
Background technique
Oily sludge is the one kind generated during oil exploitation, oil-gas gathering and transportation, refining processing and oily waste water treatment
The solid waste being made of crude oil, water, heavy metal, sludge.In sludge in addition to containing a large amount of residual oils, also contain benzene series
The poisonous and harmful substances such as the noxious materials such as object, anthracene, phenols, pyrene and heavy metal, Polychlorinated biphenyls, two uh English, radioactive element.
One side petroleum enters the Nomenclature Composition and Structure of Complexes that soil changes the soil organism, causes carbon-nitrogen ratio and carbon-phosphorus ratio to change, leads to soil
Earth microbal flora, while different kinds of petroleum hydrocarbon has certain toxic to microorganism, to a variety of edaphons and soil
Enzymatic activity has a degree of inhibiting effect.On the other hand, oil-polluted soils change soil property to hinder plant roots
System's breathing and absorption, while high concentration petroleum pollution object has acute and chronic toxicity to plant growth, influences the metabolism of plant
And physiological function, it directly results in Plant death or influences plant growth.
Biologic treating technique is a kind of effective contained Mud Treatment Technology.The natural microbial being present in soil environment
Population is one of the Basic Ways that petroleum hydrocarbon and other hydrocarbons pollutants are eliminated from soil to the degradation of petroleum hydrocarbon.It is micro-
Biological utilisation biology self-regulatory mechanism and to the petroleum hydrocarbon class pollutant in the comprehensive purifying function treatment soil of pollutant,
Make more thoroughly conversion and the degradation obtained in the metabolic processes of biology, and finally generates CO2And water.Thus biology
Processing technique is the developing direction of Sludge in Oilfields processing from now on.
Sludge degradation bacterium can stimulate proliferation in oil polluted environment, but the efficiency for relying on endogenous indigenous bacterium proliferation degradation is difficult to
Realize the fast degradation of oily sludge petroleum hydrocarbon.By the separation screening and industrial fermentation of high activity petroleum hydrocarbon, batch production is dirty
Mud degradation bacteria adds the content for increasing sludge degradation bacterium in oily sludge by external source, and improving degradation efficiency is to realize greasy filth sheet
The key of native biological reinforcing technology.
Be in place of the deficiencies in the prior art: the biochemical degradation speed of current oily wastewater or sludge is slow, degradation efficiency
Low, the culture process of bacterium is complicated.
Summary of the invention
The purpose of the present invention is provide a kind of petroleum hydrocarbon for the shortcoming in current oily sludge biodegrading technology
Degradation bacteria keeps it high to the degradation efficiency of the oil in greasy filth, and speed is fast, and equipment investment is few, process is simple, operation is easy, power
Consumption less, cost of material it is low.
For this purpose, the petroleum hydrocarbon degradation bacterium is a type Brevibacillus brevis the present invention provides a kind of petroleum hydrocarbon degradation bacterium
(Latin name is Brevibacillus parabrevis), strain name: GWN-2 is preserved in Chinese microorganism strain guarantor
Hide administration committee's common micro-organisms collection, deposit number: CGMCC No.13903.
Through being sequenced, gene order is as shown in sequence table.
Such Brevibacillus brevis is obtained from Jiangsu oilfield oil well greasy filth pond, goes out strain sludge degradation through separation screening
Bacterium is accredited as class Brevibacillus brevis, has obvious degradation metabolism to greasy filth petroleum hydrocarbon.Promoted by bio-enhancer
The fast degradation of oily sludge may be implemented in the greasy filth biodegrade microbial inoculum that greasy filth degradation bacteria activity is developed, and handles greasy filth
Oil content decline 80% or so in 20 days, provides new approach for the processing of Sludge in Oilfields.
The present invention also provides a kind of fermentation process of petroleum hydrocarbon degradation bacterium, include the following steps:
1) by the sterile streak inoculation of class Brevibacillus brevis of claims 1 or 2 on sterile solid culture medium, 33~35 DEG C quiet
2~3 d of culture is set, the single colonie of the class Brevibacillus brevis of activation is obtained；
2) single colonie of activation is seeded in the culture medium slant in the sterile square bottle of 250mL capacity, 33~35 DEG C
4~6 d of static gas wave refrigerator is to form bacterial spore；Solid culture primary surface gemma in 3~4 rectangular bottles is done the wash with sterile water,
The microorganism or spore suspension for obtaining sludge degradation bacterium are used as seed liquor, sediments microscope inspection count brood cell's content greater than 1 ×
1010cfu/mL；
3) seed liquor obtained in step 2 is inoculated in the aseptic liquid nutrient medium in fermentor；Fermentation condition is temperature:
35 DEG C of inoculation temperature；It is inoculated with the spore germination phase of 1~8h, controls 34 DEG C of temperature；It is inoculated with the logarithmic growth phase of 8~15h, control temperature
33 DEG C of degree is inoculated with the lag phase of 16~35h, controls 32 DEG C of temperature；Ventilatory capacity: inoculation 1~8h fermentation ventilatory capacity be 0.1 ±
0.02m3/ min, fermentor tank pressure are maintained at 0.2 ± 0.02MPa, and the ventilatory capacity of 9~35h of inoculation is 3 ± 0.02m3/ min, tank
Pressure is maintained at 0.3 ± 0.02MPa；Constant agitation, revolving speed 230rpm are used in fermentation process；
4) 35h is cultivated in the fluid nutrient medium of step 3), microscope dyeing is viewed as that density is higher, the consistent ellipse of form
Spore, brood cell leads when being 90% or more, collects fermentation culture and obtains petroleum hydrocarbon degradation bacterium product.
Above-mentioned solid medium includes mass percent by following component: yeast extract 0.4~0.6%, and peptone 0.8~
1.2%, agar powder 1.5~2.0%, surplus is water；Each component is integrally mixed into solidification state, and pH 7.0~7.2, culture medium goes out
Bacterium condition are as follows: 120 DEG C, 0.11 ± 0.02Mpa, continue 25~35min.
It is as a further improvement of the present invention, spore suspension is through 70~75 DEG C of 10~15min of water-bath to be sent out
Bud gesture is strong, develops consistent spore suspension as seed liquor.
Above-mentioned steps 3) in fluid nutrient medium include mass percent by following component: beancake powder 1.8~2.5%, starch
1.0~1.5%, corn pulp 0.4~0.6%, magnesium sulfate 0.05~0.1%, sodium chloride 0.1~0.2%, dipotassium hydrogen phosphate 0.05~
0.1%, zinc sulfate 0.01~0.015%, GPE 0.02~0.03%, surplus is water, pH value 7.0~7.2.
The volume of fermentor is 10000L in step 3), and fluid nutrient medium loadings are 5000L.
Class Brevibacillus brevis of the invention is once inoculated with using the carry out gemma of industrial fermentation tank, intermittent deep liquid
Zymotechnique, have equipment investment is few, production procedure is simple, operation control is easy, fermentation period is short, power energy consumption and raw material at
This low feature, while the temperature of fermentation process and pH parameter can be used as the monitoring Con trolling index of fermentation process, instruction fermentation
Process realizes the industrialized production of sludge degradation bacterium, accelerates the field application of greasy filth biodegrade microbial inoculum.
The beneficial effect of above-mentioned fermentation process is: medium component is easy to get, is at low cost, and technological parameter is simple, fermentation week
Phase is short, and gained class Brevibacillus brevis bacterium number is high, and blood counting chamber statistics is up to 1 × 109Cfu/ml or more.
Specific embodiment
Embodiment 1
One type Brevibacillus brevis (Latin name is Brevibacillus parabrevis), strain name: GWN-2,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms collection, deposit number: CGMCC No.13903.
Its gene order is as shown in sequence table.
It is fermented using such Brevibacillus brevis, obtains petroleum hydrocarbon degradation bacterium product, specific fermentation process is according to such as
Under step carry out:
1) inciting somebody to action the class Brevibacillus brevis saved in -70 DEG C of glycerol, streak inoculation is cultivated to preparatory sterilizing in superclean bench
On solid medium in ware.Culture medium by mass percentage, is grouped as by following group: yeast extract 0.4%, peptone 0.8%, fine jade
Cosmetics 1.5%, surplus are water, and 7.0,120 DEG C of pH, 0.11Mpa persistently sterilize 30min.Culture dish is inverted in 33 DEG C of incubator
Middle culture 3d obtains the class Brevibacillus brevis single colonie of activation.
2) sterile working picking single colonie is suspended in the distilled water of 20ml sterilizing.Sterilizing pipette is drawn 1ml and is inoculated in
In culture medium slant in the sterile square bottle of 250mL capacity (culture medium by mass percentage, is grouped as by following group:
Yeast extract 0.4%, peptone 0.8%, agar powder 1.5%, surplus are water, pH 7.0), rectangular bottle is jiggled, single colonie is made to suspend
The uniform spread of liquid is in solid culture primary surface.Rectangular bottle is inclined in constant incubator, keeps the holding of solid culture primary surface horizontal,
33 DEG C of 5 d of static gas wave refrigerator form bacterial spore.The rectangular bottle of 4 culture 5d is taken, every bottle of addition 100ml sterile distilled water elution is solid
The bacterial spore of body media surface, obtains 200ml spore suspension, and the observation of microscope blood counting chamber calculates bacteria containing amount and is
1.38×1010Cfu/ml, spore suspension is through 70 DEG C of water-bath 10min as fermentation inoculation seed liquor.
3) seed liquor of the preparation in 2 is inoculated into the 10000L fermentor for loading 5000L aseptic liquid nutrient medium, control
Fermentation condition processed.Temperature: 35 DEG C of inoculation temperature；It is inoculated with 1~8h and controls 34 DEG C of temperature；8~15h controls 33 DEG C of temperature, 16~35h
Controlled at 32 DEG C；Ventilatory capacity: 0.1 ± 0.02M of ventilatory capacity is originated after inoculation3/ min, tank press 0.2 ± 0.02Mpa；Fermentation
Intermediary and later stages (9~35h) ventilatory capacity is 3 ± 0.02m3/ min, tank pressure are maintained at 0.3 ± 0.02MPa.Using perseverance in fermentation process
Fixed stirring, revolving speed 230rpm.
Culture medium by mass percentage, is grouped as just like the following group: beancake powder 1.8%, starch 1.0%, corn pulp 0.4%, sulfuric acid
Magnesium 0.05%, sodium chloride 0.1%, dipotassium hydrogen phosphate 0.05%, zinc sulfate 0.01%, GPE 0.02%, surplus are water, pH value 7.0.
4) class Brevibacillus brevis cultivates proliferation 35h in fermentation tank culture medium, and sampling microscope dyeing is viewed as density
Higher, the consistent elliptic spore of form, it is 90% or more that brood cell, which leads, and culture solution pH is 7.8, and blood counting chamber counts culture solution
Class Brevibacillus brevis bacteria containing amount is 1.2 × 109cfu/ml.It collects fermentation culture and obtains petroleum hydrocarbon degradation bacterium product.
Embodiment 2
Fermented using the class Brevibacillus brevis of embodiment 1, obtain petroleum hydrocarbon degradation bacterium product, specific fermentation process according to
It is carried out as follows in step:
1) in superclean bench by the class Brevibacillus brevis streak inoculation saved in -70 DEG C of glycerol to preparatory sterilizes culture dish
In solid medium on (culture medium by mass percentage, is grouped as by following group: yeast extract 0.5%, peptone 1%, agar powder
1.8%, surplus is water, and 7.2,120 DEG C of pH, 0.11 ± 0.02Mpa persistently sterilize 30min).Culture dish is inverted in 33 DEG C of training
It supports in case and cultivates 3d, obtain the class Brevibacillus brevis single colonie of activation.
2) picking single colonie, sterile working are suspended in the distilled water of 20ml sterilizing.Sterilizing pipette is drawn 1ml and is inoculated in
In culture medium slant in the sterile square bottle of 250mL capacity (culture medium by mass percentage, is grouped as by following group:
Yeast extract 0.5%, peptone 1%, agar powder 1.8%, surplus are water, pH 7.2), rectangular bottle is jiggled, single colonie suspension is made
Uniform spread is in solid culture primary surface.Rectangular bottle is inclined in constant incubator, keeps the holding of solid culture primary surface horizontal, and 35
DEG C 4 d of static gas wave refrigerator forms bacterial spore.The rectangular bottle of 4 culture 5d is taken, every bottle of addition 100ml sterile distilled water elutes solid
The bacterial spore of media surface obtains 200ml spore suspension, and it is 2.51 that the observation of microscope blood counting chamber, which calculates bacteria containing amount,
×1010Cfu/ml, spore suspension is through 75 DEG C of water-bath 10min as fermentation inoculation seed liquor.
3) seed liquor of the preparation in 2 is inoculated into the 10000L fermentor for loading 5000L aseptic liquid nutrient medium,
Culture medium by mass percentage, is grouped as by following group: beancake powder 2%, starch 1.2%, corn pulp 0.5%, magnesium sulfate 0.08%, chlorine
Change sodium 0.15%, dipotassium hydrogen phosphate 0.07%, zinc sulfate 0.0125%, GPE 0.025%, surplus is water, pH value 7.1.
Fermentating controling condition is temperature: 35 DEG C of inoculation temperature；It is inoculated with 1~8h and controls 34 DEG C of temperature；8~15h controls temperature
33 DEG C, 16~35h is controlled at 32 DEG C；Ventilatory capacity: starting ventilation 0.01M after inoculation3/ min, tank press 0.2Mpa；In fermentation,
Later period (9~35h) ventilatory capacity is 3 m3/ min, tank pressure are maintained at 0.3MPa.Constant agitation is used in fermentation process, revolving speed is
230rpm。
4) culture proliferation 35h, it is 90% or more that sampling microscope dyeing observation brood cell, which leads, and culture solution pH is 7.8, hemocytometer
Number plate statistics culture solution class Brevibacillus brevis bacteria containing amount is 1.71 × 109cfu/ml.It collects fermentation culture and obtains petroleum hydrocarbon
Degradation bacteria product.
Embodiment 3
Fermented using the class Brevibacillus brevis of embodiment 1, obtain petroleum hydrocarbon degradation bacterium product, specific fermentation process according to
It is carried out as follows in step:
1) in superclean bench by the class Brevibacillus brevis streak inoculation saved in -70 DEG C of glycerol to preparatory sterilizing culture
On solid medium in ware, culture medium by mass percentage, is grouped as by following group: yeast extract 0.6%, peptone 1.2%, fine jade
Cosmetics 2%, surplus are water, pH 7.2.Culture dish is inverted in 33 DEG C of incubator and cultivates 2d, obtains the short gemma of class of activation
Bacillus single colonie.
2, picking single colonie, sterile working are suspended in the distilled water of 20ml sterilizing.Sterilizing pipette is drawn 1ml and is inoculated in
In culture medium slant in the sterile square bottle of 250mL capacity, rectangular bottle is jiggled, makes single colonie uniform suspension exhibition
It is distributed in solid culture primary surface.35 DEG C of static gas wave refrigerators form bacterial spore.Take the rectangular bottle of 4 culture 6d, every bottle of addition 100ml
Sterile distilled water elutes the bacterial spore of solid culture primary surface, obtains 200ml spore suspension, and microscope blood counting chamber is seen
Examining and calculating bacteria containing amount is 4.1 × 1010Cfu/ml, spore suspension is through 75 DEG C of water-bath 10min as fermentation inoculation seed liquor.
3) seed liquor of the preparation in 2 is inoculated into the 10000L fermentor for loading 5000L aseptic liquid nutrient medium.
35 DEG C of inoculation temperature；It is inoculated with 1~8h and controls 34 DEG C of temperature；8~15h controls 33 DEG C of temperature, and 16~35h is controlled at 32 DEG C.
0.1 ± 0.02M of starting ventilation after inoculation3/ min, tank press 0.2 ± 0.02Mpa；Intermediary and later stages (9~35h) ventilatory capacity that ferments is 3
±0.02 m3/ min, tank pressure are maintained at 0.3 ± 0.02MPa.Hair constant agitation revolving speed is 230rpm.The fermentor liquid training
Feeding base consists of the following components by mass percentage: beancake powder 2.5%, starch 1.5%, corn pulp 0.6%, magnesium sulfate 0.1%, chlorine
Change sodium 0.2%, dipotassium hydrogen phosphate 0.1%, zinc sulfate 0.015%, GPE 0.03%, surplus is water, pH value 7.2.
4) culture proliferation 35h, it is 90% or more that sampling microscope dyeing observation brood cell, which leads, and culture solution pH is 8.0, hemocytometer
Number plate statistics culture solution class Brevibacillus brevis bacteria containing amount is 6.1 × 109cfu/ml.It collects fermentation culture and obtains petroleum hydrocarbon drop
Solve bacterium product.
Embodiment 4
Fermented using the class Brevibacillus brevis of embodiment 1, obtain petroleum hydrocarbon degradation bacterium product, specific fermentation process according to
It is carried out as follows in step:
1) in superclean bench by the class Brevibacillus brevis streak inoculation saved in -70 DEG C of glycerol to preparatory sterilizes culture dish
In solid medium on (culture medium by mass percentage, is grouped as by following group: yeast extract 0.45%, peptone 1.1%, fine jade
Cosmetics 1.8%, surplus are water, pH 7.2).Culture dish is inverted in 33 DEG C of incubator and cultivates 3d, obtains the short bud of class of activation
Spore bacillus single colonie.
2) picking single colonie, sterile working are suspended in the distilled water of 20ml sterilizing.Sterilizing pipette is drawn 1ml and is inoculated in
In culture medium slant in the sterile square bottle of 250mL capacity, rectangular bottle is jiggled, makes single colonie uniform suspension exhibition
It is distributed in solid culture primary surface.Rectangular bottle is inclined in constant incubator, keeps the holding of solid culture primary surface horizontal, 35 DEG C static
It cultivates 4 d and forms bacterial spore.The rectangular bottle of 4 culture 5d is taken, every bottle of addition 100ml sterile distilled water elutes solid medium
The bacterial spore on surface, obtains 200ml spore suspension, the observation of microscope blood counting chamber calculate bacteria containing amount be 1.97 ×
1010Cfu/ml, spore suspension is through 75 DEG C of water-bath 10min as fermentation inoculation seed liquor.
The solid medium by mass percentage, is grouped as by following group: yeast extract 0.45%, peptone 1.1%, agar
Powder 1.8%, surplus are water, pH 7.2
3) seed liquor of the preparation in step 2 is inoculated into the 10000L fermentor for loading 5000L aseptic liquid nutrient medium,
The fermentor liquid culture medium consists of the following components by mass percentage: beancake powder 1.8%, starch 1.5%, corn pulp
0.5%, magnesium sulfate 0.08%, sodium chloride 0.15%, dipotassium hydrogen phosphate 0.08%, zinc sulfate 0.01%, GPE 0.025%, surplus is water,
PH value 7.0.
Fermentating controling condition is temperature: 35 DEG C of inoculation temperature；It is inoculated with 1~8h and controls 34 DEG C of temperature；8~15h controls temperature
33 DEG C, 16~35h is controlled at 32 DEG C；Ventilatory capacity: 0.1 ± 0.02 m of starting ventilation after inoculation3/ min, tank pressure 0.2 ±
0.02 Mpa；9~35h ventilatory capacity is 3 ± 0.02 m3/ min, tank pressure are maintained at 0.3 ± 0.02 MPa.It is used in fermentation process
Constant agitation, revolving speed 230rpm.
4) culture proliferation 35h, it is 90% or more that sampling microscope dyeing observation brood cell, which leads, and culture solution pH is 7.5, hemocytometer
Number plate statistics culture solution class Brevibacillus brevis bacteria containing amount is 1.33 × 109cfu/ml.It collects fermentation culture and obtains petroleum hydrocarbon
Degradation bacteria product.
The petroleum hydrocarbon degradation bacterium product that above-described embodiment 1~4 obtains, can be used for the degradation of oily sludge, processing method
It carries out as follows:
1) oily sludge is added in side or round open sludge-tank；The oily sludge is the former mud of oil-containing weight content 40%；
Sludge pool volume 100m3；
2) it using wheat bran or attapulgite as bio-carrier, is added biological reinforced dose and petroleum hydrocarbon degradation bacterium product obtains band carrier
Microbial inoculum, the weight ratio of each component are bio-carrier: petroleum hydrocarbon degradation bacterium product: biological reinforced dose=100:0.1:0.04；It is biological strong
Agent is potassium phosphate or potassium nitrate；
3) the band carrier microbial inoculum that previous step obtains is added in sludge-tank, the weight proportion of addition is band carrier microbial inoculum: oil-containing
Sludge=1:10；
4) lasting 20 days Air Exposures are carried out to sludge-tank using lower part aeration mode, makes the oil in sludge by fully degraded.
Sludge oil content≤0.3% after processing, oil removal efficiency is up to 80% or more.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned reality
Applying example, invention is explained in detail, for those skilled in the art, still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features.It is all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Sinopec Group
Sinopec Co., Ltd. Jiangsu Oil Field Branch
<120>a kind of petroleum hydrocarbon degradation bacterium and its fermentation process
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1394
<212> DNA
<213>class Brevibacillus brevis (Brevibacillus parabrevis)
<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 (7)
1. a kind of petroleum hydrocarbon degradation bacterium, it is characterised in that the petroleum hydrocarbon degradation bacterium is a type Brevibacillus brevis, bacterial strain name
Claim: GWN-2 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms collection, deposit number: CGMCC
No.13903。
2. a kind of petroleum hydrocarbon degradation bacterium according to claim 1, it is characterised in that its gene order is as shown in sequence table.
3. a kind of fermentation process of petroleum hydrocarbon degradation bacterium, it is characterised in that include the following steps:
1) by the sterile streak inoculation of class Brevibacillus brevis of claims 1 or 2 on sterile solid culture medium, 33~35 DEG C quiet
2~3 d of culture is set, the single colonie of the class Brevibacillus brevis of activation is obtained；
2) single colonie of activation is seeded in the culture medium slant in the sterile square bottle of 250mL capacity, 33~35 DEG C
4~6 d of static gas wave refrigerator is to form bacterial spore；Solid culture primary surface gemma in 3~4 rectangular bottles is done the wash with sterile water,
The microorganism or spore suspension for obtaining sludge degradation bacterium are as seed liquor；
3) seed liquor obtained in step 2 is inoculated in the aseptic liquid nutrient medium in fermentor；Fermentation condition is temperature:
35 DEG C of inoculation temperature；It is inoculated with the spore germination phase of 1~8h, controls 34 DEG C of temperature；It is inoculated with the logarithmic growth phase of 8~15h, control temperature
33 DEG C of degree is inoculated with the lag phase of 16~35h, controls 32 DEG C of temperature；Ventilatory capacity: inoculation 1~8h fermentation ventilatory capacity be 0.1 ±
0.02m3/ min, fermentor tank pressure are maintained at 0.2 ± 0.02MPa, and the ventilatory capacity of 9~35h of inoculation is 3 ± 0.02m3/ min, tank
Pressure is maintained at 0.3 ± 0.02MPa；It is stirred continuously in fermentation process；Constant agitation is used in fermentation process；
4) 30~35h is cultivated in the fluid nutrient medium of step 3), when microscope dyeing is viewed as that density is higher, form is consistent
Spore, brood cell leads when being 90% or more, collects fermentation culture and obtains petroleum hydrocarbon degradation bacterium product.
4. a kind of fermentation process of petroleum hydrocarbon degradation bacterium according to claim 3, it is characterised in that the solid medium
Including mass percent by following component: yeast extract 0.4~0.6%, peptone 0.8~1.2%, agar powder 1.5~2.0% are remaining
Amount is water；Each component is integrally mixed into solidification state, pH 7.0~7.2, medium sterilization condition are as follows: 120 DEG C, 0.11 ±
0.02Mpa, continue 25~35min.
5. a kind of fermentation process of petroleum hydrocarbon degradation bacterium according to claim 3, it is characterised in that obtained in step 2
To obtain, germinating energy is strong, develops consistent spore suspension as seed through 70~75 DEG C of 10~15min of water-bath for spore suspension
Liquid.
6. a kind of fermentation process of petroleum hydrocarbon degradation bacterium according to claim 3, it is characterised in that liquid is trained in step 3)
Feeding base includes mass percent by following component: beancake powder 1.8~2.5%, starch 1.0~1.5%, corn pulp 0.4~0.6%,
Magnesium sulfate 0.05~0.1%, sodium chloride 0.1~0.2%, dipotassium hydrogen phosphate 0.05~0.1%, zinc sulfate 0.01~0.015%, GPE
0.02~0.03%, surplus is water, pH value 7.0~7.2.
7. a kind of fermentation process of petroleum hydrocarbon degradation bacterium according to claim 3, it is characterised in that fermentor in step 3)
Volume be 10000L, fluid nutrient medium loadings be 5000L.
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