CN106801025A - One plant of oil-base mud well drilling detritus degradation function bacterium and its application - Google Patents
One plant of oil-base mud well drilling detritus degradation function bacterium and its application Download PDFInfo
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Abstract
The invention belongs to field of environment microorganism, bacillus subtilis category (Bacillus subtilis strain.) bacterium CGMCC 13202 is specifically disclosed, the bacterium can effectively degrade the oil base drilling wastes such as oil base DWM landwaste.Additionally, the invention also discloses the method using the bacterial degradation oil base DWM landwaste.
Description
Technical field
The invention belongs to field of environment microorganism, and in particular to one plant of function bacterium of degradable oil-base mud well drilling detritus with
And the method for oil base DWM landwaste of being degraded using the function bacterium.
Background technology
Oil-base mud well drilling detritus are the solid pollutants of generation during oil mining.It is oil-base mud and drilling well rock
The mixture of bits, the complicated heterogeneous system for mainly having petroleum-type compound, phenolic compound, landwaste and heavy metal.
With the development of China's oil-gas exploration and development, unconventional gas well, horizontal drilling, multilateral well are more and more, the borehole wall
The problem of stabilization is increasingly protruded, and it is increasing that oil base drilling fluid technology compared to water-base drilling fluid there is obvious advantage to be subject to
Pay attention to and constantly develop, but the oil-base mud well drilling detritus discarded object that oil base drilling fluid technology is produced is more and more, and by
In the long-term field operation that drills for oil, all waste thing of job site can not in time be delivered to treatment plant's treatment, nearly all
Discarded object all discharge be stacked at waste mud storage hole in.Its COD high, petroleum hydrocarbons high are to soil, earth's surface and underground
The pollution of water.Destruction soil texture and the living environment of edaphon, have impact on the activity of soil enzyme, so as to disturb crop
Growth；The mankind are contacted by skin and edible contaminated food enters in vivo and then jeopardize the health and lives peace of the mankind
Entirely, or even the health problems such as carcinogenic, teratogenesis, mutagenesis are caused.Safely and effectively go out to explore substantial amounts of discarded oil base drilling fluid
Road, mitigates the burden in oil field, mitigates harm of the discarded object of drilling well generation to environment, therefore the nothing of oil base drilling fluid is discarded in research
Evilization treatment technology has important practical significance.
Treatment to oil-base mud well drilling detritus at present mainly has exhaust method, soil cultivating method, specified place on the spot to concentrate
The physico-chemical processes such as treatment, chemical curing method, burning method, biologic treating technique.
Exhaust method is most traditional processing method on the spot, by adding appropriate flocculation after natural subsidence or mechanical dehydration
Agent the characteristics of with low cost, but easily causes secondary pollution in the method for oil field job site emergency burial.Soil cultivating method is
By after natural subsidence or mechanical dehydration, directly mixing with soil, self-purification capacity or serike using soil reach net
The purpose of change, but easily corrode earth's surface, polluted underground water.It is by aspirating car by the discarded of construction site that specified place focuses on
Thing is transported to the place specified, and focuses on, but processing cost is too high.Chemical curing method is by adding curing agent and demulsification
Agent, adsorbent, with drilling mud effect, form it into the water resistant solid of the stabilization with some strength, by waste drilling mud
In harmful components closing, parcel wherein.With the metal ion and organic substance that can be eliminated in drilling mud to water body,
The influence and harm of soil and ecological environment；But high cost, is not suitable for carrying out solidification construction to a large amount of waste drilling muds.Burn
Burning method is that the method by oil-base mud well drilling detritus by burning processes organic matter, and the residue after burning is used for construction material
Deng.Organic matter is easily carbonized into CO at high temperature2And H2O, to organic matter treatment more thoroughly, but organic components are complicated, contain
The impurity such as N, S, easily cause the secondary pollution of air, energy consumption, relatively costly.Bioremediation technology be using particular organisms (plant,
Microorganism or protozoan) absorption, be converted removing environmental contaminants, realize the depollution of environment, ecological effect recover biology
Measure.There is low cost, non-secondary pollution, but repairing efficiency is long.
Although can be seen that physical chemistry treatment technology treatment petroleum hydrocarbon class pollutant from every treatment technology Integrated comparative
Have the advantages that effect is fast, be easily controlled management and easy to implement.It is complicated that process is there is also simultaneously, it is relatively costly, to original life
State system destruction compared with it is strong, Oil Recovery is incomplete the shortcomings of, and there is secondary pollution.At biologic treating technique and physical chemistry
Reason technology is compared, it is adaptable to all kinds of waste drilling muds, and with low cost, energy saving, the characteristics of non-secondary pollution, but
Repairing efficiency is long.
In drilling process, the microorganism of nature enters in oil-base mud landwaste, in the extreme environment of high concentration oil-containing
The microbial resources of survival, have potential application value in terms of the environment remediation of oil-base mud landwaste pollution.Therefore, from oil
Isolated and purified by enrichment culture, the culture of coating selectivity, domestication and plate streaking in base mud landwaste, obtain oil-base mud rock
The indigenous degradation function bacterium of bits.
The content of the invention
The purpose of the present invention includes：
A kind of microorganism of degradable oil-base mud well drilling detritus is provided, and the microorganism purposes；
Biological treatment or the biodegradation method of a kind of oil-base mud well drilling detritus are provided；
A kind of method of fast degradation oil-base mud well drilling detritus is provided, etc..
Bacillus subtilis Pseudomonas (the Bacillus subtilis for obtaining are screened from crude oil the invention discloses one plant
Strain.) bacterial strain.It is commonly micro- that the bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms on October 31st, 2016
Bio-Centers (CGMCC), deposit number is CGMCC 13202, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of Microorganism, Academia Sinica.The bacterial strain may be simply referred to as SCDC-3 in the present invention and experiment.The present invention discloses
The nucleotide sequence of bacterial strain 16S rDNA.Based on the sequence, it is possible to identify bacillus subtilis Pseudomonas bacterial strain CGMCC 13202.
Bacillus subtilis Pseudomonas bacterial strain CGMCC 13202 disclosed by the invention can be used for oil base drilling wastes of degrading, especially
It is applied to degraded oil base DWM landwaste etc..
Additionally, the invention also discloses a kind of method of oil base DWM landwaste of degrading, including：By claim 1 institute
Bacillus subtilis Pseudomonas bacterial strain bacterial suspension inoculation is stated to crude oil culture medium, it is incubated；
Wherein, the culture pH value of the bacillus subtilis Pseudomonas bacterial strain CGMCC 13202 is 6~8, and preferably pH value is 7
~7.2；
The cultivation temperature of the bacillus subtilis Pseudomonas bacterial strain CGMCC 13202 is 33~37 DEG C, preferably 35 DEG C；
In the culture environment of the bacillus subtilis Pseudomonas bacterial strain CGMCC 13202, the mass ratio of nitrogen and P elements
It is (1~96)：1, such as 1：1、6：1、12：1、24：1、48：1 or 96：1 etc.；
Preferably, nitrogen and the mass ratio of P elements are (6~12)：1；
Most preferably, nitrogen and the mass ratio of P elements are 12:1, wherein nitrogen element content is 700 mg/litres, phosphorus unit
Cellulose content is 58mg/ liters；
In the culture environment of the bacillus subtilis Pseudomonas bacterial strain CGMCC 13202,4~6 g/l of NaCl contents, preferably
It is 5 g/l.
One kind disclosed by the invention uses the degraded oil base DWM landwaste of bacillus subtilis Pseudomonas bacterial strain CGMCC 13202
Method, for example, following step can be used：
By the bacteria suspension of bacillus subtilis Pseudomonas bacterial strain CGMCC 13202, (the bacteria concentration order of magnitude of bacteria suspension is 107CFU/
Milliliter) crude oil culture medium accessed with the inoculum concentration of volume ratio 10%, original crude oil concentration is 1000 mg/litres, pH is 7.0~
7.2, it is placed in constant incubator, 35 DEG C are cultivated 18 days, the mass ratio 12 of nitrogen and P elements in culture environment：1, wherein nitrogen
Constituent content is 700 milligrams, and phosphorus element content is 58 mg/litres；NaCl contents are preferably 5 g/l in culture environment.Bacteria suspension
Preparation method can refer to the common method that this area prepares bacteria suspension.
Beneficial effect
From the angle of environmental protection, cost is selected in the harmless treatment for oil-base mud well drilling detritus pollutant
Low, the biotechnology of simple operations non-secondary pollution has important social economy for the treatment of oil-base mud well drilling detritus
Benefit.From microorganism of the invention, degradation effect can be improved, shorten repair time, reach the purpose of quick reparation.
Bacillus subtilis Pseudomonas (Bacillus subtilis strain.) bacterial strain of the present invention is CGMCC 13202, a side
Organic macromolecule in the crude oil pollution thing such as oily sludge is degraded to the relatively lower small molecule of molecular weight by face, on the other hand may be used
Small molecule after degraded is further converted to the intermediates such as ester, alcohol, is conducive to further degraded and is eliminated.Function of the present invention
Bacterium especially has preferable degradation effect to C15~C19 linear paraffins and C21 and C24 linear paraffins, can produce branched alkane hydro carbons,
The intermediate products such as esters, alcohols and cyclohexanes.
Microbial material preservation
Bacillus subtilis Pseudomonas (Bacillus subtilis strain.) bacterium of the present invention, in October, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC) was preserved in 31st, deposit number is
CGMCC 13202, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
Brief description of the drawings
Fig. 1 SCDC-3 colonial morphologies
Fig. 2 SCDC-3 microscope forms
Fig. 3 SCDC-3 Gram's staining microscope forms
Fig. 4 SCDC-3 growth curves
Fig. 5 SCDC-316S rDNA pcr amplification product electrophoresis patterns
In Fig. 5, Line M：DL 2000；Line 1,2,3：SCDC-3 16S rDNA；Target stripe is in 1000-
1200bp positions
Fig. 6 SCDC-3 development tree graph spectrums
SCDC-3 degradeds situation under Fig. 7 differences pH
SCDC-3 degradeds situation under Fig. 8 different temperatures
Fig. 9 difference N-P are than lower SCDC-3 degraded situations
SCDC-3 degraded situations under Figure 10 difference NaCl concentrations
Degraded situations of Figure 11 SCDC-3 to oil-base mud well drilling detritus
Figure 12 oil-base mud well drilling detritus before processing (blank control group) organic principle GC-MS spectrograms
Figure 13 oil-base mud well drilling detritus organic principle GC-MS spectrograms after SCDC-3 treatment
Specific embodiment
The invention relates to oil degradation rate computing formula it is as follows：
Crude content is measured by undispersed infrared method；The shadow of microorganism adsorption in ignoring degradation process when calculating
Ring.
Embodiment one, the screening of efficient degradation function stem
Enriched medium：LB culture mediums, beef extract 3g, peptone 10g, sodium chloride 5g, distilled water 1000ml, pH is adjusted to
7.0-7.2, such as matches somebody with somebody solid medium, need to add agar 2%.It is standby after 121 DEG C of sterilizing 20min of high-pressure steam sterilizing pan.
Minimal medium：NaCl 1.0g, (NH4)2·SO40.617g, KH2PO40.50g, K2 HPO41.0g,
MgSO40.50g, CaCl20.1g, KCl 0.10g, FeSO4·7H2O0.01g, distilled water 1000ml, pH are adjusted to 7.0-7.2, high
It is standby after 121 DEG C of sterilizing 20min of pressure steam sterilization pan.
Crude oil origin：The crude oil extracted from the oil-base mud well drilling detritus in Sichuan Yibin Gongxian County shale gas oil field.
Crude oil culture medium：Inorganic salts add a certain amount of crude oil, standby after sterilizing.
Crude oil solid selection medium：Agar 2% is added in minimal medium.121 DEG C of sterilizings of high-pressure steam sterilizing pan
After 20min, pour into flat board, add concentration 50mg/L crude oil to smear when culture medium is just cooled down uniform, crude oil treats culture medium
Fully absorb completely standby.
Oil-base mud well drilling detritus are originated：Certain oil field oil-base mud well drilling detritus discarded object.
Screening step：10g oil-base mud well drilling detritus are taken in 90ml aseptic deionized waters, after dissolving vibration 10min, is taken
10% in 200ml enriched mediums, 35 DEG C, cultivate 1d under the conditions of 190rpm/min.Take 10% nutrient solution and access crude oil concentration
It is former in the crude oil culture medium of 100mg/L, continuation cultivates 5d under the conditions of 35 DEG C, 190rpm/min, repeats the above steps 3 times
Oil concentration is followed successively by 200mg/L, 500mg/L, 1000mg/L.Draw the appropriate nutrient solution after repeatedly taming and coat crude oil
On solid selection medium flat board, after colony growth is good, growth selection is good, and lawn is larger and the discrepant bacterium colony of form,
Rule on solids enrichment culture medium and isolated and purified, well-grown is obtained by multiple isolating and purifying, the bacterium colony for coming in every shape,
Then crude oil concentration is inoculated in determine its degradation capability to crude oil in the crude oil culture medium of 1000mg/L, finally obtains one plant
Efficient degradation function stem SCDC-3.
The preparation of embodiment two, bacteria suspension
Degradation function bacterial strain SCDC-3 is inoculated in enrichment culture in LB culture mediums, by the bacterium solution in exponential phase with
5000rpm/min is centrifuged 20min, washs three times with PBS (pH=7.0) and is diluted to suitable concentration.By blood
It is 8.62 × 10 that ball count plate calculates bacteria suspension concentration8CFU/ml。
Embodiment three, Morphological Identification
With the efficient degradation function stem SCDC-3 that crude oil is obtained as sole carbon source screening, seen in the culture of LB plating mediums
Its colonial morphology (Fig. 1) is examined, its individual morphology (Fig. 2) and its Gram's staining (Fig. 3) are observed in LB fluid nutrient medium cultures, half is solid
Its motility is observed on body culture medium and Anaerobic culturel observes whether it is amphimicrobian bacterial strain.Result shows that bacterial strain SCDC-3 is long
Shaft-like, gram-positive bacteria, sports type is medium, facultative anaerobic bacteria；Its bacterium colony size 3-5mm, rounded offset flat shape, edge is whole
Together, the smooth moistening in bacterium colony surface, matt, opaque, yellow-white.
Example IV, bacterial growth cycle experimental
The optical density (OD values) in nutrient solution is determined using photoelectric turbidimetry to determine the increment of bacterium indirectly.
The function bacterium for obtaining will be screened to be inoculated on beef extract-peptone solid medium using four zoning collimation methods, at 35 DEG C
In activation 3-4 generations, then select the single colony inoculation for having grown in fresh beef cream peptone from flat board in constant incubator
In fluid nutrient medium, after cultivating 24h in 35 DEG C, 190rpm/min shaking tables, 4 DEG C of Refrigerator stores are used as seed liquor.
Seed liquor is linked into fresh sterilized beef extract-peptone fluid nutrient medium by 2% inoculum concentration, 35
DEG C, 190rpm/min cultures.
The wavelength of 754- UV, visible light spectrophotometers is transferred to 600nm, start preheating 20min.
Think that the beef-protein medium of inoculation, for reference, takes a sample at regular intervals, determine nutrient solution
OD600。
With the OD of bacteria suspension600It is ordinate, incubation time is abscissa, draws the growth curve (Fig. 4) of dominant strain.
The growth of microorganism by deadtime, logarithmic phase, stationary phase and decline phase a process.Understand the life of microorganism
Production practices are had great directive significance by rule long.Growth rhythm according to logarithmic phase can obtain being contracted during culture strain
The method of casual labourer's phase：The strain of logarithmic phase is inoculated with, using most suitable cell age.As shown in Figure 4, the deadtime of SCDC-3 is 1h, then
Into logarithmic phase, terminate to enter the stage of stable development in 15h.
The molecular biology identification of embodiment five, oil-base mud well drilling detritus oil degradation function bacterium SCDC-3
(1) the extraction of genomic DNA
After by culture in bacterial strain liquid medium within to certain concentration, using (the purchase of bacterial genomes DNA extraction kit
From Tiangeng life biochemical technology Co., Ltd) extract bacterium total genomic dna.
(2) PCR amplifications
It is template with the bacterial genomes DNA for extracting, it is amplimer to use bacterial 16 S rDNA universal primers, enters performing PCR
Amplified reaction.
Preceding primer 27F:5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇
Primer 1492R afterwards:5 ＇-GGTTACCTTGTTACGACTT-3 ＇
PCR reaction systems are 50ul, and specific it is as shown in the table：
The PCR reaction systems of table 1
PCR cycle system is as shown in the figure
The PCR cycle system of table 2
| Temperature (DEG C) | Time | Cycle-index | Program |
| 95 | 5min | 1 | Predegeneration |
| 94 | 30sec | 29 | Denaturation |
| 55 | 30sec | 29 | Annealing |
| 72 | 1:40min | 29 | Extend |
| 72 | 5min | 1 | After extend |
| 12 | ∞ | - | Insulation |
(3) gel electrophoresis
The PCR primer that will be obtained carries out gel electrophoresis test, and test method is as follows：
The comb of predetermined dimension is placed in glue channel mould first, then with 1 × TAE running buffers in conical flask
Liquid prepares 1% agarose solution 25ml, is put into micro-wave oven and is heated to agarose dissolving, and taking-up shakes up.Treat that agarose is cooled down
To feel it is micro- boiling hot when, add 5ul GoldView, mix.Gel is poured into glue groove, thickness is about 5mm or so, glue mistake
Note avoiding bubble in journey.After gelling is solid, removes comb and be put into electrophoresis tank, to addition TAE buffer solutions in electrophoresis tank, it is ensured that
Liquid level did not had gel, and then appropriate pcr amplification product is added in glue hole, and reference is made with 100bpDNA Ladder, opened electricity
Swimming instrument, after electrophoretic band moves 20min in 131V, closes power supply, and removal gel is in observation electrophoresis knot on gel imaging system
Really.
(4) purifying is reclaimed
DNA purpose bands needed for the cutting of PCR primer electrophoretic band, (day is purchased from using Ago-Gel DNA QIAquick Gel Extraction Kits
Root biochemical technology Co., Ltd) purifying reclaim after be routed directly to Shanghai life work biology Co., Ltd sequencing.
(5) the structure of phylogenetic tree
The sequence for obtaining will be sequenced to be spliced using Chromas softwares, splicing gained sequence is submitted to GenBank numbers again
According to storehouse, and identify analyses are carried out with Blast softwares, analysis result carries out Multiple Sequence Alignment using Clustal W softwares.
Finally application MEGA 5.0 softwares are based on Neighbour-joinjing using the development of Bootstrap analysis constructing systems
Tree, building parameter is：Neighbour-joinjing (Kimura 2-parameter model, Bootstrap
Replications of 1000)。
The online BLAST alignment sites of sequence：http://blast.ncbi.nlm.nih.gov/Blast.cgi
After the gene of the oil-base mud well drilling detritus oil degradation function bacterium with round pcr to newly filtering out is expanded,
Analyzed using agarose gel electrophoresis, as a result as shown in Figure 5.
16S rDNA genes 1053bp, the PCR amplification of oil-base mud well drilling detritus oil degradation function bacterium SCDC-3 is obtained
Sequence as shown in sequence table SEQ ID NO.1.
Above-mentioned sequence submission Genbank databases are carried out into BLAST contrasts to preserve, and using the MEGA5.0 sequence of calculations
Phyletic evolution distance, Fig. 6 is shown in using ortho position phase connection phylogenetic tree construction, is bacillus subtilis through analyzing bacterial strain SCDC-3
Category (Bacillus subtilis strain.), homology is 98%
The oil degradation performance measurement of embodiment six, oil-base mud well drilling detritus degradation function bacterium SCDC-3 under different pH
By the bacteria suspension of bacterial strain SCDC-3 bacterial strains, (the bacteria concentration order of magnitude of bacteria suspension is 107CFU/ml 10% (v/v)) is pressed
Inoculum concentration access crude oil culture medium in, original crude oil concentration be 1000mg/L.With equivalent phosphate buffer (pH=7.0) generation
For bacteria suspension, as control.5 groups of different initial pH are set to be respectively：3,5,7,9,11, investigate different initial pH degradation function bacterium
To the degradation capability of crude oil.Sample is placed in 190rpm/min in constant incubator, after 35 DEG C of culture 7d, in determination sample
Crude content, calculates degradation rate.3 Duplicate Samples of every group of Setup Experiments.
Requirement of the different microorganisms to pH value condition is different, and they can grow in certain pH value range, therefore
Adaptability of the microorganism to environment can be reflected to a certain extent.Such as Fig. 7, function bacterium SCDC-3 degrade in pH=7
Rate reaches maximum, and pH is below or above 7, and degradation rate significantly decreases trend, because pH value is too small or excessive can make
Zymoprotein denaturation is even inactivated, so as to influence degradation rate.
Embodiment seven, oil-base mud well drilling detritus degradation function bacterium SCDC-3 oil degradation performance at different temperatures are surveyed
It is fixed
By the bacteria suspension of bacterial strain SCDC-3 bacterial strains, (the bacteria concentration order of magnitude of bacteria suspension is 107CFU/ml 10% (v/v)) is pressed
Inoculum concentration access crude oil culture medium in, original crude oil concentration be 1000mg/L, pH=7.0~7.2.With equivalent phosphate-buffered
Liquid (pH=7.0) replaces bacteria suspension, used as control.5 groups of different temperatures are set to be respectively：15 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40
DEG C, degradation capability of the function bacterium of being degraded under investigation different temperatures to crude oil.Sample is placed in constant incubator with 190rpm/
Min, after 35 DEG C of culture 7d, the crude content in determination sample calculates degradation rate.3 Duplicate Samples of every group of Setup Experiments.
Result such as Fig. 8.The degradation rate of function bacterium SCDC-3 increases first increases and then decreases, the highest at 35 DEG C with temperature.Drop
Solution rate is influenceed by relatively low and higher temperature, because low temperature can be suppressed the activity of enzyme, cell and metabolic activity
Weaken, the inhibitory action of enzymatic activity is released from the rising of temperature, cell and metabolic activity are strengthened, but more than most
After preference temperature, the activity reduction of enzyme, or even inactivation, cause degradation rate to reduce.
The oil degradation of embodiment eight, oil-base mud well drilling detritus degradation function bacterium SCDC-3 under different N-P element ratios
Performance measurement
By the bacteria suspension of bacterial strain SCDC-3 bacterial strains, (the bacteria concentration order of magnitude of bacteria suspension is 107CFU/ml 10% (v/v)) is pressed
Inoculum concentration access crude oil culture medium in, original crude oil concentration be 1000mg/L, pH=7.0~7.2.With equivalent phosphate-buffered
Liquid (pH=7.0) replaces bacteria suspension, used as control.Set 5 groups of different N element contents respectively 75mg/L, 350mg/L,
700mg/L, 1400mg/L, 2800mg/L, 5600mg/L, P elements concentration are 58mg/L (equivalent PO4 3-Concentration be about
177mg/L), different N-P are investigated than lower degradation capability of the function bacterium to crude oil of degrading.By sample be placed in constant incubator with
190rpm/min, after 35 DEG C of culture 7d, the crude content in determination sample calculates degradation rate.Every group of Setup Experiments 3 are parallel
Sample.
The deficiency of N, P nutrient limits the growth and breeding of microorganism, directly influences the degraded effect of the oily function bacterium of drop
Really, but the nutrient of excess can suppress the growth and breeding of microorganism again.Such as Fig. 9.Function bacterium SCDC-3 is constant in P content
In the case of, the Different adding amount of N element, the degradation rate for showing is different.During N element 350~700mg/L of content, degradation rate is most
Greatly, and excursion less, the degradation rate highest at 2~3% or so, N element 700mg/L, by calculating N, P ratio of quality (6
~12):Between 1.The addition for continuing to increase N element improves N:P, degradation rate is presented downward trend, because nutriment
It is superfluous, it is suppressed that the growth and breeding of degradation function bacterium, and then cause degradation rate to decline.
The oil degradation performance of embodiment nine, oil-base mud well drilling detritus degradation function bacterium SCDC-3 under different salinity
Determine
By the bacteria suspension of bacterial strain SCDC-3 bacterial strains, (the bacteria concentration order of magnitude of bacteria suspension is 107CFU/ml 10% (v/v)) is pressed
Inoculum concentration access crude oil culture medium in, original crude oil concentration be 1000mg/L, pH=7.0~7.2.With equivalent phosphate-buffered
Liquid (pH=7.0) replaces bacteria suspension, used as control.Set 5 groups of difference NaCl contents be respectively 0%, 3%, 6%, 10%,
15%th, 20%, 30%, degradation capability of the function bacterium of being degraded under the different salinity of investigation to crude oil.Sample is placed in incubated
With 190rpm/min in case, after 35 DEG C of culture 7d, the crude content in determination sample calculates degradation rate.Every group of Setup Experiments 3
Duplicate Samples.
Inorganic salts are the important components of cell and enzyme, are the activator and confactor of bacterium some enzymes, and are fitted
Suitable salinity can maintain osmotic pressure constant in organism.Such as Figure 10.With the increase of NaCl contents, function bacterium SCDC-3 drops
Solution rate is presented the trend for first increasing and reducing afterwards, and when NaCl contents are 0.5%, growth is optimal, and its degradation rate reaches highest；
With the increase of salinity, its degradation rate is presented downward trend, and when NaCl contents are 10%, strain cannot survive substantially,
Illustrate to increase with the increase of salinity, somatic cells exosmosis pressure, cause somatic cells dehydration, suppress the growth of strain, very
To causing strain dead, the degradation rate of function bacterium declines.
The degraded of embodiment ten, oil-base mud well drilling detritus degradation function bacterium SCDC-3 to oil-base mud well drilling detritus prepares
250ml conical flasks, conical flask weighs 50g oil-base mud well drilling detritus, 200ml minimal mediums is added, by bacterial strain SCDC-3
(the bacteria concentration order of magnitude of bacteria suspension is 10 to the bacteria suspension of bacterial strain7CFU/ml) in by the inoculum concentration access culture medium of 10% (v/v),
PH=7.0~7.2, replace bacteria suspension, as control with equivalent phosphate buffer (pH=7.0).Obtained in above-mentioned experiment
Optimal pH, temperature, N-P ratios under conditions of salinity, cultivate, per surveying at regular intervals in 190rpm/min constant-temperature tables
Fixed its degradation rate to oil-base mud well drilling detritus, draws degradation curve.According to pH=7.0,35 DEG C of preference temperature, N, P compare 12:
1, the optimum condition of salinity 0.5%, its degradation curve that function bacteria strain SCDC-3 is measured at optimum conditions, experimental result is such as
Figure 11, after degradation reaction 18d, degradation rate is 46.1%.
Degraded contrast experiment of the SCDC-3 function bacteriums of embodiment 11 to oil-base mud well drilling detritus organic principle
Experimental technique
SCDC-3 function bacterium degradation experiment groups：Prepare 5 500ml conical flasks, 50g oil-base mud drilling well rocks are weighed respectively
Bits, every group of conical flask adds a certain amount of minimal medium, keeps moisture content 86% or so, each bacterial strain in its optimal pH,
Optimum temperature, most suitable N-P when under most suitable NaCl concentration, are respectively connected to function bacterium SDCD-3, in 190rpm/ by inoculum concentration 10%
Min, after cultivating 15d in 35 DEG C of shaking tables, oil-base mud well drilling detritus 5000rpm/min is centrifuged 10 minutes, removes supernatant, oil
Mud natural air drying.
Greasy filth after air-drying is placed in 250ml conical flasks, addition 100ml petroleum ethers (30-60 DEG C) is 20 DEG C in shaking table
Extraction 2h, static rear taking-up supernatant, good seal is placed in 4 DEG C of refrigerators, is repeated 5 times.By 5 extract mixing 5000rpm from
Heart 5min, petroleum ether and the oil product after being degraded are reclaimed using Rotary Evaporators.The petroleum-type organic matter mixture that will be obtained
Delivering to Sichuan University's test analysis center carries out GC-MS component analyses.
Blank control group：Identical oil base DWM landwaste is taken, is only not to be inoculated with SDCD-3 functions with identical experiment step
Bacterium, repeats above-mentioned experimental technique, obtains the oil product after the treatment of oily sludge blank control group.The petroleum-type organic matter that will be obtained is mixed
Compound delivers to Sichuan University's test analysis center and carries out GC-MS component analyses.
GC-MS analyzes experiment condition
Chromatographic condition:Chromatographic column for 30m × 0.32mm OV-101 quartz capillary columns, 40 DEG C of column temperature, stop 5min again with
10 DEG C/min rises to 150 DEG C, stops 2min and rises to 290 DEG C, 290 DEG C of vapourizing temperature, 270 DEG C of detection temperature, carrier gas with 5 DEG C/min again
N2, 0.4kg, detector FID are pressed before post.
Mass Spectrometry Conditions:Ion gun be electron bombardment ionization source (EI), electron energy 70eV, accelerating potential 3kV, resolution ratio 1000,
Vacuum 1.33 × 10-4Pa, 190 DEG C of sampling system temperature, 250 DEG C of ionization chamber temp, sweep limits m/z 22-600.
Result and analysis
Oil-base mud well drilling detritus before processing (blank control group) organic principle GC-MS spectrum analysis (Figure 12) and table 3 can
Know：The component of oil has peak to occur at 16.9~46.0min of retention time in oil-base mud well drilling detritus, illustrates oil-base mud
Organic components are complicated in well drilling detritus discarded object, totally 14 kinds of organic constituentses.Between retention time 16.9min~39min
Organic constituentses key component be C15~C19 linear paraffins and 2- hexyl -1- decyl alcohol, single 2-ethyl hexyl ester and 14- first
11 kinds of organic matters of base -8- hexadecenes organic matter and C21 and C24 linear paraffins and butyl heptadecane base ester etc.；In retention time
Organic constituentses between 39min~46min are mainly C31, C43, C44 macromolecule linear paraffin 3 kinds of organic matters of class.
Each component molecular formula and title in the oil-base mud well drilling detritus blank organic matter of table 3
| Molecular formula | Title | Molecular formula | Title |
| Pentadecane | Nonadecane | ||
| Hexadecane | Butyl heptadecane base ester | ||
| 2- hexyl -1- decyl alcohol | Heneicosane | ||
| Single 2-ethyl hexyl ester | Lignocerane | ||
| Heptadecane | Hentriacontane | ||
| 14- methyl -8- hexadecenes | Tritetracontane | ||
| Octadecane | Tetratetracontane |
The oil-base mud well drilling detritus of table 4 are through T-3 before processing organic component chemical formulas and title
Oil-base mud well drilling detritus organic principle GC-MS spectrograms (Figure 13) and table 4 after being processed through SDCD-3 understand：
The retention time of the component of oil has peak to occur in 20~46min in oil-base mud well drilling detritus after SDCD-3 treatment, says
Bright organic matter fraction is complicated, and 15 kinds of organic matter fractions are detected altogether.The main organic matter fraction between 18~39min of retention time
12 kinds of organic matters such as alkanes, acids, cycloalkane, esters for C11~C24；Between retention time 39min~46min
Organic constituentses be mainly C31, C43 and C44 macromolecule linear paraffin 3 kinds of organic matters of class.Collection of illustrative plates 13 and table 4 are compared to spectrogram
12 and table 5 understand, retention time when occurring peak in collection of illustrative plates 13 has elapsed 4min backward；In 16.9~39min retention times
The peak height and peak area of appearance, collection of illustrative plates 13 are below collection of illustrative plates 12；The peak height occurred in 39~46min retention times and peak face
Product, collection of illustrative plates 13 is not changed in substantially with collection of illustrative plates 12；And table 4 is relative to acids, esters, the alcohol that C11~C16 is increased in table 3
The organic matter such as class and cyclohexanes, has lacked pentadecane, hexadecane and octadecane, correspondence atlas analysis heptadecane, nonadecane, two
Hendecane, lignocerane are reduced, and functions bacterium SDCD-3 has to C15~C19 linear paraffins and C21 and C24 linear paraffins
Preferable degradation effect, produces the intermediate products such as acids, esters, alcohols and cyclohexanes.
Sequence table
<110>Chengdu University of Technology
<120>One plant of oil-base mud well drilling detritus degradation function bacterium and its application
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1053
<212> DNA
<213>Bacillus subtilis Pseudomonas（Bacillus subtilis）
<400> 1
tgcaatggcg gcagctatac atgcagtcga gcgaactgat tagaagcttg cttctatgac 60
gttagcggcg gacgggtgag taacacgtgg gcaacctgcc tgtaagactg ggataacttc 120
gggaaaccga agctaatacc ggataggatc ttctccttca tgggagatga ttgaaagatg 180
gtttcggcta tcacttacag atgggcccgc ggtgcattag ctagttggtg aggtaacggc 240
tcaccaaggc aacgatgcat agccgacctg agagggtgat cggccacact gggactgaga 300
cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 360
gacggagcaa cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg 420
aagaacaagt acgagagtaa ctgctcgtac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 540
gcgtaaagcg cgcgcaggcg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg 600
gagggtcatt ggaaactggg gaacttgagt gcagaagaga aaagcggaat tccacgtgta 660
gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcggctt tttggtctgt 720
aactgacgct gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960
gtcttgacat cctctgacac tctagagata gagcgttccc cttcggggga cagagtggac 1020
aggtgggttg catgggttgt cgtcagcctc gtg 1053
Claims (10)
1. a bacillus subtilis belong to (Bacillus subtilis strain.) bacterium, and microbial preservation numbering is CGMCC
13202。
2. bacillus subtilis Pseudomonas bacterium according to claim 1, the nucleotide sequence such as sequence table SEQ of its 16S rDNA
Shown in ID NO.1.
3. the bacillus subtilis Pseudomonas bacterium described in claim 1 is used for the purposes of oil base drilling wastes of degrading.
4. the purposes according to right 3, it is characterised in that the oil base drilling wastes are oil-base mud well drilling detritus.
5. it is a kind of degrade oil base DWM landwaste method, including：After oil-base mud well drilling detritus add minimal medium,
Bacillus subtilis Pseudomonas bacterium bacteria suspension described in inoculation claim 1, it is incubated.
6. method according to claim 5, it is characterised in that the bacillus subtilis Pseudomonas bacterium CGMCC's 13202
Culture pH value is 6~8, and preferably pH value is 7~7.2.
7. method according to claim 5, it is characterised in that the bacillus subtilis Pseudomonas bacterium CGMCC's 13202
Cultivation temperature is 33~37 DEG C, preferably 35 DEG C.
8. method according to claim 5, it is characterised in that the bacillus subtilis Pseudomonas bacterium CGMCC's 13202
In culture environment, nitrogen is (1~96) with the mass ratio of P elements: 1；Preferably, nitrogen is with the mass ratio of P elements
(6~12): 1；Most preferably, nitrogen and the mass ratio of P elements are 12:1, wherein nitrogen element content is 700 mg/litres, phosphorus
Constituent content is 58mg/ liters.
9. method according to claim 5, it is characterised in that the training of the bacillus subtilis Pseudomonas bacterium CGMCC13202
In foster environment, 4~6 g/l of NaCl contents, preferably 5 g/l.
10. method according to claim 5, it is characterised in that the method includes:Oil-base mud well drilling detritus are added into nothing
Machine salt culture medium, the bacteria suspension of bacillus subtilis Pseudomonas bacterium bacterial strain, described as described in 10% volume ratio inoculation claim 1
The bacteria concentration order of magnitude of bacteria suspension is 107CFU/ milliliters, pH is 7.0~7.2, is placed in constant incubator and stirs, 35 DEG C of cultures
18 days, nitrogen and the mass ratio of P elements were 12: 1 in culture environment, and wherein nitrogen element content is 700 mg/litres, P elements
Content is 58 mg/litres；NaCl contents are preferably 5 g/l in culture environment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486726A (en) * | 2018-12-27 | 2019-03-19 | 黄河三角洲京博化工研究院有限公司 | The bacterial strain of one plant of degradable petroleum hydrocarbon and its application |
| CN110468082A (en) * | 2019-09-19 | 2019-11-19 | 四川农业大学 | A kind of A Shi bacillus OCB-6 and its application |
| CN110747141A (en) * | 2019-11-04 | 2020-02-04 | 重庆市生态环境科学研究院 | Oil-based drilling cutting degrading strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182093A (en) * | 2007-12-07 | 2008-05-21 | 陈五岭 | Microbe harmless treatment method for oil-gas field waste slurry |
| CN104694439A (en) * | 2015-03-24 | 2015-06-10 | 东华大学 | Crude oil degradation bacterium and application thereof |
| CN105907679A (en) * | 2016-05-11 | 2016-08-31 | 陈五岭 | Composition or composite bacterium agent for treating waste well drilling mud |
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2017
- 2017-01-12 CN CN201710022627.0A patent/CN106801025A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182093A (en) * | 2007-12-07 | 2008-05-21 | 陈五岭 | Microbe harmless treatment method for oil-gas field waste slurry |
| CN104694439A (en) * | 2015-03-24 | 2015-06-10 | 东华大学 | Crude oil degradation bacterium and application thereof |
| CN105907679A (en) * | 2016-05-11 | 2016-08-31 | 陈五岭 | Composition or composite bacterium agent for treating waste well drilling mud |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486726A (en) * | 2018-12-27 | 2019-03-19 | 黄河三角洲京博化工研究院有限公司 | The bacterial strain of one plant of degradable petroleum hydrocarbon and its application |
| CN110468082A (en) * | 2019-09-19 | 2019-11-19 | 四川农业大学 | A kind of A Shi bacillus OCB-6 and its application |
| CN110747141A (en) * | 2019-11-04 | 2020-02-04 | 重庆市生态环境科学研究院 | Oil-based drilling cutting degrading strain and application thereof |
| CN110747141B (en) * | 2019-11-04 | 2021-05-18 | 重庆市生态环境科学研究院 | Oil-based drilling cutting degrading strain and application thereof |
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Application publication date: 20170606 |