CN110699297B - Alcaligenes faecalis subspecies and application thereof - Google Patents
Alcaligenes faecalis subspecies and application thereof Download PDFInfo
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- CN110699297B CN110699297B CN201911102882.1A CN201911102882A CN110699297B CN 110699297 B CN110699297 B CN 110699297B CN 201911102882 A CN201911102882 A CN 201911102882A CN 110699297 B CN110699297 B CN 110699297B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/05—Alcaligenes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
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- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention provides an alcaligenes faecalis subspecies and application thereof. The classification of the subspecies of the alcaligenes faecalis is Alcaligene sfaecalis subsp.Phenolicus GFB-14, and the subspecies of the alcaligenes faecalis are preserved in China Center for Type Culture Collection (CCTCC) in 8-19 days of 2019, and the preservation number is CCTCC NO: m2019646. The alcaligenes faecalis subspecies can efficiently degrade water-based drilling solid waste and organic additives, can be used as excellent degradation bacteria for biological treatment of the water-based drilling solid waste and environmental pollution thereof, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, in particular to an alcaligenes faecalis subspecies and application thereof.
Background
Natural gas (conventional gas and shale gas) is an important energy source for national economic development, but drilling base fluid must be used in the natural gas exploration and exploitation process to lubricate and cool drilling tools, protect well walls and balance formation pressure. In order to meet various requirements of drilling, the water-based drilling base fluid is usually added with some polymer organic matters to improve the thermal stability, high temperature resistance, filtration reduction and other performances of the drilling fluid, and currently used polymer additives mainly comprise sulfonated lignite, sulfonated phenolic resin, CMC and PAM, wherein the addition amount is 1% -5%. The polymers are polymerized by various organic matters and are an important source of water-based drilling solid waste COD; and simultaneously has the characteristic of difficult degradation. Therefore, if the polymer additive enters water environment or soil, the growth of animals and valued objects is seriously affected, even the health of human bodies is endangered, and the research significance of carrying out the harmless treatment of the solid waste of the water-based drilling is great.
At present, the water-based drilling solid waste harmless treatment technology comprises the steps of preparing sintered bricks, producing cement and the like. However, these treatment techniques have problems such as high energy consumption, low digestion, and susceptibility to geographical constraints. The microorganism can convert complex organic matters into simple organic matters, thereby achieving the harmless effect, and the method has the advantages of low investment and operation cost, low energy consumption, large digestion amount and no regional limitation, so the water-based drilling solid waste microorganism treatment is more and more concerned in recent years. The key point of the microbial treatment is to screen out high-efficiency degradation strains, more than 100 genera and more than 200 species of bacteria, actinomycetes, yeasts, molds, algae and other microbial groups are reported to degrade organic pollutants, and part of strains are already applied. However, in the field of petroleum and natural gas exploration, most of the treatment technologies of drilling solid waste/slurry microorganisms relate to petroleum hydrocarbon degradation microorganisms, and the research on degradation microorganism resources of high polymer organic additives is less. Therefore, the resources of the high-efficiency water-based drilling solid waste degradation strain are further enriched.
Disclosure of Invention
An object of the present invention is to solve one or more of the problems occurring in the prior art, in view of the disadvantages of the prior art. For example, one of the purposes of the invention is to provide a strain which efficiently degrades water-based drilling fluid additives, and a subspecies of alcaligenes faecalis which is relatively resistant to stress.
In order to achieve the above object, in one aspect, the present invention provides a subspecies of alcaligenes faecalis, which is classified and named as Alcaligene sfaecalis subsp.phenolicus GFB-14, and has been preserved in the China center for type culture collection (CCTCC NO) at 8.19 of 2019: m2019646.
In an exemplary embodiment of the present invention, the subspecies of alcaligenes faecalis are gram positive bacteria, have spores, and the thallus is rod-shaped, motile, and obligatory aerobic.
In an exemplary embodiment of a strain of alcaligenes faecalis subspecies of the present invention, the 16S rDNA sequence of the alcaligenes faecalis subspecies is shown in SEQ ID NO. 1.
In an exemplary embodiment of the invention, the colony formed by the alcaligenes faecalis subspecies on the beef extract peptone culture medium after 24 hours is round or irregular, and the colony after 48 hours is round, white, 0.5-1 mm in diameter, neat in edge and flat and moist.
In another aspect of the present invention there is provided a degrading biological agent which may comprise the above-described alcaligenes faecalis subspecies or a bacterial suspension thereof or a culture broth thereof or a fermentation product thereof or a fermentation supernatant thereof.
In a further aspect, the invention provides the use of said subspecies alcaligenes faecalis and said degrading biological agent for degrading water-based drilling solid wastes and organic additives.
In a further aspect, the invention provides the use of said alcaligenes faecalis subspecies and said degrading biological agent for degrading water-based drilling fluid additives sulfonated lignite, sulfonated phenolic resins, carboxymethyl cellulose and polyacrylamide.
In a further aspect, the invention provides the use of said alcaligenes faecalis subspecies and said degrading biological agent for degrading total organic carbon of water-based drilling solid wastes.
In a further aspect, the invention provides the use of the alcaligenes faecalis subspecies and the degraded biological agent in the remediation of a water-based drilling solid waste polluted environment.
Compared with the prior art, the invention has the beneficial effects that: the alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14 can effectively degrade water-based drilling solid waste and main organic additives; after the inoculation and cultivation are carried out for 7 days, the degradation rate of the alcaligenes faecalis subspecies on COD (chemical oxygen demand) of the water-based drilling solid waste reaches 64.34 percent, the degradation rate of the sulfonated lignite reaches 26.12 percent, the degradation rate of the sulfonated phenolic resin reaches 5.65 percent, the degradation rate of sodium carboxymethylcellulose (CMC) reaches 32.0 percent, the degradation rate of Polymethyl Acrylate (PAM) reaches 45.61 percent, and the treatment effect is obvious; the alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14 has strong stress resistance, can be used as excellent degradation bacteria for biological treatment of water-based drilling solid waste and polluted environment thereof, and has good application prospect.
Drawings
The foregoing and other objects and features of the invention will become more apparent from the following description taken in conjunction with the accompanying drawings in which:
FIG. 1 shows the morphology of thalli and colonies of the hypocrete subspecies of the present invention on beef extract peptone medium;
FIG. 2 shows a phylogenetic diagram of the 16S rDNA sequence of the alcaligenes faecalis subspecies of an exemplary embodiment of the invention.
Detailed Description
Hereinafter, a fecal alciferol subspecies and the use thereof according to the present invention will be described in detail with reference to the drawings and exemplary embodiments.
In one aspect of the invention, a sub-species of Alcaligenes faecalis is provided, which is classified under the name Alcaligene sfaecalis subsp. The strain has been preserved, preservation unit: china center for type culture Collection; address: in the Wuhan university of No. 299 of Wuhan district of Wuhan, hubei province; preservation date: 8 months and 19 days in 2019, the preservation number is CCTCC NO: m2019646.
In this example, the Alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 was gram positive with spores, and the cells were rod-shaped, motile, and obligatory aerobic.
In this example, total DNA of Alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 was extracted, a 16S rDNA fragment was amplified, and the 16S sequence was determined as shown in SEQ ID NO. 1. The measured 16S sequences were aligned in the National Center for Biological Information (NCBI) database and GFB-14 was most similar (99.4%) to the model strain Alcaligenes faecalis subspecies (Alcaligenes faecalis subsp. Phenolicus) J T in GenBank, thereby determining that GFB-14 belongs to Alcaligenes faecalis subspecies (Alcaligenes faecalis subsp. Phenolicus GFB-14) in classification.
In this example, the colony formed by culturing the Alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 on beef extract peptone medium for 24 hours is round or irregular, and the colony after 48 hours is round, milky white (white), 2 mm-3 mm in diameter, irregular in edge and flat and moist.
The alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 has strong degradation capability, can degrade water-based drilling solid waste and organic pollutants, and is particularly suitable for degrading water-based drilling fluid additives such as sulfonated lignite, sulfonated phenolic resin, carboxymethyl cellulose (CMC) and Polyacrylamide (PAM). And the alcaligenes faecalis subspecies can repair the medium polluted by the solid waste of the water-based drilling fluid, wherein the medium is soil, water and air. The Alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 can degrade the total organic carbon (total organic carbon) TOC of water-based drilling solid wastes.
The bacterial suspension of the alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 or the culture solution or the fermentation product or the fermentation supernatant thereof has strong degradation capability, can degrade water-based drilling solid waste and organic pollutants, can degrade sulfonated lignite, sulfonated phenolic resin, carboxymethyl cellulose (CMC) and Polyacrylamide (PAM), and degrade the total organic carbon (totalorganic carbon) TOC of the water-based drilling solid waste, and can repair the medium polluted by the water-based drilling solid waste. In the degradation of the above-mentioned substances, the addition ratio of the Alcaligenes faecalis subsp (Alcaligene sfaecalis subsp. Phenolicus) GFB-14 may be 0.5% or more of the mass of the treated matter. For example, for soil remediation, the alkaline manure mycophenolic subspecies may be added in an amount of 0.7% of the soil mass. The temperature of the treatment may be 20℃to 28 ℃.
In another aspect of the invention, a degradation biologic is provided. In an exemplary embodiment of the degraded biological agent of the present invention, the active ingredient of the degraded biological agent comprises GFB-14 or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof or a fermentation supernatant thereof of the above-described alcaligenes faecalis subspecies (Alcaligene sfaecalis subsp. Phenolicus).
Similarly, the manure degradation biological agent has strong degradation capability, can degrade water-based drilling solid waste and organic pollutants, can degrade sulfonated lignite, sulfonated phenolic resin, carboxymethyl cellulose (CMC) and Polyacrylamide (PAM) and total organic carbon (total organic carbon) TOC of the water-based drilling solid waste, and can repair medium polluted by the water-based drilling liquid solid waste.
For a better understanding of the above-described exemplary embodiments of the present invention, they are further described below in conjunction with specific examples.
Example 1
(1) Isolation, purification and preservation of Alcaligenes faecalis GFB-14 (abbreviated as "strain GFB-14").
The water-based drilling solid waste sample of the potassium polysulfonate system is collected from the natural gas drilling site in the parallel market of Sichuan province, stored at a low temperature and brought back to a laboratory.
And (3) respectively preparing:
beef extract peptone medium: beef extract 3g, peptone 10g, sodium chloride 5g, agar 20g, natural pH and water 1000mL.
Taking 5kg of water-based drilling solid waste, adding straw powder into the drilling solid waste according to 10% (w: w, mass ratio concentration), adding ammonium bicarbonate, adjusting the C/N of a mixed matrix to be 15:1-20:1, and adjusting the water content to be 28% -30% by using distilled water; the mixed substrate was placed in a plastic pot (standard: length×width×height=60 cm×35cm×10 cm) with white water, sealed with a preservative film, and incubated at 30 ℃. Periodically agitating and oxygenating, and controlling the water content at 28% -30%. After 30d of culture, sampling is carried out, and the mixture is coated on a beef extract peptone culture medium by adopting a gradient dilution method and cultured at a constant temperature of 30 ℃. After bacterial colonies grow out, colonies with different forms are selected from the plates and streaked on the beef extract peptone plates. After 16h of incubation, a loop was picked for repeated streak incubation, combined with microscopic observation, until purification. And (5) inoculating the purified strain into a test tube slant culture medium of a beef extract peptone culture medium for preservation.
As shown in FIG. 1, the isolated and purified strain GFB-14 of this example was cultured on a beef extract peptone medium, and after 48 hours of culture, the colony was round, white, 0.5mm to 1mm in diameter, and flat and moist with a neat edge, wherein FIG. 1 (a) shows the colony morphology and FIG. 1 (b) is a schematic diagram of the colony after streaking on a beef extract peptone plate.
(2) Amplification and phylogenetic analysis of 16S rDNA of Strain GFB-14
The total DNA of the strain is extracted, the total DNA is used as a template, the 16S fragment is amplified by using 27F and 1492R as primers, and a PCR reaction is performed by using a Bio-RADMyCyclerTM instrument.
Reaction system (50 μl): 2 XPCRMIX 25. Mu.l, 1. Mu.l each of primers 27F and 1492R (10. Mu.M), 1. Mu.l of DNA template, and make up to 50. Mu.l with ultrapure water; the nucleotide sequences of primers 27F and 1492R are shown in SEQ ID No.2 and SEQ ID No. 3.
PCR reaction conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 54℃for 1min, extension at 72℃for 2min, and cycling for 30 times; finally, the extension is carried out for 8min at 72 ℃.
The PCR amplified product was detected by electrophoresis on a 1.0% agarose gel and sent to Shanghai Biotechnology Co., ltd. The calculation of the similarity of the gene sequences was performed using the software DNA man 6.0. The sequencing result is shown as SEQ ID No.1 (shown in a sequence table).
The sequence results obtained were aligned in the National Center for Biological Information (NCBI) database to find the 16S rDNA gene sequence of GFB-14 and the model strain Alcaligenes faecalis subspecies (Alcaligenes faecalis subsp. Phenolicus) J in the database T The highest similarity of (2) was 99.4%. Based on NCBI alignment results, the model strain with highest similarity was selected as the reference strain, and a phylogenetic tree (as shown in FIG. 2) was constructed by the Neighbor-joining method (MEGA 6.0 software), and the bootstrap value (boottrap) was 1000.
Based on the above characteristics, the strain GFB-14 was identified as a sub-species of Alcaligenes faecalis (Alcaligenes faecalis subsp. Phenolicus). The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 8 and 19 days, and the preservation number is CCTCC NO: m2019646.
(3) Alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14 ability experiments to degrade sulfonated Brown coal, sulfonated phenolic resin, CMC and PAM
GFB-14 is inoculated on beef extract peptone liquid culture medium for activating culture, when the bacterial suspension OD600 = 1.0 for activating culture, 1.5mL of bacterial liquid is taken, the bacterial liquid is centrifuged at 3000rpm for 5min, the supernatant is discarded, bacterial cells are washed 2 times by sterile physiological saline, 1.5mL of sterile water is used for suspending, and the bacterial cells are respectively inoculated into sterilized sulfonated lignite, sulfonated phenolic resin, CMC and MS culture medium with the concentration of 1% of PAM, and shake culture is carried out at 30 ℃ and 140rpm, and the bacterial culture is repeated for 3 times by taking non-inoculation as a control.
Measurement of the ability of the strain to degrade sulfonated lignite: drying the filter paper to constant weight at 80 ℃, weighing and recording (M1), filtering the culture solution after 7d of test culture by using the weighed filter paper, drying the filter paper and solid residues to constant weight at 80 ℃, weighing and recording (M2); the control residue was weighed in the same manner and recorded (M3). The degradation rate (%) = (M2-M1) of sulfonated lignite is 100/(M3-M1).
Measurement of the ability of the strain to degrade sulfonated phenolic resin/CMC/PAM: after 7d of culture in the above test, the culture broth was collected, centrifuged at 1000rpm for 5min, the supernatant was collected, the TOC content thereof was measured and recorded (T1), and the initial TOC content of the treatment broth was measured and recorded (T2) in the same manner. Sulfonated phenolic resin/CMC/PAM degradation rate (%) = (T2-T1) ×100/T1.
The measurement results show that after 7d of culture, the degradation rates of the sulfonated lignite, the sulfonated phenolic resin, the CMC and the PAM treated by the GFB-14 strain reach 26.12%, 5.65%, 32.0% and 45.61%, respectively.
(4) Resistance test of Alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14
Acid or alkali resistance determination: the pH of the medium was measured and adjusted to 4,5,6,7,8,9, 10 with 10mol/L NaOH solution and 10mol/L HCl solution. After sterilization at 121℃for 30min, the bacterial suspension was added to the freshly prepared medium at 1% (v: v), three replicates were set, placed in a 30℃incubator and shake-cultured at 150r/min for 48h, and the number of bacteria was determined by UV spectrophotometry at wavelength λ=600.
Salt tolerance determination: preparing a beef extract peptone culture medium, and regulating the pH value to 7; naC1 was added to the basal beef extract peptone medium to prepare respective media having salt concentrations of 0.5%, 1%, 2%, 4%, 8% and 10%. After sterilization at 121℃for 30min, the bacterial suspension was added to the freshly prepared medium at 1% (v: v), three replicates were set, placed in a 30℃incubator and shake-cultured at 150r/min for 48h, and the number of bacteria was determined by UV spectrophotometry at wavelength λ=600.
And (3) measuring a growth temperature range: preparing beef extract peptone culture medium, adjusting pH value to 7, sterilizing at 121deg.C for 30min, adding bacterial suspension to the newly prepared culture medium according to 1% (v: v), placing the inoculated culture medium into constant temperature incubator at 4deg.C, 15deg.C, 30deg.C, 45 deg.C and 55deg.C respectively, shake culturing at 150r/min for 48 hr, repeating each temperature for 3 times, and measuring bacterial quantity at wavelength lambda=600 with ultraviolet spectrophotometer.
The result shows that the alcaligenes faecalis subspecies Alcaligenes faecalis subsp.Phenolicus GFB-14 has good stress resistance, and the initial pH value of the growth is=5.0-10.0; the salt tolerance is strong, and the plant can grow in a beef extract peptone culture medium containing 4% NaCl; the growth temperature range is wider, and the growth can be carried out within the temperature range of 15-45 ℃.
(5) Experiment of Alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14 on treatment of solid waste from Water-based drilling
According to the treatment water-based drilling solid waste amount of 1% (m/m), inoculating GFB-14 microbial inoculum (OD 600nm approximately equal to 1.6) to the treated matter (water-based drilling solid waste: corn straw=9:1), fully mixing uniformly, taking non-inoculation as a control, and repeating for 3 times. Each treated drilling solid waste weighed 5.0kg. The treatment was carried out under natural conditions for 45d (from 6 months in 2019 to 7 months in 2019), and the humidity of the treatment system was kept at about 25%. TOC content and leachate COD of the initial treated material and the inoculated and non-inoculated treated samples of treatment 45d were measured, respectively.
The results show that after 45d treatment, the TOC removal rate of GFB-14 in water-based drilling solid waste reaches 22.09%, the COD degradation rate of the leaching solution reaches 80.35%, which is remarkably higher than that of the control 1.23% and 20.67%, and good degradation potential is shown.
TABLE 1 Effect of Strain GFB-14 on treatment of Water-based drilling solid wastes
Note that: different letters represent a significant level of difference (p < 0.05)
In conclusion, the alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14 can effectively degrade water-based drilling solid wastes and main organic additives; after the inoculation and cultivation are carried out for 7 days, the degradation rate of the alcaligenes faecalis subspecies on COD (chemical oxygen demand) of the water-based drilling solid waste reaches 64.34 percent, the degradation rate of the sulfonated lignite reaches 26.12 percent, the degradation rate of the sulfonated phenolic resin reaches 5.65 percent, the degradation rate of sodium carboxymethylcellulose (CMC) reaches 32.0 percent, the degradation rate of Polymethyl Acrylate (PAM) reaches 45.61 percent, and the treatment effect is obvious; the alcaligenes faecalis subsp Alcaligenes faecalis subsp.Phenolicus GFB-14 has strong stress resistance, can be used as excellent degradation bacteria for biological treatment of water-based drilling solid waste and polluted environment thereof, and has good application prospect.
Although the present invention has been described above by way of the combination of the exemplary embodiments, it should be apparent to those skilled in the art that various modifications and changes can be made to the exemplary embodiments of the present invention without departing from the spirit and scope defined in the appended claims.
Sequence listing
<110> China oil and gas group Co., ltd, which is a drilling engineering Co., ltd
<120> a strain of alcaligenes faecalis subspecies and use thereof
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<213> Alcaligenes faecalis subspecies (Alcaligenes faecalis subsp. Phenolicus)
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gcgtcgccag ctttaccatg cagtcgaacg gcagcgcgag agagcttgct ctcttggcgg 60
cgagtggcgg acgggtgagt aatatatcgg aacgtgccca gtagcggggg ataactactc 120
gaaagagtgg ctaataccgc atacgcccta cgggggaaag ggggggatcg caagacctct 180
cactattgga gcggccgata tcggattagc tagttggtgg ggtaaaggct caccaaggca 240
acgatccgta gctggtttga gaggacgacc agccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatttt ggacaatggg ggaaaccctg atccagccat 360
cccgcgtgta tgatgaaggc cttcgggttg taaagtactt ttggcagaga agaaaaggta 420
tcccctaata cgggatactg ctgacggtat ctgcagaata agcaccggct aactacgtgc 480
cagcagcssc ggtaatacgt agggtgcaag cgttaatcgg aattactggg cgtaaagcgt 540
gtgtaggcgg ttcggaaaga aagatgtgaa atcccagggc tcaaccttgg aactgcattt 600
ttaactgccg agctagagta tgtcagaggg gggtagaatt ccacgtgtag cagtgaaatg 660
cgtagatatg tggaggaata ccgatggcga aggcagcccc ctgggataat actgacgctc 720
agacacgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccctaaacg 780
atgtcaacta gctgttgggg ccgttaggcc ttagtagcgc agctaacgcg tgaagttgac 840
cgcctgggga gtacggtcgc aagattaaaa ctcaaaggaa ttgacgggga cccgcacaag 900
cggtggatga tgtggattaa ttcgatgcaa cgcgaaaaac cttacctacc cttgacatgt 960
ctggaaagcc gaagagattt ggccgtgctc gcaagagaac cggaacacag gtgctgcatg 1020
gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080
tcattagttg ctacgcaaga gcactctaat gagactgccg gtgacaaacc ggaggaaggt 1140
ggggatgacg tcaagtcctc atggccctta tgggtagggc ttcacacgtc atacaatggt 1200
cgggacagag ggtcgccaac ccgcgagggg gagccaatct cagaaacccg atcgtagtcc 1260
ggatcgcagt ctgcaactcg actgcgtgaa gtcggaatcg ctagtaatcg cggatcagaa 1320
tgtcgcggtg aatacgttcc cgggtcttgt acacaccgcc cgtcacacca tgggagtggg 1380
tttcaccaga agtaggtagc ctaaccgtaa ggagggcgct accacggtga tcgtg 1435
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (27F)
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agagttgatc ctggctcag 19
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (1492R)
<400> 3
cggttacctt gttacgactt 20
Claims (9)
1. The alcaligenes faecalis subspecies is characterized in that the classification of the alcaligenes faecalis subspecies is Alcaligenes faecalis subsp. Phenolicus GFB-14, and the alcaligenes faecalis subspecies are preserved in China Center for Type Culture Collection (CCTCC) on 8 months 19 days 2019, and the preservation number is CCTCC NO: m2019646.
2. The subspecies of alcaligenes faecalis of claim 1, wherein the subspecies of alcaligenes faecalis are gram positive bacteria, have spores, and the thalli are rod-shaped, motile, and obligate aerobic.
3. The alcaligenes faecalis subspecies according to claim 1, wherein the 16S rDNA sequence of the alcaligenes faecalis subspecies is shown in SEQ ID No. 1.
4. The alcaligenes faecalis subspecies according to claim 1, wherein the colony formed after the alcaligenes faecalis subspecies are cultured on the beef extract peptone culture medium for 24 hours is round or irregular, and the colony after 48 hours is round, white, 0.5-1 mm in diameter, neat in edge and flat and moist.
5. Use of the alcaligenes faecalis subspecies or its bacterial suspension or its culture broth of claim 1 for degrading water-based drilling solid waste containing sulfonated lignite, sulfonated phenolic resin, carboxymethyl cellulose or polyacrylamide.
6. A degradation biological agent comprising the alcaligenes faecalis subspecies or bacterial suspension or culture thereof of claim 1.
7. Use of the alcaligenes faecalis subspecies of claim 1 or the degraded biological agent of claim 6 for degrading water-based drilling solid waste and organic additives including water-based drilling fluid additives sulfonated lignite, sulfonated phenolic resins, carboxymethyl cellulose and polyacrylamide.
8. Use of the alcaligenes faecalis subspecies of claim 1 or the degrading biological agent of claim 6 for degrading total organic carbon of water-based drilling solid waste.
9. Use of the alcaligenes faecalis subspecies of claim 1 or the degraded biological agent of claim 6 for remediation of a water-based drilling solid waste contaminated environment.
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