CN100494350C - Clostridium Daqing clostridium and application thereof - Google Patents
Clostridium Daqing clostridium and application thereof Download PDFInfo
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Abstract
The present invention relates to clostridium 'daqing' clostridium and its uses. The present invention solves the problem it is difficult to remove or degrade polypropylene of oil field sewage. The cloThe present invention relates to clostridium 'daqing' clostridium and its uses. The present invention solves the problem it is difficult to remove or degrade polypropylene of oil field sewage. The clostridium 'daqing' clostridium -WL is preserved at CGMCC on January 10, 2007, preserving number being CGMCC No. 1911. The 'daqing' clostridium is Gram-positive bacilli, with endo-spore, facultative anastridium 'daqing' clostridium -WL is preserved at CGMCC on January 10, 2007, preserving number being CGMCC No. 1911. The 'daqing' clostridium is Gram-positive bacilli, with endo-spore, facultative anaerobic, grows under pH 5.0-11.0,10-65 degree. The strain is variable in length, breadth being 0.4um-0.8um, length being 1.4um-4um, free of flagellum. The inventive 'daqing' clostridium -WL is a new sterobic, grows under pH 5.0-11.0,10-65 degree. The strain is variable in length, breadth being 0.4um-0.8um, length being 1.4um-4um, free of flagellum. The inventive 'daqing' clostridium -WL is a new strain, and can use polypropylene as unique carbon source which can be used in polypropylene biodegradation. 10ml 'daqing' clostridium -WL of concentration 6x10<6> per ml is added in oil field sewage wrain, and can use polypropylene as unique carbon source which can be used in polypropylene biodegradation. 10ml 'daqing' clostridium -WL of concentration 6x10<6> per ml is added in oil field sewage with polypropylene concentration being 500mg/L and the polypropylene degradation rate is 35%-76% after 20 days. ith polypropylene concentration being 500mg/L and the polypropylene degradation rate is 35%-76% after 20 days.
Description
Technical field
The present invention relates to a kind of microorganism and application thereof.
Background technology
Water-soluble polymers polyacrylamide (HPAM) is widely-used in the tertiary oil production in oil field, Daqing oil field in China is extensive use of the tertiary oil recovery of " ternary composite driving " oil recovery technique now, and " ternary " mainly comprises super high molecular weight polyacrylamide (HPAM), alkali and tensio-active agent.Super high molecular weight polyacrylamide (HPAM) aqueous solution is used as oil-displacing agent and injects oil reservoir, make that mobility ratio descends between crude oil (composition is mainly stable hydrocarbon, aromatic hydrocarbons, colloid and bituminous matter) with big viscosity and the water, weaken the viscous fingering of water, thereby improve oil recovery factor.Not only contain crude oil in the extraction liquid of polymer flooding, but also contain a large amount of polyacrylamides, cause the sewage that contains polyacrylamide to roll up, the polyacrylamide amine content also significantly increases in the sewage.The sewage that the oil field contains polyacrylamide is relatively more complicated, the special sewage of a class, exists oil-containing many, and therefore the characteristics that viscosity is big are difficult to remove or degrade polypropylene of oil field sewage.
Summary of the invention
The objective of the invention is to be difficult at present to remove or the problem of degrade polypropylene of oil field sewage in order to solve, and a kind of clostridium Daqing clostridium and the application thereof that provide.
Clostridium Daqing clostridium-WL provided by the invention (Clostridium Daqing WL) belongs to genus clostridium (Clostridium) through evaluation, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the preservation address is the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, preservation date is on January 10th, 2007, and preserving number is CGMCC No.1911; Grand celebration clostridium-WL is a Gram-positive bacillus, endogenous spore, and amphimicrobian is grown under the condition of pH5.0～11.0,10～65 ℃, and bacterial strain length is changeable, and wide is 0.4 μ m～0.8 μ m, and length is 1.4 μ m～4 μ m, atrichia; Grand celebration clostridium-WL is grown to the circular bacterium colony of black, smooth surface, neat in edge on solid medium; Grand celebration clostridium-WL can be sole carbon source with the polyacrylamide.Above-mentioned clostridium Daqing clostridium-WL can be a sole carbon source with the polyacrylamide, can be used for the biological degradation polyacrylamide.
Clostridium Daqing clostridium-WL provided by the invention (Clostridium Daqing WL) is 94% by the similarity of the 16S rDNA gene order the most close kind Si Shi clostridium with it of compare of analysis (Clostridiumsticklandii), and 16S～23S rDNA transcribed spacer sequence alignment result shows that conservative region only is tRNA
AlaAnd tRNA
IleSequence, the variation zone does not have the homology zone with the 16S～23S rDNA transcribed spacer sequence that belongs to known other kind brood cell clostridium bacterium together.According to " Oren A, Ventosa A, GrantWD.Proposed minimal standards for the description of new taxa in the OrderHalobacteriales.Int J Syst Bacteriol, 1997,47:233-2381 " in 16S rDNA sequence homology both can regard as the theory that belongs to not of the same race less than 97%, clostridium Daqing clostridium-WL provided by the invention belongs to a novel bacterial---clostridium Daqing clostridium kind (ClostridiumDaqing).
Clostridium Daqing clostridium-WL provided by the invention can be sole carbon source with the polyacrylamide.With the 10mL bacterial concentration is 6 * 10
6The clostridium Daqing clostridium of individual/mL-WL adds cultivates in the oilfield sewage that the 90mL concentration of polyacrylamide is 500mg/L after 20 days that the degradation rate of polyacrylamide is 35%～76% in the oilfield sewage.
Clostridium Daqing clostridium-WL (Clostridium Daqing WL) belongs to genus clostridium (Clostridium), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the preservation address is the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, preservation date is on January 10th, 2007, and preserving number is CGMCC No.1911.
Description of drawings
Fig. 1 is the saturating look electron microscopic observation figure of clostridium Daqing clostridium-WL, Fig. 2～Fig. 4 is the atomic force microscope observation figure of clostridium Daqing clostridium-WL, Fig. 5 is the NJ phylogeny tree graph of the 16S rDNA sequence construct of clostridium Daqing clostridium-WL and close bacterial strain, Fig. 6 is the scanning electron microscopic observation figure of polyacrylamide pressed powder dry plate, Fig. 7 is the scanning electron microscopic observation figure of polyacrylamide solution dry plate, and Fig. 8 is the scanning electron microscopic observation figure through the clostridium Daqing clostridium-polyacrylamide solution dry plate of WL degraded after 7 days.
Embodiment
Embodiment one: present embodiment clostridium Daqing clostridium-WL (ClostridiumDaqing WL) belongs to a novel species-grand celebration clostridium of genus clostridium (Clostridium) through evaluation, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on January 10th, 2007, and preserving number is CGMCC No.1911; Grand celebration clostridium-WL is a Gram-positive bacillus, endogenous spore, and amphimicrobian is grown under the condition of 5.0～11.0,10～65 ℃ of pH, and bacterial strain length is changeable, and wide is 0.4 μ m～0.8 μ m, and length is 1.4 μ m～4 μ m, atrichia; Grand celebration clostridium-WL is grown to the circular bacterium colony of black, smooth surface, neat in edge on solid medium; Grand celebration clostridium-WL can be sole carbon source with the polyacrylamide.
Clostridium Daqing clostridium-WL's in the present embodiment is different in size, and bacterial strain is wide to be that 0.4 μ m～0.8 μ m, length are 1.4 μ m～4 μ m.Fig. 1 is the saturating look electron microscopic observation figure of clostridium Daqing clostridium-WL, Fig. 2～4th, and the atomic force microscope observation figure of clostridium Daqing clostridium-WL can observe clostridium Daqing clostridium-WL atrichia from Fig. 1～4.
Clostridium Daqing clostridium-WL in the present embodiment is that electron acceptor(EA) is reduced into H with it with vitriol, sulphite and reductibility sulfuration thing
2S, and amphimicrobian.Clostridium Daqing clostridium-WL in the present embodiment can be a carbon source with Sodium.alpha.-hydroxypropionate, sodium formiate, oxysuccinic acid, glucose, succsinic acid, yeast extract paste and peptone, is the nitrogenous source growth with sulfate of ammoniac, urea.
Embodiment two: present embodiment clostridium Daqing clostridium-WL (ClostridiumDaqing WL) bacterial strain is to record by following steps:
(1) separation, purifying and the screening of clostridium Daqing clostridium-WL bacterial strain:
A. the aqueous solution 5～10mL that gets polyacrylamide injection station mother liquor tank puts into the anaerobism bottle that is full of nitrogen, contains the anaerobism sterilized water, and adds 5～20 granulated glass spherees, the 4 ± 0.1h that vibrates under 30～38 ℃ condition;
B. use anaerobism sterilized water doubling dilution bacterium liquid, again bacterium liquid is inoculated in the improvement Starkey solid medium, adopt " rolling pipe " to cultivate 7～10 days;
C. carry out the separating for several times purifying until confirm as pure bacterial strain with Electronic Speculum;
(2) select efficient degrading bacterial strain: with being inoculated in the polyacrylamide by identical inoculum size after the pure bacterial strain enrichment cultivation is that sole carbon source prepares substratum, therefrom selects the highest bacterial strain of polyacrylamide degradation rate (clostridium Daqing clostridium-WL);
(3) bacterial strain (physiological and biochemical analysis of clostridium Daqing clostridium-WL): (bacterial strain is identified with reference to " the 8th edition operation of the outstanding Bacteria Identification handbook of uncle, qualification result are that clostridium Daqing clostridium-WL is that electron acceptor(EA) is reduced into H with it with vitriol, sulphite and reductibility sulfuration thing by the Physiology and biochemistry of clostridium Daqing clostridium-WL)
2S, and amphimicrobian; Clostridium Daqing clostridium-WL can be a carbon source with Sodium.alpha.-hydroxypropionate, sodium formiate, oxysuccinic acid, glucose, succsinic acid, yeast extract paste and peptone, is the nitrogenous source growth with sulfate of ammoniac, urea; Improvement Starkey liquid nutrient medium liquid phase end products is ethanol, acetate, propionic acid and butyric acid through gas chromatographic detection;
(4) bacterial strain (classification of clostridium Daqing clostridium-WL) is identified:
A. use precious biotechnology (Dalian) company limited test kit to extract the bacterial strain (DNA of clostridium Daqing clostridium-WL), the performing PCR of going forward side by side amplification, upstream primer the Eubac27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' of 16S rDNA sequence, downstream primer 1492R:5 '-GGTTACCTTGTTACGACTT-3 ', upstream primer the P1:5 '-GGGGTGAAGTCGTAACAAGGTA-3 ' of 16S～23S rDNA transcribed spacer, downstream primer P2:5 '-CCTCTGACTGCCAGGGCATCC-3 '; The PCR reaction system is 50 μ L, comprise dna profiling 40ng in the 50 μ LPCR reaction systems, final concentration is the TagDNA polysaccharase of 0.3U, concentration is the dNTP of 0.3mmol/L, and concentration is that primer Eubac27F, the concentration of 0.1 μ mol/L is that primer 1492R, the concentration of 0.1 μ mol/L is that primer P1 and the concentration of 0.1 μ mol/L is the primer P2 of 0.1 μ mol/L; The pcr amplification program is: 94 ℃ of preheating 5min, and 94 ℃ of sex change 30s, 58 ℃ of renaturation 45s, 72 ℃ are extended 90s, circulate 30 times, and last 72 ℃ are extended 10min;
B. amplified production is in 1.0% agarose electrophoresis detection;
The c.DNA gel reclaims and is connected with pGEM-T (Promega) carrier after test kit is cut the glue recovery, is transformed into intestinal bacteria TOP10 competent cell;
Add penbritin AMP (5 μ g/mL) and X-gal (5-bromo-4-hydrogen-3-indoles-β-D-galactoside) in the d.LB solid medium, screen blue hickie transformant;
E. extract plasmid and order-checking, examining order is finished by Shanghai biotechnology company limited; Clostridium Daqing clostridium-WL16S rDNA gene order is shown in SEQ ID NO.1, and 16S～23S rDNA transcribed spacer portion gene sequence is shown in SEQ ID NO.2.
Improvement Starkey solid medium is pressed 0.5g HPAM, 0.5gK in the present embodiment step ()
2HPO
4, 1.0g NH
4Cl, 2g Na
2SO
4, 0.1g CaCI
22H
2O, 2.0g MgSO
47H
2O, 1.0g yeast extract paste, 6mL concentration are the ratio preparation of 60% sodium lactate solution, 2% resazurin solution 1mL, 0.5g L-half light hydrochloride, 12g agarose and 1000mL water, and behind 121 ℃ of substratum sterilization 20min, add 0.1mL concentration separately and be 3% FeSO
4(NH
4)
2SO
4Solution is made; Wherein substratum boils the back and adds L-half light hydrochloride, and feeds high pure nitrogen and drive oxygen 30 ± 2min, and it is anaerobic state that substratum becomes the faint yellow substratum that shows by redness; Add separately 0.1mL concentration behind 121 ℃ of sterilization 20min and be 3% FeSO
4(NH
4)
2SO
4Solution.Improvement Starkey liquid nutrient medium only is not add agarose with the difference of improvement Starkey solid medium in the present embodiment step (three).
" rolling pipe " in the present embodiment step () and cultivating is to use the anaerobic operation technology that Hungate proposes, bacterium liquid is injected the solid medium that melts, in cold water, roll, make substratum under centrifugal action, on tube wall, the cultural method that bacterium is grown from the teeth outwards.
PCR primer in the present embodiment step (four) is synthetic by the precious biotechnology in Dalian company limited.Clostridium Daqing clostridium-WL gene order that present embodiment is obtained compares, and the 16S rDNA gene similarity of the most close kind Si Shi clostridium with it of clostridium Daqing clostridium-WL (Clostridiumsticklandii) is 94%; 16S～23S rDNA transcribed spacer sequence alignment result shows that conservative region only is tRNA
AlaAnd tRNA
IleSequence, the variation zone does not have the homology zone with the 16S～23S rDNA transcribed spacer sequence that belongs to known other kind brood cell clostridium bacterium together.Clostridium Daqing clostridium-WL belongs to a kind of novel bacterial by analysis---clostridium Daqing clostridium kind (Clostridium Daqing), according to the NJ phylogenetic tree of the 16SrDNA sequence construct of clostridium Daqing clostridium-WL and close bacterial strain as shown in Figure 5.
Embodiment three: present embodiment clostridium Daqing clostridium-WL can be a sole carbon source with the polyacrylamide, can be used for the biological degradation polyacrylamide.
Ability to clostridium Daqing clostridium-WL biological degradation polyacrylamide detects:
Getting 10mL under 10～38 ℃ condition, contain colony number is 6.0 * 10
6The clostridium Daqing clostridium of individual/mL-WL nutrient solution adds cultivates in the oilfield sewage that the 90mL concentration of polyacrylamide is 500mg/L after 20 days that the degradation rate of polyacrylamide is 35%～76% in the oilfield sewage.Change through clostridium Daqing clostridium-WL biological degradation polyacrylamide surface tissue, the amide group on the molecular chain is hydrolyzed into carboxyl, the side chain degraded, and part functional group changes, and solution viscosity descends more than 70%.
Analyze polyacrylamide generation chain rupture through gas chromatograph mass spectrometer (GC-MS): the degradation product of clostridium Daqing clostridium-WL degradation of polypropylene acid amides is checked through the retrieval of mass spectrum computer system and with the pure compound spectrogram, determine retention time be 9.368min be the positive palmitamide (C of substrate
16H
33N), be the product after the C-C chain rupture under the polyacrylamide biological degradation; Also has 3-methyl-4-health alcohol (C
8H
18O) retention time is 1.505min, 2-pyrovinic acid isobutyl ester (C
13H
24O
4) retention time is 2.418min, and in addition degradation product and the meta-bolites of 14 kinds of clostridium Daqing clostridium-WL arranged in addition, wherein removes the low molecular weight polyacrylamide (C that minority contains two keys, epoxy and carbonyl
4～C
30) outside the fragment, great majority are derivatives of acrylamide oligomer.
Polyacrylamide pressed powder, polyacrylamide solution and the polyacrylamide solution after clostridium Daqing clostridium-WL degraded 7 days are made dry plate, carry out metal spraying (Gatan 682 ion etching plated film instrument) for increasing the dry plate electric conductivity, observe the structure of polyacrylamide then down in scanning electron microscope (HITACHI S-4700), scanning electron microscopic observation figure is as a result gone into respectively as Fig. 6, Fig. 7 and shown in Figure 8.Scanning electron microscopic observation found that polyacrylamide pressed powder majority is block, is rough and uneven in surface; Polyacrylamide structure in the aqueous solution becomes the flakey of rule, marshalling, even structure; Polyacrylamide is after clostridium Daqing clostridium-WL degraded, the polyacrylamide amine structure becomes not have in a jumble and opens, gestalt takes place significantly to change and becomes many tiny random strips branch in structure, the polyacrylamide surface tissue changes, and illustrates that clostridium Daqing clostridium-WL has Degradation to polyacrylamide.
With polyacrylamide pressed powder, polyacrylamide solution with after clostridium Daqing clostridium-polyacrylamide solution of WL degraded after 7 days made dry plate, carry out infrared spectra and detect, the infrared spectra detected result as shown in Figure 9, curve A is a polyacrylamide solution dry plate infrared spectra curve among the figure, curve C is a polyacrylamide pressed powder dry plate infrared spectra curve among the figure, and curve B is through the clostridium Daqing clostridium-polyacrylamide solution dry plate infrared spectra curve of WL degraded after 7 days among the figure; The present worth that goes out of 3198.8299 absorption peaks is represented the N-C bond rupture among the figure; Occur 615.2946 to 1116.7572 the different benzene ring structure of absorption peak representative in the drawings through the clostridium Daqing clostridium-polyacrylamide solution dry plate infrared spectra curve of WL degraded after 7 days, these benzene ring structures are clostridium Daqing clostridium-WL degraded or meta-bolites.It is carbon source and nitrogenous source with the polyacrylamide that infrared spectra detects proof clostridium Daqing clostridium-WL, and the degraded of polyacrylamide amine side chain, part functional group change, and cause the polyacrylamide chain rupture.
Sequence table
＜110〉Harbin Institute of Technology
＜120〉a kind of clostridium Daqing clostridium and application thereof
<160>2
<210>1
<211>1695
<212>DNA
＜213〉genus clostridium (Clostridium)
<220>
＜223〉clostridium Daqing clostridium-WL 16S rDNA
<400>1
<210>2
<211>436
<212>DNA
＜213〉genus clostridium (Clostridium)
<220>
＜223〉clostridium Daqing clostridium-WL 16S～23S rDNA transcribed spacer portion gene sequence
<400>2
Claims (2)
1, a kind of clostridium Daqing clostridium, it is characterized in that clostridium Daqing clostridium-WL (Clostridium Daqing WL) belongs to genus clostridium (Clostridium) through evaluation, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on January 10th, 2007, and preserving number is CGMCC No.1911; Grand celebration clostridium-WL is a Gram-positive bacillus, endogenous spore, and amphimicrobian is grown under the condition of pH5.0～11.0,10～65 ℃, and bacterial strain length is changeable, and wide is 0.4 μ m～0.8 μ m, and length is 1.4 μ m～4 μ m, atrichia; Grand celebration clostridium-WL is grown to the circular bacterium colony of black, smooth surface, neat in edge on solid medium; Grand celebration clostridium-WL can be sole carbon source with the polyacrylamide.
2, the application of clostridium Daqing clostridium according to claim 1 is characterized in that clostridium Daqing clostridium-WL can be a sole carbon source with the polyacrylamide, can be used for the biological degradation polyacrylamide.
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| CN102452760B (en) * | 2010-10-19 | 2013-07-03 | 中国石油化工股份有限公司 | Treatment method for recycle of oil field produced water |
| CN106635863B (en) * | 2016-07-19 | 2018-09-11 | 桂林理工大学 | Anaerobic degradation handles the cultural method of the Clostridium strain YB-7 of oil extraction waste water |
| CN106477718B (en) * | 2016-07-19 | 2019-04-12 | 桂林理工大学 | A method of oil extraction waste water is handled using YB-7 bacterial strain anaerobic degradation |
| CN106635864B (en) * | 2016-07-19 | 2018-11-06 | 桂林理工大学 | A method of handling oil extraction waste water using YB-6 bacterial strain anaerobic degradations |
| CN106479911B (en) * | 2016-07-19 | 2018-09-11 | 桂林理工大学 | Anaerobic degradation handles the cultural method of the Clostridium strain YB-6 of oil extraction waste water |
| CN111235057B (en) * | 2020-02-01 | 2022-06-07 | 烟台大学 | Biological agent for treating polyacrylamide wastewater and preparation method and application thereof |
| CN113773986B (en) * | 2021-08-26 | 2023-07-18 | 郑州轻工业大学 | Microbial inoculum for rapid temperature rise of organic solid waste aerobic fermentation in winter and preparation method thereof |
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