CN106701626A - Phenol degradation bacteria and application thereof - Google Patents
Phenol degradation bacteria and application thereof Download PDFInfo
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- CN106701626A CN106701626A CN201710005990.1A CN201710005990A CN106701626A CN 106701626 A CN106701626 A CN 106701626A CN 201710005990 A CN201710005990 A CN 201710005990A CN 106701626 A CN106701626 A CN 106701626A
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The invention discloses marine bacillus Oceanobacillus sp.LCB-20, wherein the collection number is CGMCC No.13422. Through separation from Xinjiang saline alkali soil, screening, physiological and biochemical identification and the like, the bacteria are determined to be aerobic bacteria which are Gram-positive and mobile with a short rod shape, the cell size is (0.2-0.4)mu m*(1.0-1.5)mu m, and the bacterial colony is of cream color. Oxidase and catalase are positive. The pH is 6.5-11, and the NaCl concentration is 1-20%. The bacteria have the enzyme activities of amylolytic enzyme, lipase, beta-glucuronidase, alpha-glucosidase, beta-glucosidase and the like as well as ability of efficiently degrading high-concentration phenol (figure 1), and can be widely used for developing phenol degradation enzymes and treating phenol-containing industrial wastewater. The figure shows a degradation process of phenol of different initial concentration by Oceanobacillus sp.LCB-20 under a condition of TSB+5%NaCl.
Description
Technical field
The present invention relates to phenol degrading microorganism and its application, specifically, the present invention relates to a kind of energy degradation of phenol
Bacillus marinus and its technical field of application.
Background technology
With the continuous improvement continued to develop with people's living standard of science and technology, problem of environmental pollution is also increasingly dashed forward
Go out, global water pollution is on the rise, seriously threaten the sustainable development of social economy or even mankind itself.The water of China
Pollution is essentially from industrial pollution source discharge of wastewater.Set about from the investigating of the origins of industrial pollution and environmental monitoring, China is determined in water
All controls pollutant " blacklist ", wherein, aldehydes matter is in the row impressively.Phenol is important industrial chemicals, is widely used in
In the production of phenolic resin, oil refining, coke, dyestuff, weaving, insecticide, agricultural chemicals and medicine, and as in these industrial wastewaters
One of major pollutants.
Phenolic compound in phenol wastewater is a kind of prototype matter poisonous substance, has toxic action to organism, and be difficult
It is degraded.Blood circulation can be directly entered without liver detoxification by the contact of skin, mucous membrane, cause cytoclasis and lose work
Power, it is also possible to human body is invaded by oral cavity, the damage of cell is caused.When phenol content reaches 5 ~ 10mg/L in water, fish can be caused
Mortality.Additionally, using phenol wastewater irrigated farmland, can also make crop production reduction or withered.Therefore, the discharge of phenol wastewater is not
Serious pollution is only caused to environment, the growth and breeding of human health and biology is also endangered.
The processing method to phenol wastewater mainly has Physical, chemical method and bioanalysis at present.The conventional absorption of Physical, mistake
Filter, extraction, precipitation etc., its advantage are that processing method is simple, maintenance cost is few, but long using the method process time, and often
The emission request of country is extremely difficult to, therefore can only typically be used as the pretreatment of sewage.Chemical method is often anti-with redox, neutralization
Should, chemical precipitation etc., its advantage is short process time, efficiency high, but investment in equipment and maintenance is costly, thereby increases and it is possible to existed secondary
Pollution.And bioanalysis mainly use can be by the use of aldehydes matter as the microbial degradation sewage of carbon source for growth in nature in
Phenolic comp ' ds pollution, its whole process is substantially the biochemical reaction occurred in microbial enzyme presence, with wide adaptability, energy
Consumption is few, and treatment effeciency is high, and secondary pollution is few, is relatively advanced and important in current field of waste water treatment the advantages of small investment
Method.
In recent years, the phenol degrading microorganism of separated identification both at home and abroad mainly includes bacterium and the major class of fungi two.Bacterium
Mainly include pseudomonas（Pseudomonas sp.）, bacillus（Bacillus sp.）, Alcaligenes
（Alcaligens sp.）And Rhod（Rhodococcus sp.）；Fungi mainly includes Blastocystis
（Saccharomyces sp.）, candida（Candida sp.）And acinetobacter（Acinetobacter sp.）.Drop
Solution activity is more in 300-1000mg/L, but under high salt conditions, its degrading activity can be greatly reduced.Salinity is to microbial degradation
Influence is larger, and salinity high can be such that the growth rate and degradation rate of microorganism declines rapidly.However, weaving, leather chemical industry and agriculture
High-salt wastewater can be produced in the production process of the chemical industries such as medicine, these sewage with high salinity can greatly suppress the work of microorganism
Property, application of the limitation most of microbe in sewage disposal.Therefore, filter out and be both resistant to high salt degree condition, and with height
The microorganism for imitating degradation of phenol ability is significant.
The content of the invention
For present Research both at home and abroad about Phenol-degrading Bacteria Strains, the present invention provides a kind of new phenol degrading microorganism.
Described Phenol-degrading Bacteria Strains have oxidizing ferment, catalase, amylolytic enzyme, lipase, lipase, GRD beta-glucuronidase,
The enzymatic activitys such as alpha-glucosidase, beta-glucosidase, and with the ability of fast degradation phenol.
The present invention provides one plant of bacillus marinusOceanobacillus sp. LCB-20。
The present invention by with different salinity, phenol concentration and Screening of Media as enrichment condition, from Xinjiang salt affected soil
Middle sampling, isolates and purifies and obtains multi-strain bacteria, and one plant of phenol degradation by bacterial strain with high efficiency is determined by multistage screening（Oceanobacillus sp. LCB-20）, it is named as bacillus marinus LCB-20.The bacterial strain is preserved in the micro- life of budapest treaty in application a few days ago
Thing International Depository Authority：China Committee for Culture Collection of Microorganisms's common micro-organisms center（CGMCC）, preservation time difference
It it is in December, 2016 No. 5, preserving number is that CGMCC No.13422, CGMCC addresses are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number Institute of Microorganism, Academia Sinica, postcode is 100101.
To the bacterial strainOceanobacillus sp. The morphologic observation of LCB-20, Physiology and biochemistry and the equal reference of cultural character
《Actinomyces systematics --- method, principle and practice》Carry out.The bacterium is aerobic bacteria, Gram-positive, motility, in quarter butt
Shape, cell size is 1.0-1.5 μm of 0.2-0.4 μ ms, and colony colour is cream color.Oxidizing ferment and catalase positive, urea
Enzyme is negative, with nitrate reduction ability, can hydrolysis starch.With lipase, lipase, GRD beta-glucuronidase, phlorose
The enzymatic activitys such as glycosides enzyme, beta-glucosidase.
To the bacterial strainOceanobacillus sp. The phenol degrading ability of LCB-20, TSB trainings are connected to by strain LCB-20
Support in base, add the NaCl of various concentrations, adjust pH to 8.0,30oC, 200rpm concussion and cultivate, at the beginning of being separately added into difference after 24h
The phenol of beginning concentration 200,500,700,1000 and 1200mg/L, sampling detection phenol content.The sampling detection thalli growth per 24h
Situation and remaining phenol content.Thalli growth state testing method：Sampling, is existed with UV spectrophotometer measuring bacteria suspension
Absorbance during 600nm.Remaining phenol content detection method：Sampling, 12000rpm centrifugation 10min, using 0.22 μm of filter membrane mistake
Filter, sample is detected using HPLC.Test in triplicate, results averaged.Experimental result shows that LCB-20 is in various concentrations
Under the conditions of NaCl, when energy degradation of phenol, and NaCl concentration is relatively low, the degradation efficiency of phenol is higher, meanwhile, initial phenol concentration
Lower, degradation efficiency is higher.LCB-20 under the conditions of 5%NaCl, in 96h can degradable 1200mg/L phenol, 10%
Under the conditions of NaCl, the phenol of the degradable 1000mg/L of energy in 216h.
By implementing particular technique index of the invention, following Expected Results is can reach.
It is of the inventionOceanobacillus sp. LCB-20 is aerobic bacteria, and Gram-positive, bacterium colony is in cream color.Oxygen
Change enzyme and catalase positive.PH growth scopes are 6.5 to 11, and NaCl concentration growth scope is 3% to 20%.The bacterium has forms sediment
The enzymatic activitys such as powder hydrolase, lipase, lipase, GRD beta-glucuronidase, alpha-glucosidase, beta-glucosidase, while tool
There is the ability of efficient degradation high concentration phenol, the exploitation of phenol degrading enzyme and the place containing phenol industrial wastewater can be widely used in
In reason.
Brief description of the drawings：Fig. 1 is bacterial strainOceanobacillus sp. LCB-20 under the conditions of TSB+5%NaCl, to difference
The degradation process of initial concentration phenol.Fig. 2 is to do canonical plotting using HPLC Pyrogentisinic Acid's standard items.
The embodiment explanation present invention is named, but the present invention is not limited to following embodiments.
Specific implementation method
Embodiment one：Oceanobacillus sp. The screening and separation of LCB-20
Collecting soil sample is from Xinjiang Uygur autonomous region Shache County.1g pedotheques are weighed to add equipped with 50ml sterilized waters
In shaking flask, concussion and cultivate.Pregnant solution is made into 10 times of gradient dilutions with sterilized water, 200 μ l 10 are taken-1-10-5Multiple dilutions liquid is distinguished
It is applied on different salinity, the TSB of different phenol concentrations, beef extract-peptone, Gause I culture medium and cultivates, 30oC is biochemical
7 d are cultivated in incubator.Observation upgrowth situation and morphological feature, it is 200 mg/L that the bacterium colony that will be grown is forwarded to phenol concentration
On TSB agar plates, TSB slant mediums are forwarded to through repeatedly line, then picking single bacterium colony, put 4oIt is standby that C makees preservation.Finally
One plant of bacterial strain for being resistant to high concentration phenol is screened, LCB-20 is named as.
Embodiment two：Different salinity pairOceanobacillus sp. The influence of LCB-20 growths
Influence that different NaCl concentrations grow to bacterial strain LCB-20 experiment, configuration NaCl concentration is respectively 0%, 3%, 5%, 7%, 10%,
12%th, 15%, 17%, 20% TSB fluid nutrient mediums, adjust pH to 8.0, fresh bacterium solution are inoculated into shaking flask, 30oC、200rpm
Lower concussion and cultivate 24h.Its light absorption value is detected under 600nm using ultraviolet specrophotometer.Result shows that the NaCl of LCB-20 is dense
It is 1-20% to spend, and most suitable NaCl growth concentrations are 7-10%.
Embodiment three：Oceanobacillus sp. The enzymatic property of LCB-20
Oxidizing ferment is tested：1% dimethyl-p-phenylenediamine's hydrochloride is added drop-wise on cultured bacterium colony, if bacterium colony immediately becomes powder
Red, then becomes laking, then be oxidase positive；It is oxidizing ferment if occurring blueness after without obvious color change or 60s
It is negative.Catalase is tested：Picking cultivates the thalline to exponential phase on filter paper on a small quantity, and the peroxide of 3% concentration is added dropwise
Change hydrogen solution, be catalase positive if occurring a large amount of bubbles in 30s, conversely, being then feminine gender.Bacterial strain LCB-20 its
He is identified enzymatic property using API ZYM reagent strips.Experimental result shows that bacterial strain LCB-20 has following enzymatic activity：Oxidizing ferment,
Catalase, amylolytic enzyme, lipase, lipase, GRD beta-glucuronidase, alpha-glucosidase, beta-glucosidase.
Example IV：Under the conditions of 5% NaClOceanobacillus sp. The degradation experiment of LCB-20 Pyrogentisinic Acids
Strain LCB-20 is connected in TSB culture mediums, addition 5%（Mass fraction）NaCl, adjust pH to 8.0,30oC、
200rpm is cultivated, and second day same time added the phenol of different initial concentrations 200,500,700,1000 and 1200mg/L, took
Sample detects phenol content.Sampling detection thalli growth situation and remaining phenol content per 24h.Thalli growth state testing method：
Sampling, absorbance of the bacteria suspension in 600nm is surveyed with ultraviolet specrophotometer.Remaining phenol content detection method：Sampling,
12000rpm is centrifuged 10min, crosses 0.22 μm of filter membrane, and sample is detected using HPLC.Test in triplicate, results averaged.Knot
Fruit shows that LCB-20, can be degradable by the phenol of concentration 1200mg/L in 96h under the conditions of 5% NaCl.
Embodiment five：Under the conditions of 10% NaClOceanobacillus sp. The degradation experiment of LCB-20 Pyrogentisinic Acids
Strain LCB-20 is connected in TSB culture mediums, addition 10%（Mass fraction）NaCl, adjust pH to 8.0,30oC、
200rpm is cultivated, and second day same time added the phenol of different initial concentrations 200,500,700,1000 and 1200mg/L, took
Sample detects phenol content.Sampling detection thalli growth situation and remaining phenol content per 24h.Thalli growth state testing method：
Sampling, absorbance of the bacteria suspension in 600nm is surveyed with ultraviolet specrophotometer.Remaining phenol content detection method：Sampling,
12000rpm is centrifuged 10min, crosses 0.22 μm of filter membrane, and sample is detected using HPLC.Test in triplicate, results averaged.Knot
Fruit show, LCB-20 under the conditions of 10% NaCl, can in 216h by concentration for 1000mg/L phenol it is degradable.
Claims (2)
1. one plant of bacillus marinusOceanobacillus sp.LCB-20, its deposit number is CGMCC No. 13422.
2. bacillus marinus as claimed in claim 1Oceanobacillus sp.LCB-20, it is characterized by aerobic bacteria, leather
Lan Shi is positive, motility, and in rod-short, cell size is 1.0-1.5 μm of 0.2-0.4 μ ms, and colony colour is cream color, oxidation
Enzyme and catalase positive, pH growth scopes are 6.5 to 11, and NaCl concentration growth scope is 1% to 20%, and the bacterium has starch
The enzymatic activitys such as hydrolase, lipase, lipase, GRD beta-glucuronidase, alpha-glucosidase, beta-glucosidase, while having
The ability of efficient degradation phenol, the phenol of the degradable 1200mg/L of energy in 96h.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146342A (en) * | 2011-01-11 | 2011-08-10 | 山东潍坊润丰化工有限公司 | Halophilic bacterial agent and preparation method thereof as well as biological treatment system fixed with bacterial agent and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146342A (en) * | 2011-01-11 | 2011-08-10 | 山东潍坊润丰化工有限公司 | Halophilic bacterial agent and preparation method thereof as well as biological treatment system fixed with bacterial agent and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XIUFENG LONG等: "Oceanobacillus damuensis sp. nov. and Oceanobacillus rekensis sp. nov., isolated from saline alkali soil samples", 《ANTONIE VAN LEEUWENHOEK》 * |
| 向红灯: "生物净水工艺中降解有机污染物优势菌的筛选初探", 《环境科学与技术》 * |
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