CN103409340A - Microbial strain and application of bactericide thereof in biological treatment of phenolic wastewater - Google Patents
Microbial strain and application of bactericide thereof in biological treatment of phenolic wastewater Download PDFInfo
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Abstract
The invention discloses a microbial strain and application of a bactericide thereof in biological treatment of phenolic wastewater. The microbial strain is a degrade phenolic strain (coryne bacterium lubricantis phe-05); the preservation number is CCTCCNO:M2012031; and the bactericide can be directly used for treatment of industrial wastewater containing phenol with high efficiency and low cost, and does not cause secondary pollution to the environment. The microbial strain can be used for providing a specific dephenolized bactericide to related sewage treatment enterprises so as to culture novel activated sludge which is widely applied to treatment of various water bodiesand reduction of phenol residue. The microbial strain can be used for providing a specific dephenolized bactericide to related sewage production enterprises, so that the population quantity of phenol-degrading bacteria in sewage treatment systems of the enterprises is improved; and the biological treatment process of the phenolic wastewater, which is efficient, is low in cost, is simple to operate and manage, and does not cause secondary pollution to the environment, is realized.
Description
Technical field
The present invention relates to the application of phenol degradation by bacterial strain with high efficiency on phenolic wastewater is processed, belong to environmental protection technical field, also belong to the category of biological reinforcing technology.
Background technology
Phenolic wastewater is one of important pollutent of water body.The method of domestic and international industrial improvement phenolic wastewater generally is divided into physico-chemical process, chemical method, biochemical process three major types at present.According to long-term trial effect, show, physico-chemical process, chemical method and biochemical process have been obtained appreciable theoretical result and actual effect in the processing of industrial phenolic wastewater, as: vaporizing extract process, tower extraction process, absorption method, chemical precipitation method, the ordinary activated sludge method of purification, electrolytic cleaning method etc. all have the removal effect to the aldehydes matter in trade effluent.Yet, also existing the problems that can not be ignored, often cost is higher on the input of disposing of sewage for physico-chemical process, chemical method, and can cause secondary pollution to environment; For biochemical process, only can process and contain the sewage that phenol concentration is lower, therefore to the now still unrealized less investment at present of industrial phenolic wastewater of high density, effective processing intent.
Be directed to above problem, in conjunction with physics, chemistry and biological treatment, containing the relative merits of phenol method for industrial waste water, show on bioremediation, to have wide development space through decades of research.The biological pathway of this understanding of hexyl microbiological deterioration phenol mainly contains two at present: 1. aerobic microbiological utilizes phenol hydroxylase that oxidation of phenol is to pyrocatechol, acetyl-CoA, succsinic acid, pyruvic acid etc. can enter tricarboxylic acid cycle, continue to be utilized by microorganism; 2. by anaerobion, be 4-HBA by the phenol carboxylation, then pass through the effect phenylformic acid approach of relevant enzyme, generate acetyl-CoA, finally change into acetic acid, continue to be utilized by microorganism.From this path analysis, can find out, adopt the phenol in microbiological deterioration sewage under anaerobism or aerobic conditions, to carry out, therefore, microorganism falls phenol and from principle, possesses wider Technological adaptability.
However, set up an efficient microorganism phenolic wastewater treatment process, still need to carry out a large amount of previous works, how by separating, the good naturalized strain of screening acquired character, and further to develop the microbiobacterial agent that adapts to production technique be crucial.And at present, the great majority report fall the phenol bacterium can tolerate the concentration of phenol mostly lower (<1500mg/L); Fall the phenol rate and generally be no more than 90%, few for the research report of high-concentration phenolic wastewater microorganism.
Summary of the invention
The present invention is intended to screen, tame and develop the microorganism of aldehydes matter in relevant efficient degradation industrial sewage
Agromyces soliPhe-03 with
Corynebacterium lubricantisThe bacterial classification of Phe-05, microbial inoculum, and for the processing of industrial phenolic wastewater, to realize the high-level efficiency of processing, low cost, pollute little effect.
The present invention is achieved through the following technical solutions above-mentioned technological invention purpose.
(1) utilize chemical environment waste water and near soil extract microbial bacterial group, by only having phenol, being to cultivate on the substratum of carbon source, then through in the high-concentration industrial phenol-containing wastewater, obtaining bacterial strain Phe-03 and Phe-05 after screening for several times and domestication.Through molecular biology identification, Phe-03 is accredited as
Agromyces soliBacterial strain; Phe-05 is accredited as
Corynebacterium lubricantisBacterial strain.Two bacterial strains all on February 22nd, 2012 " Chinese Typical Representative culture collection " center " has carried out effective preservation, and (CCTCC is called for short at Chinese Typical Representative culture collection center; also cry Wuhan University's preservation center; address is: Wuhan City Wuhan University Life Science College, postcode: 430072).Deposit number is respectively:
Agromyces soliPhe-03 CCTCC NO:M 2012030,
Corynebacterium lubricantisPhe-05 CCTCC NO:M 2012031; By the content of Artificial Control phenol, directed toward bacteria
Agromyces soliPhe-03 with
Corynebacterium lubricantisThe experimental model of Phe-05 degradation of phenol ability, efficiency and related detecting method Erecting and improving.
(2) by further adopting the domestication of actual industrial phenolic wastewater, obtain the mixed microbial inoculum of suitable Sewage Environment, and process for pilot scale.The common Sewage treatment systems result of take is blank, relatively the effect of microbial inoculum on industrial phenolic wastewater is processed.
Embodiment
Example 1
Agromyces soliPhe-03,
Corynebacterium lubricantisThe strain improvement of Phe-05 and bacterial preparation process
(1) minimal medium preparation:
By 0.5g KH
2PO
4, 0.5g K
2HPO
4, 0.2g MgSO
4, 0.2g CaCL
2, 0.2g NaCl, micro Fe SO
4With water, be placed in beaker and mix, and adjust between its pH to 7.4-7.6 with NaoH, stir and namely make minimal medium.
(2) preliminary screening of phenol bacterium:
By the liquid minimal medium of 1000ml, with the 200ml/ bottle, divide (without sterilizing) in the triangular flask that installs to 5 500ml, and be numbered: P
5,P
10, P
15, P
20, P
25, and to P
5,P
10, P
15, P
20, P
25The middle phenol that adds respectively 0.5g/l, 1.0g/l, 1.5g/l, 2.0g/l, 2.5g/l, with in backward each triangular flask, adding the mud sample of 5ml, be positioned in 28 ℃, the shaking table of 210rmp and cultivate 48h, then respectively from P
5,P
10, P
15, P
20, P
25Respectively get a sample and be applied on slide, with methylene blue, dye after drying, examine under a microscope bacterial growth situation in each triangular flask.Observations shows: with the phenol concentration incremental, observe P
10, P
15Bacterial growth quantity is more, and is evenly distributed; And P
25On substantially without bacterial growth.
(3) separation and purification of phenolic wastewater bacterium
The inorganic salt nutrient solution for preparing and culture dish are placed in to sterilizing 30min under 121 ℃ of high-pressure sterilizing pots, take out nutrient solution and plate, respectively to P
10, P
15, P
20, P
25The phenol solution that adds 1ml, 1.5ml, 2ml, 2.5ml 50%, and mix with nutrient solution, with the 30ml/ ware, be down flat ware, take out P
10, P
15, P
20, P
25On bacterial strain carry out separation and purification.With the 200ml sterilized water respectively by P
10, P
15, P
20, P
25Bacterium liquid is diluted to 10
-3, 10
-4, 10
-5Three concentration gradients, and coat plate according to each concentration gradient 4 ware, amount to 4 * 3 * 4=48 plate, cultivates 48h in 28 ℃, 210rmp incubator.Through observations, show:
1. bacterial strain kind, growing way, quantity, color, shape all along with the difference of phenol concentration and extension rate and different, along with the rising of phenol concentration, the bacterial strain kind is more single, quantity is fewer; Also bacterial strain quantity is fewer along with the rising of extension rate.
2. by isolation medium plate observed and recorded, difference by phenol concentration and extension rate, selected 38 strain bacterium colonies carry out next step separation and purification, it is respectively phenol concentration is 30 strains of cultivating under 1.0mg/ml, phenol concentration is 3 strains of cultivating under 1.5mg/ml, phenol concentration is 4 strains of cultivating under 2.0mg/ml, and phenol concentration is 1 strain of cultivating under 2.5mg/ml.
(4) the further optimization screening of phenolic wastewater bacterium
Method, equipment, reagent and step are identical with " separation and purification of Phenol-degrading Bacteria Strains ", to 38 strain bacterium selected after " separation and purification of Phenol-degrading Bacteria Strains ", adopt higher phenol concentration substratum to carry out the adaptability domestication and cultivate.Through observing, result shows:
1. phenol concentration is when 2000mg/ml, P
10Concentration gradient is 10
-3With 10
-2The bacterial strain growing way good than other concentration gradients, and bacterial classification is identical, apparent is orange in flakes, and unified called after phe-01;
2. phenol concentration is when 2000mg/ml, p
15Concentration gradient is 10
-3The bacterial strain growing way good than other concentration gradients, apparent is orange in flakes, called after phe-02;
3. phenol concentration is when 2500mg/ml, p
20Concentration gradient is 10
-2The bacterial strain growing way good than other concentration gradients, apparent is white micro-Huang Cheng's sheet, called after
Agromyces soliPhe-03;
4. phenol concentration is when 2000mg/ml, P
20Concentration gradient is 10
-2With 10
-4The bacterial strain growing way good than other concentration gradients, and bacterial classification is identical, apparent is that white particle is in blocks, and unified called after phe-04;
5. phenol concentration is when 2000mg/ml, P
20Concentration gradient is 10
-2Have a bacterial strain with 4. in bacterium for of the same race, and growing way is good than other concentration gradients, apparent is white micro-Huang Cheng's sheet called after
Corynebacterium lubricantisPhe-05.
(5) phenolic wastewater bacterium phenol degrading quantitative screening
1. the phenol Standard curve is set up
With electronics sky chessboard weighing 5.00g phenol, be placed in the volumetric flask of 500ml, add the fixed molten 500ml of arriving of pure water dilution as mother liquor, and then with liquid-transfering gun accurately draw respectively mother liquor 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, 3.0ml, 3.5ml, 4.0ml, 4.5ml, 5.0ml join in the volumetric flask of 10 50ml cleanings, and be dissolved to 50ml with pure water, be numbered 100mg/ml, 200mg/ml, 300mg/ml, 400mg/ml, 500mg/ml, 600mg/ml, 700mg/ml, 800mg/ml, 900mg/ml, 1000mg/ml; Surely solvent phenol solution is placed in after refrigerator and cooled is hidden 24h and measures with high performance liquid chromatograph, do the typical curve of phenol concentration and peak area relation.The HPLC solvent systems of high performance liquid chromatography is second eyeball: pure water=8:2; The chromatographic column model is Japanese YMC phenol dedicated analysis post (hydrosphere C18.250 * 4.6 mmI.D. S-5um, 12nm), column temperature is 37 ℃.Result draws typical curve equation: y=4382.7X+24055, R
2=0.9969; " y " measures peak area for phenol HPLC, and " X " is phenol concentration.
2. the phenol screening quantitatively falls in phe-01 ~ phe-05 shake-flask culture
The minimal medium compound method is the same.After medium sterilization, add the phenol mother liquor, make phenol concentration in culture system reach 1500mg/ml.Under aseptic condition, with inoculating needle, from the inclined-plane of keeping, provoke respectively phe-01 ~ phe-05 bacterium lawn in shaking flask, be placed in 28.5 ℃, under the condition of 200rpm, cultivate.In this process, phenol nutrient solution blank is set, and (phenol concentration is 1500mg/ml; Inoculated bacteria not).After cultivating 8h, start sampling, at every turn with the standard sampling container 100ul that takes a sample, then use the 100ul distilled water diluting, phenol concentration in HPLC quantitative assay culture system, measuring method is the same, and calculates the phenol degrading rate.Sampling time interval 8h, i.e. 8h, 16h, 24h, 32h, 40h and 48h sampling.The sampling determination result shows: phe-03 falls the phenol rate and reaches 81.08%, phe-05 and fall the phenol rate and reach 83.02% in 32h; Phe-01, phe-02, phe-04 fall the phenol rate and are respectively 37.06%, 36.51% and 48.13%; The phenol nature evaporation rate of blank is only 1.28%.By above work, determine that phe-03 and phe-05 are best selected bacterial strain.
(6) selected bacterial strain electron-microscope scanning is observed
With transfer pipet, pipette the 5ml glutaraldehyde in every centrifuge tube, and be numbered phe-03 and phe-05.In between ultraviolet ray inoculation under the spirit lamp conditions for sterilization the appropriate lawn of picking be placed in 2 centrifuge tubes that glutaraldehyde is housed, and mix, build lid and seal up for safekeeping, after spending the night, pipette appropriate bacterium liquid to slide with transfer pipet, metal spraying is processed after drying, and then uses electron microscope observation.Observations shows: two strain bacterium are spherical bacterium.The about 0.5um of thalline diameter, length 0.5-1.0um does not wait.
(7) phe-03, phe-05 identification of strains
Bacterial strain phe-03 and phe-05 are by total DNA extraction, and bacterial 16 S rDNA universal primer increases.Amplification purpose fragment is utilized by order-checking is rear
BlastSoftware carries out similarity searching at GenBank etc. in the GenBank database, select the 16S rDNA sequence of the typical strain that homology is higher as the reference object, then uses
CLUSTALX software
]Carry out Multiple Sequence Alignment and calculate the sequence similarity between examination bacterial strain and reference bacterial strain, the employing adjacent method (
Neighbor-joining) use
MAGE4 software buildings be take 16S rDNA sequence and are basic for the phylogenetic tree between examination bacterium and reference bacterium, and analytical results shows: phe-03 belongs to
Agromyces soliBacterial strain, phe-05 is
CorynebacteriumlubricantisBelong to bacterial strain.
Two strain bacterium all on February 22nd, 2012 " Chinese Typical Representative culture collection " center " has carried out effective preservation.Deposit number is respectively:
Agromyces soliPhe-03 CCTCC NO:M 2012030,
Corynebacterium lubricantisPhe-05 CCTCC NO:M 2012031.
Example 2 phe-03, the single bacterial strain of phe-05 are used on the industry high-concentration phenolic wastewater
(1) coal chemical industrial waste water water-quality determination
At first from the coal chemical industrial waste water source respectively water sampling amount to 50L, mixing is measured respectively the indexs such as phenol content in coal chemical industry sewage, CODcr, ammonia nitrogen, sulfide by methods such as high performance liquid chromatograph, potassium dichromate processs, and result is shown as: C(phenol)=750.0mg/L, CODcr=5035.10mg/L, C (ammonia nitrogen)=800.0mg/L, C(sulfide)=40.0mg/L, ph=9.5, SS=650.0mg/L, oils=200 mg/L.
(2) the single bacterial strain domestication of phe-03, phe-05
Coal chemical industrial waste water 2000ml, after adopting aseptic membrane filtration, be distributed into 20 bottles (100ml/ bottles).Every 10 bottles are accessed respectively phe-03 and phe-05 strain cultured solution (nutrient solution is cultivated 48h in advance, and composition is that salt+1000mg/ml phenol is cultivated on basis) in 10% ratio, and are placed in shaking table, 28.5 ℃, cultivate under the condition of 200rpm.Timing sampling and observation.After cultivating 24h, start sampling, sampling time interval 24h, i.e. 24h, 48h, 72h, 96h, 108h and 132h sampling.Observe and the sampling determination result shows: phe-03 bacterial strain growing way after 96h is fine, and a large amount of flockss appear in the shaking flask bottom, and bacterium liquid muddiness is fallen the phenol rate and reached from 32.34% and be increased to rapidly 87.15% in 108h ~ 132h.Phe-05 bacterial strain growing way after 96h is fine, and bacterium liquid muddiness is fallen the phenol rate and reached from 24.51% and be increased to rapidly 90.05% in 96h ~ 132h.
(3) the single bacterial strain of phe-03, phe-05 is further tamed
Coal chemical industrial waste water 1000ml, after adopting aseptic membrane filtration, be distributed into 10 bottles (100ml/ bottles).Every 5 bottles are accessed respectively phe-03 and phe-05 bacterial strain 96h domestication liquid (the acclimation process is the same) in 10% ratio, add in proportion inorganic salt, are placed in shaking table, 28.5 ℃, cultivate under the condition of 200rpm.Timing sampling and observation.After cultivating 24h, start sampling, sampling time interval 24h, i.e. 24h, 48h, 72h, 96h, 108h and 132h sampling.Observation and sampling determination result show: the phe-03 bacterial strain continues domestication through 7 generations, and growing way is fine after 24h, and bacterium liquid muddiness is fallen the phenol rate and reached 86.15% in 48h.The phe-05 bacterial strain continues domestication through 11 generations, and growing way is fine after 24h, and bacterium liquid muddiness is fallen the phenol rate and reached 80.78% in 48h.
(4) the active microbial inoculum of the single bacterial strain of phe-03, phe-05 is cultivated
By the pattern of amplifying step by step, phe-03, phe-05 domestication bacterium liquid is amplified in the open aerated culture tank of 200l from 1000ml cultivation amount.Culture tank adopts hot water circulation heated, and universal blower ventilates, and general-purpose machine stirs.28 ℃, 120rpm cultivation, air flow is 50L/min.Incubation time 48h.
Example 3 phe-03, the application on the industry high-concentration phenolic wastewater is processed of phe-05 hybrid bacterial strain
(1) Sewage treatment systems is set up
According to phenolic wastewater, produce enterprise's production area Sewage treatment systems technical process and build a set of day processing 5m
3Pilot scale Sewage treatment systems model, that is: production waste through grid, enters coagulation basin, then, to MESB (acid adding), then enters efficient sedimentation tank, flows into hydrolysis acidification pool, then to the MBBR pond, the PAC pond, then flow into the oxidative decoloration pond, last water outlet.
(2) phenolic wastewater produces enterprise's phenolic wastewater water-quality determination
Select the coal chemical industrial waste water source to measure respectively the indexs such as phenol content in coal chemical industry sewage, CODcr, ammonia nitrogen, sulfide through methods such as high performance liquid chromatograph, potassium dichromate processs, result is shown as: C(phenol)=810.0mg/L, CODcr=5416.05mg/L, C (ammonia nitrogen)=810.21mg/L, C(sulfide)=51 mg/L, ph=9.13, SS=712.0mg/L, oils=152 mg/L.
(3) practical application of phe-03, phe-05 mixt bacteria
Every 168h mixes thin agent (each 100L) by phe-03, phe-05 and joins the MBBR pond in the pilot scale Sewage treatment systems, and after the bacterium adaptive process in early stage of 30 days, the microorganism fixation is in Sewage treatment systems.
After the microorganism fixation completes, carry out the test of mix bacterium agent degradation of phenol.Every 8h sampling once.Sampling time interval 8h, i.e. 8h, 16h, 24h, 32h, 40h and 48h sampling.The effluent quality that produces the enterprise practical sink drainage with phenolic wastewater contrasts.Result shows: phe-03, phe-05 mixt bacteria have reached the expectation effect to the degraded that contains the aldehydes matter in the phenol processing wastewater in the pilot scale model, in the wastewater treatment effluent quality of pilot scale model 24h, containing phenol concentration is 0.05mg/l, the phenol clearance has reached more than 95%, improves 23% than contrast.Sink drainage water quality has reached one-level sewage drainage standard from three grades of discharges.
Claims (2)
1. degraded phenol bacterial strain that deposit number is CCTCC NO:M 2012031
Corynebacterium lubricantisPhe-05 is characterized in that: utilize chemical environment waste water and near the microbial bacterial group of soil sample, by take phenol, be the Screening of Media of sole carbon source, then adopt high density to contain the domestication of phenol industrial sewage, the bacterial strain Pyrogentisinic Acid has good degradation property.
2. degraded phenol bacterial strain that deposit number is CCTCC NO:M 2012031
Corynebacterium lubricantisThe application of Phe-05 on wastewater containing phenol is processed is characterized in that:
Corynebacterium lubricantisThe microbial inoculum of Phe-05 is processed for the high-performance bio of wastewater containing phenol.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105712498B (en) * | 2016-04-28 | 2018-05-08 | 河海大学 | A kind of biological treatment device of phenol wastewater |
| CN109097435A (en) * | 2018-09-01 | 2018-12-28 | 长沙理工大学 | A method of screening malaga carbohydrate oxidase strain from air |
| CN111471630A (en) * | 2020-06-11 | 2020-07-31 | 鲁东大学 | Corynebacterium Ytld-phe09 and application thereof |
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| CN104845890B (en) * | 2015-02-09 | 2017-10-31 | 暨南大学 | Applications of earth mould (Agromyces sp.) the MT E in a variety of phthalic acid esters of degrading |
| CN104845891B (en) * | 2015-02-09 | 2017-11-24 | 暨南大学 | A kind of earth mould bacteria suspension of a variety of phthalic acid esters of degrading and its application |
| CN106566768A (en) * | 2016-11-04 | 2017-04-19 | 上海昭泉环境技术有限公司 | Combined type bio-membrane reactor combined with mycelium pellet and method for phenolic waste water treatment by bio-membrane reactor |
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| CN105712498B (en) * | 2016-04-28 | 2018-05-08 | 河海大学 | A kind of biological treatment device of phenol wastewater |
| CN109097435A (en) * | 2018-09-01 | 2018-12-28 | 长沙理工大学 | A method of screening malaga carbohydrate oxidase strain from air |
| CN111471630A (en) * | 2020-06-11 | 2020-07-31 | 鲁东大学 | Corynebacterium Ytld-phe09 and application thereof |
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| CN102776137A (en) | 2012-11-14 |
| CN103409340B (en) | 2016-06-01 |
| CN102776137B (en) | 2014-06-18 |
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