CN104293725A - Nitrile degradation biofilm formed genetically engineered bacterium and application of genetically engineered bacterium in nitrile-containing wastewater treatment - Google Patents
Nitrile degradation biofilm formed genetically engineered bacterium and application of genetically engineered bacterium in nitrile-containing wastewater treatment Download PDFInfo
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Abstract
The invention provides a nitrile degradation biofilm formed genetically engineered bacterium and application of the genetically engineered bacterium in nitrile-containing wastewater treatment, and belongs to the bioengineering technology. The genetically engineered bacterium Bacillus subtilis N4-pHT01-Nitr with the preservation number of CGMCC No.9484 is preserved in the China General Microbiological Culture Collection Center on July 25, 2014. The bacterium is prepared by inserting a nitrilase gene in Rhodococcus rhodochrous BX2 into Bacillus subtilis N4 for expressing by means of gene engineering. The bacterium is capable of generating a biofilm and degrading acetonitrile, the OD570nm value of biofilm formation capacity is 1.45 when the bacterium is culture in synthetic wastewater at 37 DEG C and 60r/min for 48 hours; and the residual concentration of acetonitrile is 23.56mg/L after the bacterium is oscillated and cultured in 800mg/L acetonitrile-containing synthetic wastewater at 37 DEG C and 160rpm for 48 hours. The bacterium has strong acetonitrile impact resistance, acetonitrile in synthetic wastewater is reduced from 800mg/L to 0.32mg/L when the wastewater is output, and the COD value of the synthetic wastewater is reduced from 487mg/L to 16.39mg/L when the wastewater is output. The bacterium provides a novel method to nitrile-containing wastewater treatment by utilizing a biofilm method.
Description
Technical field
The invention belongs to technical field of bioengineering, relate generally to a bifunctional genetically engineered bacterium of strain nitrile degraded-biofilm formation and containing the application in nitrile wastewater treatment.
Background technology
Acetonitrile is the Organic Chemicals of a kind of cyano-containing (R-CN), is widely used in pharmacy, synthon, field of petrochemical industry, is containing one of main component in nitrile waste water.The large quantities of death of the hydrobionts such as fish can be caused after entering natural water area containing the acetonitrile in nitrile waste water, serious destruction is caused to ecotope.Acetonitrile can by suction, eat and skin absorb and enter in body, be converted into highly toxic substance in vivo---prussic acid and acetaldehyde, threaten human and livestock health.Nitrilase (nitrilase) is one of important degrading enzyme of nitrile degradation bacteria metabolism nitrile compounds, nitrile can be converted into corresponding carboxylic acid and ammonia by single step reaction.
Microbial film (Biofilms) refers to the structural bacterial flora be made up of the hydratability matrix of the bacterial cell and parcel bacterium that are attached to inertia or active entities surface.Form the most idealized model that microbial film is most microorganism growth, microbial film can stop the injury of antiseptic-germicide, sterilant, heavy metal, host defense mechanism etc. effectively, improves bacterium adaptability in the environment.Research finds, bacterium is stronger than the metabolic activity under floating state in microbial film, and the frequency that transfer occurs dissimilation plasmid is higher, is conducive to the biological treatment of waste water.But in its natural state, in microbial film, microorganism species often can not bear high density toxic substance, thus when process high-concentration hardly-degradable waste water time efficiency lower, even lost efficacy.Therefore, the microorganism of certain pollutent of screening degraded targetedly, it being added together with biofilm-forming bacterium enhanced sewage improvement in bio-reactor is the effective method of one solved the problem.But this method also exists some problems, mainly comprise: 1. likely there is competitive growth between contaminant degradation bacterium and film forming bacterium; If 2. contaminant degradation bacterium cannot grow in film forming bacterium well surely, then degradation bacteria easily runs off, and water treatment effect is not good.If gene pollutent to degradation capability is proceeded in biofilm-forming bacterium, just can solve the problem to a certain extent, thus reduce processing cost, decreasing pollution.Therefore genetic engineering means is utilized to build multifunctional engineering strain, develop and both there is degraded nitrile compounds ability, possess again the double-function bacterial strain of biofilm formation ability simultaneously, for containing in the biomembrance process process of nitrile waste water, will there are higher economic benefit, social benefit and ecological benefits.
Summary of the invention
The object of the invention is to structure one plant height efficient expression nitrilase and can form biomembranous genetic engineering bacterium subtilis Bacillus subtilis N4-pHT01-Nitr simultaneously, and this bacterial strain is applied to degraded acetonitrile and the process containing nitrile waste water.
The object of the invention is to be achieved through the following technical solutions:
Nitrile degradation biological film provided by the present invention forms bifunctional genetically engineered Strains B. subtilis Bacillus subtilis N4-pHT01-Nitr, be preserved on 07 25th, 2014 " China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) ", its preserving number is CGMCCNo.9484.Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.
The original strain that the present invention utilizes is acetonitrile efficient degrading bacteria Rhodococcus rhodochrous BX2 and the Bacillus subtilis N4 with strong microbial film Forming ability.The present invention utilizes genetic engineering means to be inserted in Bacillus subtilis N4 by the nitrilase gene (nitrilase degradable acetonitrile) in Rhodococcus rhodochrous BX2 and expresses, and obtains nitrile degraded-biofilm formation double functions yeast bacillus subtilis Bacillus subtilis N4-pHT01-Nitr.
Described genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr builds by the following method: using the STb gene of Rhodococcus rhodochrous BX2 as template, according to the gene order of nitrilase gene and the polyclone restriction enzyme site design primer of subtilis expression vector pHT01, go out nitrilase gene DNA fragmentation by pcr amplification.Nitrilase gene fragment is cloned on pMD18-T carrier, the positive colony identified through PCR checks order, successful for order-checking PCR primer nitrilase gene fragment is connected on prokaryotic expression carrier pHT01 by restriction enzyme site, proceed in bacillus coli DH 5 alpha, through plasmid PCR and the qualification of XbaI/AatII double digestion, screening positive recombinant, success builds subtilis prokaryotic expression carrier pHT01-Nitr, to be proceeded to by expression vector pHT01-Nitr have in the wild-type Bacillus subtilis N4 of biofilm formation ability by electrotransformation.Filtering out Positive recombinant clones through extracting plasmid PCR, double digestion and order-checking, building the subtilis Bacillus subtilis N4-pHT01-Nitr double functions yeast containing nitrilase gene.
Described genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr can produce microbial film: subtilis Bacillus subtilis N4-pHT01-Nitr in synthetic wastewater 37 DEG C, 60r/min cultivates in 48h, biofilm formation amount OD
570nmvalue can reach more than 1.4.
Described genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr can degrade acetonitrile: in the synthetic wastewater containing 800mg/L acetonitrile, after inoculation double functions yeast subtilis Bacillus subtilis N4-pHT01-Nitr, acetonitrile concentration declines rapidly, and after 48h, the residual concentration of acetonitrile is 23.56mg/L.
Described genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr is containing the application in nitrile wastewater treatment: in synthetic wastewater, the interpolation concentration of acetonitrile is 800mg/L, in impact resistance experiment, the anti-acetonitrile impact capacity of subtilis Bacillus subtilis N4-pHT01-Nitr is strong, change after synthetic wastewaters at continuous six times, its finally in induction 8 hours to synthetic wastewater in the degradation rate of acetonitrile reach 49%; In bioreactor for treatment containing in nitrile Wastewater, the MBBR reactor adding active sludge and subtilis Bacillus subtilis N4-pHT01-Nitr is 0.32mg/L running 35d acetonitrile concentration, and water outlet COD value is 16.39mg/L.
Described original strain Rhodococcus rhodochrous BX2 and Bacillus subtilis N4 is separating obtained by this laboratory.(Sun Jing, Xiong Minghua, Cheng little Song, Li Yue, Zang Hailian, Li Chunyan.Rhodococcus sp.BX2 bacterium is to the degradation characteristic of acetonitrile and degradation pathway research [J] thereof. ACTA Scientiae Circumstantiae, 2012, 32 (5): 1041-1048.Chunyan Li, Yue Li, Xiaosong Cheng, Liping Feng, Chuanwu Xi, Ying Zhang.Immobilization of Rhodococcus rhodochrous BX2 (an acetonitriledegrading bacterium) with biofilm-forming bacteria for wastewater treatment, Bioresource Technology 131 (2013) 390-396.)
E.coli competent cells DH5a is purchased from TransGen.
Plasmid vector pMD18-T simple vector, purchased from precious bio tech ltd, Dalian.
Bacillus subtilus expression vector pHT01 is purchased from German molecular biotechnology (Molecular Biotechnology) company.
Bacterial strain provided by the invention, the Microbial resources of acetonitrile degraded are enriched on the one hand, on the other hand, film forming bacterium is made not only to have film forming ability by gene clone means, also there is nitrile degradation function simultaneously, further research its for MBBR (MBBR) process containing the effect of nitrile waste water, for the exploitation containing nitrile wastewater treatment novel method is laid a good foundation.
Accompanying drawing explanation
The agarose gel electrophoresis figure of Fig. 1 nitrilase gene.
The PCR primer agarose gel electrophoresis figure of Fig. 2 plasmid pMD18-T-Nitr.
Fig. 3 pHT01-Nitr carrier double digestion result figure.
The double digestion result figure of recombinant plasmid pHT01-Nitr in Fig. 4 subtilis.
Fig. 5 subtilis Bacillus subtilis N4-pHT01-Nitr SDS-PAGE electrophorogram.
The biofilm formation amount change curve of Fig. 6 subtilis Bacillus subtilis N4-pHT01-Nitr.
Fig. 7 subtilis Bacillus subtilis N4-pHT01-Nitr is to the tolerance situation map of acetonitrile.
Fig. 8 subtilis Bacillus subtilis N4-pHT01-Nitr is to the degradation capability figure of acetonitrile.
The anti-acetonitrile striking energy of microbial film of Fig. 9 subtilis Bacillus subtilis N4-pHT01-Nitr is tried hard to.
The simulation of Figure 10 MBBR biofilm reactor contains in nitrile wastewater treatment process, water outlet acetonitrile residue change curve.
The simulation of Figure 11 MBBR biofilm reactor contains in nitrile wastewater treatment process, water outlet COD change curve.
Embodiment
The structure of example 1, genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr
(1) pcr amplification of nitrilase gene and subclone in Rhodococcus rhodochrous BX2
Using the STb gene of Rhodococcus rhodochrous BX2 as template, according to the primer 5 ' GCG that the gene order of nitrilase gene and the polyclone restriction enzyme site of subtilis expression vector pHT01 design
tCTAGAaTGGTCGAATACACAAACAATTTC3 ' and 5 ' ATT
gACGTCtCAGATGGAGGCTGTCGC3 ' (underscore place is followed successively by XbaI enzyme cutting site and AatII restriction enzyme site) is primer, the gene fragment of the long nitrilase gene DNA for 1101bp is gone out by pcr amplification, the agarose gel electrophoresis figure of this nitrilase gene as shown in Figure 1, wherein 1,2 is the PCR primer of nitrilase gene, M is the DNA Marker of 10000bp, consistent with expection object clip size.Nitrilase gene is added after A tail completes and be connected with pMD18-T simple vector, by pMD18-T-Nitr Transformed E .coli DH5 α after successful connection, screening positive recombinant pMD18-T-Nitr, obtains the plasmid pMD18-T-Nitr (as Fig. 2) of purifying.
(2) structure of pHT01-Nitr expression vector
By the E.coli DH5 α containing pMD18-T-Nitr and pHT01 plasmid respectively containing in 100 μ g/mL penbritins and 50 μ g/mL penbritin LB substratum 35 DEG C, 160rpm incubated overnight.Extract pMD18-T-Nitr and pHT01 plasmid, institute's upgrading grain carries out double digestion with restriction enzyme XbaI and AatII respectively, after enzyme cuts end, carry out 1% agarose gel electrophoresis detection immediately, then reclaim enzyme with OMEGA test kit cut successful object fragment and connect, connect after terminating, Transformed E .coli DH5 α Competent cell.Obtain recon pHT01-Nitr after amplification cultivation, carry out pcr amplification with this recon for template, obtain the fragment that size is 1101bp, conform to nitrilase gene fragment size.Recon obtains 1101bp and 7955bp two bar segment through restriction enzyme Xba I and Aat II double digestion, correspond respectively to the size of nitrilase gene and pHT01, confirm that nitrilase gene and pHT01 carrier are successfully recombinated, as shown in Figure 3, wherein 1,2 is pHT01-Nitr carrier double digestion result; M is 15000bp DNA Marker.
(3) electricity conversion expression vector pHT01-Nitr proceeds in Bacillus subtilis N4
Preparation B.subtilis N4 Electroporation-competent cells, the expression vector pHT01-Nitr built is proceeded to B.subtilis N4 by electroporated method, cultivate in the LB solid medium containing paraxin (5 μ g/mL), picking list bacterium colony is cultivated and extracts plasmid, carry out plasmid PCR and double digestion qualification, double digestion result as shown in Figure 4, obtain 2 bands conformed to pHT01 carrier size with nitrilase gene respectively, prove that plasmid successfully proceeds in B.subtilis N4, the double functions yeast called after subtilis Bacillus subtilis N4-pHT01-Nitr of restructuring.
(4) genetic stability of recombinant plasmid pHT01-Nitr and the expression at protein level
The uneven distribution of plasmid in daughter cell is the major cause of plasmid loss, often makes part daughter cell not containing plasmid.In order to prove that can recombinant plasmid carry out genetic stability in B.subtilis N4, by subtilis Bacillus subtilis N4-pHT01-Nitr exponential phase with 1% inoculum size transfer continuously after 25 generations and on selectivity LB substratum (containing 5 μ g/mL paraxin) and non-selective LB substratum (not containing paraxin), be coated with cultivation respectively, calculate the colony number on two flat boards, by following formulae discovery plasmid loss rate.
After cultured continuously 25 generation after continuous passage cultivated for 25 generations, calculating plasmid loss rate is 5.52%, shows that recombinant plasmid pHT01-Nitr has good genetic stability in B.subtilis N4.
To be inoculated in 1% inoculum size in exponential phase by subtilis Bacillus subtilis N4-pHT01-Nitr and to be equipped with in 50mL (containing 5 μ g/mL paraxin) LB substratum, 37 DEG C, 200r/min is cultured to OD
600nmfor 0.7-0.8 adds inductor IPTG (volumetric concentration is 1mM), after induction 24h, get nutrient solution 10mL, carry out ultrasonication after centrifuge washing and obtain crude enzyme liquid.Get 20 μ L crude enzyme liquids and add equal-volume 2 × SDS sample-loading buffer, carry out protein electrophorese.
SDS-PAGE protein electrophorese result shows, Strains B. subtilis Bacillus subtilis N4-pHT01-Nitr is after IPTG induction, give expression to a size and be about 40kDa protein band, obvious protein band, the albumen size expressed with nitrilase gene conforms to.Shown in Fig. 5, wherein M is standard protein; 2 is the crude enzyme liquid of subtilis B.subtilis N4-pHT01-Nitr.
Above result shows, nitrilase gene successfully proceeds in subtilis B.subtilis N4, and recombinant bacterial strain can be expressed by genetic stability.
Example 2 genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr film forming ability
Biological film culture device is as follows: 150mL triangular flask, includes 50mL synthetic wastewater, 20 polyvinyl chloride packings and 2 pieces of slide glasss.1mL subtilis Bacillus subtilis N4-pHT01-Nitr is inoculated, not connect bacterium in contrast in biological film culture device.Arrange at identical conditions three parallel, get its mean value.37 DEG C, 60r/min shaking culture, adds 1mM/L IPTG in subtilis Bacillus subtilisN4-pHT01-Nitr after cultivating 4h, surveys biofilm formation amount in its 48h.
Described synthetic wastewater is by following proportions: glucose 0.3g/L, NH
4cl 0.0401g/L, KH
2pO
40.0092g/L, CaCl
20.0107g/L, MgSO
40.008g/L, FeSO
40.00011g/L, is dissolved in tap water, and acetonitrile addition is 800mg/L.
Described biological membrane biological amount measures as follows: after cultivating different time, discard the synthetic wastewater containing suspension cell.Then distilled water rinsing twice is used, all the other Adherent bacteria 50mL0.1% (w/v) violet staining 30 minutes.Thorough cleaning triangular flask, dried overnight.Remaining Viola crystallina is dissolved in the ethanol-acetone (4: 1, v/v) of 10mL, measures the absorbancy at 570nm place.Using the OD value at 570nm place as biofilm formation figureofmerit.
Subtilis Bacillus subtilis N4-pHT01-Nitr cultivates in 48h under these conditions, biofilm formation amount OD
570nmvalue reaches 1.45 (Fig. 6), shows that this bacterial strain has good film forming ability and carrier adhesive capacity, and after illustrating that recombinant plasmid pHT01-Nitr successfully proceeds to Bacillus subtilis N4, bacterial strain retains good filming ability.
Example 3 genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr is to the degraded of acetonitrile
(1) subtilis Bacillus subtilis N4-pHT01-Nitr is to the tolerance of acetonitrile and utilization power
Inoculation 1mL (OD
600nm=1.0) bacteria suspension of subtilis Bacillus subtilis N4-pHT01-Nitr contains in 50mL in the synthetic wastewater of 800mg/L acetonitrile, the synthetic wastewater of acetonitrile is not added in contrast with inoculation, 37 DEG C of 160r/min shaking table shaking culture, 1mM/L IPTG is added in subtilis Bacillus subtilis N4-pHT01-Nitr after cultivating 4h, measure the light absorption value at 600nm place in 48h, observe the raw situation of bacterial strain, arrange at identical conditions three parallel, get its mean value; The bacteria suspension of another inoculation 1mL subtilis Bacillus subtilis N4-pHT01-Nitr contains in 50mL in the inorganic salt basic medium of 800mg/L acetonitrile, with the substratum do not inoculated for contrast, 37 DEG C, 160r/min shaking table shaking culture, 1mM/L IPTG is added in subtilis Bacillus subtilis N4-pHT01-Nitr after cultivating 4h, measure the light absorption value at 600nm place in 48h, whether detect bacterial strain can utilize acetonitrile for the growth of sole carbon nitrogenous source, arrange at identical conditions three parallel, get its mean value.
Described inorganic salt basic medium is by following proportions: KH
2pO
41.7g/L, Na
2hPO
49.8g/L, MgSO
47H
2o 0.1g/L, CaCO
30.002g/L, ZnSO
47H
2o 0.00144g/L, FeSO
47H
2o 0.0009g/L, CuSO
45H
2o 0.00025g/L, H
3bO
30.00006g/L, natural pH7.2.
Containing 800mgL respectively
-1after the synthetic wastewater of acetonitrile and inorganic salt base culture base 48h, the growing state of Strains B. subtilis Bacillus subtilisN4-pHT01-Nitr as shown in Figure 7, as seen from the figure, Strains B. subtilis Bacillus subtilis N4-pHT01-Nitr well-grown in synthetic wastewater, 30h 600nm place light absorption value is 1.092, and in inorganic salt basic medium, 36h 600nm value is 0.715.Show that genetic engineering bacterium can utilize acetonitrile for the growth of sole carbon nitrogenous source, also demonstrate nitrilase gene simultaneously and successfully to proceed in Bacillus subtilis N4 and to express, can be used for carrying out the process containing nitrile waste water.
(2) genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr is to the degradation capability of acetonitrile
Reaction unit and cultural method: 150mL triangular flask, be equipped with synthetic wastewater, 20 polyvinyl chloride packings and the 2 piece slide glasss of 50mL containing 800mg/L acetonitrile.1mL (OD is inoculated in triangular flask
600nm=1.0) subtilis Bacillus subtilis N4-pHT01-Nitr bacteria suspension, separately establishes a blank not connecing bacterium, to determine the natural volatile quantity of acetonitrile.37 DEG C, 160rpm shaking culture, adds 1mM/L IPTG in subtilis Bacillus subtilis N4-pHT01-Nitr after 4h, continues to cultivate 48h.Arrange at identical conditions three parallel, get its mean value.Timing sampling 1mL, measures the residual quantity of acetonitrile with gas spectrometry.
The mensuration of acetonitrile residue: gas chromatographic detection condition: GC-14C type gas chromatograph, fid detector; Chromatographic column: interior painting 14%OV-1701 wide bore capillary column (30m × 0.53mm); Temperature condition: injection port 200 DEG C, fid detector 220 DEG C, column temperature 80 DEG C; Gas flow: carrier gas is high-purity N
2, flow 300kPa (dividing potential drop 50kPa), hydrogen 50kPa, air 50kPa; Sample size is 1 μ L, with quantified by external standard method. degradation rate calculation formula is:
X=(C
cK-C
x)/C
cKin × 100% formula, X is the biological degradation rate of acetonitrile, C
xfor connecing acetonitrile concentration (mg/L) in bacterium process nutrient solution, C
cKfor not connecing acetonitrile concentration (mg/L) in bacterium contrast.
Strains B. subtilis Bacillus subtilis N4-pHT01-Nitr is in the synthetic wastewater containing 800mg/L acetonitrile, after cultivation 48h, the residual concentration of acetonitrile is 23.56mg/L, and acetonitrile residual concentration is about about 650mg/L after blank 48h, illustrate that, after IPTG induction, subtilis Bacillus subtilis N4-pHT01-Nitr can utilize acetonitrile to carry out own metabolism thus reduce the concentration (as shown in Figure 8) of acetonitrile in environment.
Example 4 genetic engineering bacterium bacillus subtilis Bacillus subtilis N4-pHT01-Nitr process is containing nitrile waste water
(1) Strains B. subtilis Bacillus subtilis N4-pHT01-Nitr forms biomembranous impact resistance
Reaction unit and cultural method are with example 3 (2), after triangular flask inoculation 1mL subtilis Bacillus subtilis N4-pHT01-Nitr bacteria suspension cultivates 24h, discard the synthetic wastewater containing bacterioplankton in bottle, with aqua sterilisa rinsing 2 times, biomembranous bacterium is not adsorbed in remove on filler and bottle wall, again add synthetic wastewater 50mL to continue to cultivate, every 8h changes single sintering waste water (24-72h), measures microbial film to the continuous degradation ability of acetonitrile with gas spectrometry.The synthetic wastewater acetonitrile concentration of each interpolation is 800mg/L.By the testing conditions of example 3 (2) and formulae discovery Strains B. subtilis B.subtilis N4-pHT01-Nitr to the degradation rate of acetonitrile.
From acetonitrile residual concentration figure (Fig. 9), can find out, at 24-72h in the repeat impact of 8h, the residual concentration of primary stage of inoculation acetonitrile is higher, this is because twice microbial film is not formed before impingement, and subtilis Bacillus subtilis N4-pHT01-Nitr cannot be fixed on membrane carrier, changing in the process of synthetic wastewater continuously, the increase acetonitrile residual concentration of number of shocks significantly reduces, after continuous six replacing synthetic wastewaters, subtilis Bacillus subtilis N4-pHT01-Nitr finally in induction 8 hours to synthetic wastewater in the degradation rate of acetonitrile reach 49%, this has played its strong film forming advantage owing to extending subtilis Bacillus subtilis N4-pHT01-Nitr along with incubation time, on membrane carrier, self forms microbial film and fixes, anti-shock loading level is higher, be suitable for being applied to moving-bed bioreactor (Moving Bed Biofilm Reactor, containing in nitrile wastewater treatment MBBR).
(2) biofilm reactor (MBBR) simulation is containing nitrile wastewater treatment
Adopt MBBR (MBBR), seed sludge mixed solution and filler put into reactor simultaneously, static aeration 24h, and aeration rate is adjusted to and just made the complete fluidisation of filler.Then drain supernatant liquor and suspended sludge, 2d starts intermittent water inflow, and hydraulic detention time is 24h, and operating temperature is 20 DEG C-25 DEG C.Timing sampling adopts gas spectrometry and potassium dichromate process to measure ethane nitrile content and COD value in water.
Described MBBR useful volume is 10L, and mud is aerobic activated sludge, takes from peaceful sewage work A/O (Anoxic/Oxic) pond, Harbin oxygen supply section; Filler is modified poly ethylene, and true density, close to water, is 0.94-0.98gcm before biofilm
-3, after biofilm, approximate 1gcm
-3, its filling ratio is 30%, and flowing into the initial COD concentration of synthetic wastewater is 400mgL
-1, acetonitrile starting point concentration is 800mgL
-1.
Adding biological total amount in reactor is 3 000mg/L, and No. 1 reactor only adds active sludge (30g); No. 2 reactors add active sludge and double functions yeast bacillus subtilis Bacillus subtilis N4-pHT01-Nitr (24g, 6g).
COD adopts standard method to measure and sees APHA, AWWA, WEF.Standard Methods for the Examination of Water and Wastewater, 21st ed.American Public Health Association, Washington, DC.2005
Acetonitrile concentration and COD value change that reactor is running water outlet in 35d process are shown in Figure 10 and Figure 11.Only add No. 1 reactor of active sludge owing to lacking the bacterial strain of acetonitrile of can degrading, before operation, 13d acetonitrile concentration is higher.After running 13d, in reactor water outlet successively there is downtrending in acetonitrile concentration, infers that its reason is due to long domestication, occurred in the reactor decomposing the microorganism utilizing acetonitrile.These microorganisms have played effect in acetonitrile degradation process, make acetonitrile concentration remain on lower level.Reactor runs 35d, and in water outlet, acetonitrile concentration is 58.5mg/L, and COD value is 84.12mg/L.
No. 2 reactors acetonitrile concentration after the vexed 24h of exposing to the sun adding active sludge subtilis Bacillus subtilis N4-pHT01-Nitr is down to 346mg/L.Afterwards may be not yet ripe due to short period of time microbial film, subtilis Bacillus subtilis N4-pHT01-Nitr does not accumulate in a large number on reactor carrier, show as reactor and run front 10d, acetonitrile decline concentration is also not obvious, and bacterial strain is all lower to acetonitrile degradation rate; After operation 10d, in reactor, significantly reducing appears in acetonitrile residual concentration, and this is because microbial film is ripe gradually, impels the nitrilase of expressing in subtilis Bacillus subtilis N4-pHT01-Nitr to play its degradation function.After reactor runs 35d, in No. 2 reactors, acetonitrile concentration is 0.32mg/L, and COD value is 16.39mg/L.Run after 35d at reactor, acetonitrile is almost removed completely, illustrates that genetic engineering bacterium subtilis Bacillus subtilis N4-pHT01-Nitr has played the difunctional of the degraded of its nitrile and biofilm formation, thus raising acetonitrile degradation rate.
This genetic engineering bacterium subtilis Bacillus subtilis N4-pHT01-Nitr there is the degraded of stronger nitrile and biofilm formation ability, environmental compatibility strong, have great potential containing in the biological restoration of nitrile waste water, this is applied to biomembrance process process for it and has great importance containing nitrile waste water.
Claims (4)
1. a strain nitrile degradation biological film forms bifunctional genetically engineered bacterium subtilis Bacillus subtilis N4-pHT01-Nitr, this bacterial strain was preserved on 07 25th, 2014 " China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) ", and preserving number is CGMCCNo.9484.
2. a strain nitrile degradation biological film as claimed in claim 1 forms bifunctional genetically engineered bacterium subtilis Bacillus subtilis N4-pHT01-Nitr and produces microbial film.
3. a strain nitrile degradation biological film as claimed in claim 1 forms bifunctional genetically engineered bacterium subtilis Bacillus subtilis N4-pHT01-Nitr for acetonitrile of degrading.
4. a strain nitrile degradation biological film as claimed in claim 3 forms bifunctional genetically engineered bacterium subtilis Bacillus subtilis N4-pHT01-Nitr containing the application in nitrile wastewater treatment.
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| CN201410519862.5A CN104293725A (en) | 2014-09-23 | 2014-09-23 | Nitrile degradation biofilm formed genetically engineered bacterium and application of genetically engineered bacterium in nitrile-containing wastewater treatment |
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| CN201410519862.5A CN104293725A (en) | 2014-09-23 | 2014-09-23 | Nitrile degradation biofilm formed genetically engineered bacterium and application of genetically engineered bacterium in nitrile-containing wastewater treatment |
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Cited By (4)
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| CN105154464A (en) * | 2015-10-28 | 2015-12-16 | 天津亿利科能源科技发展股份有限公司 | Method for utilizing beneficial film-forming bacteria for restraining corrosion caused by sulfate reducing bacteria in site |
| CN107162175A (en) * | 2017-05-12 | 2017-09-15 | 南京大学 | A kind of method that training degraded penicillin activated sludge is tamed and dociled by co-substrate of glucose |
| CN110773562A (en) * | 2019-11-05 | 2020-02-11 | 北京高能时代环境技术股份有限公司 | Microbial remediation method for polycyclic aromatic hydrocarbon in heavy metal-polycyclic aromatic hydrocarbon combined contaminated soil |
| CN114874036A (en) * | 2022-04-27 | 2022-08-09 | 眉县食品药品安全检验检测中心 | Method for enzymatic treatment of acetonitrile in residue after laboratory agricultural product detection |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154464A (en) * | 2015-10-28 | 2015-12-16 | 天津亿利科能源科技发展股份有限公司 | Method for utilizing beneficial film-forming bacteria for restraining corrosion caused by sulfate reducing bacteria in site |
| CN107162175A (en) * | 2017-05-12 | 2017-09-15 | 南京大学 | A kind of method that training degraded penicillin activated sludge is tamed and dociled by co-substrate of glucose |
| CN107162175B (en) * | 2017-05-12 | 2020-06-02 | 南京大学 | Method for domesticating and degrading penicillin activated sludge by taking glucose as co-matrix |
| CN110773562A (en) * | 2019-11-05 | 2020-02-11 | 北京高能时代环境技术股份有限公司 | Microbial remediation method for polycyclic aromatic hydrocarbon in heavy metal-polycyclic aromatic hydrocarbon combined contaminated soil |
| CN114874036A (en) * | 2022-04-27 | 2022-08-09 | 眉县食品药品安全检验检测中心 | Method for enzymatic treatment of acetonitrile in residue after laboratory agricultural product detection |
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