Short Communication
Degradation pathway,toxicity and kinetics of 2,4,6-trichlorophenol with different co-substrate by aerobic granules in SBR
Mohammad Zain khan a ,Pijush Kanti Mondal b ,⇑,Suhail Sabir a ,⇑,Vinod Tare c
a
Environmental Rearch Laboratory,Department of Chemistry,Aligarh Muslim University,Faculty of Scie
nce,Aligarh 202002,UP,India呕心沥血是什么意思
b
Environmental Rearch Laboratory,Department of Applied Chemistry,Faculty of Engineering and Technology,Aligarh Muslim University,Aligarh 202002,UP,India c
Environmental Engineering and Management,Department of Civil Engineering,Indian Institute of Technology,Kanpur 208016,UP,India
a r t i c l e i n f o Article history:
Received 3February 2011
意义英文
Received in revid form 18April 2011Accepted 19April 2011
Available online 23April 2011Keywords:
Bioremediation TCP Gluco Acetate
Genotoxicity
a b s t r a c t
starting from 10to 360mg L À1with more than 90%efficiency was achieved.Sludge volume index decreas as the operation proceeds to stabilize at 35and 30mL g À1while MLVSS increas from 4to 6.5and 6.2g L À1for R1(with gluco as co-substrate)and R2(with sodium acetate as co-substrate),respectively.FTIR,GC and GC/MS spectral studies shows that the biodegradation occurred via chlorocate-chol pathway and the cleavage may be at ortho -position.Haldane model for inhibitory substrate was applied to the system and it was obrved that gluco fed granules have a high specific degradation rate and efficiency than acetate fed granules.Genotoxicity studies shows that effluent coming from SBRs was non-toxic.
Ó2011Elvier Ltd.All rights rerved.
山东省普通高中学业水平考试成绩查询1.Introduction
available什么意思2,4,6-Trichlorophenol (TCP)is a potential water pollutant widely ud in the preparation of biocides,flame retardants (Belchik et al.,2010),chemicals reagents and prervatives and caus
vere toxicity to humans and aquatic life (Gaitan et al.,2011).It is carcinogenic in animals,causing lymphomas,leuke-mia,and liver cancer via oral exposure and classified as Group B2(probable human carcinogen)by USEPA.Biodegradability of chlorophenols varies depending on the position and the number of chlorine groups and usually biodegradability decreas and toxicity increas with increasing number of chlorine groups (Eker and Kargi,2009).Several techniques such as physical,chemical,electrochemical and biological methods have been pro-pod for efficient wastewater treatment,most of them prent-ing some limitations such as poor capacity,generate waste products,incomplete mineralization or high operating cost (Gonzalez et al.,2010;Mondal et al.,2010).Nowadays aerobic granulation treatments are considered to be the most effective route (Khan et al.,2010;Wang et al.,2007).Aerobic granulation is a process of microbial lf-immobilization,resulting into a cell-structured shape characterized by den biomass (Khan
et al.,2011).Aerobic granules have been cultivated in a quenc-ing batch reactor (SBR).SBR has a number of advantages over other reactors such that it provides equalization,aeration and dimentation steps in a time rather than in a space quence.Aerobic bacteria degrade xenobiotic pollutant (such as TCP)and,in veral cas,grow on it as the sole carbon source.How-ever,veral findings suggested that the addition of some conven-tional carbon sources (gluco,fructo or acet
ate etc.)might aid in reducing the toxicity and growth inhibition of xenobiotics on cells,thereby increasing the transformation rates of xenobiotics.The prence of easily degradable carbon sources stimulates the growth of microbial population which then enhances the biodegra-dation of target contaminants like TCP.The additional carbon sources may also acts as an inducing agent or provide reducing power for degradation of recalcitrant organic compounds.The same strategy was ud by many rearchers in order to prevent unfavorable impacts on cells metabolism.
Most of the available literature deals with biodegradation of chlorophenols using isolated pure culture even though mixed microbial cultures are chiefly ud for large scale wastewater sys-tems.However,a very few studies are concerned with mixed cul-ture (Eker and Kargi,2009;Snyder et al.,2006;Farrell and Quilty,1999).Therefore the prent work focud on comparative study of TCP degradation in prence of gluco and acetate as co-substrate by mixed culture and characterization of the biodegrada-tion products including the kinetics and toxicity.
0960-8524/$-e front matter Ó2011Elvier Ltd.All rights rerved.doi:10.1016/j.biortech.2011.04.057
⇑Corresponding authors.Tel.:+919358251520.
E-mail address: (P.K.Mondal),sabirsuhail09@gmail.-com (S.Sabir).
2.Methods
潮孩2.1.Chemicals
2,4,6-Trichlorophenol,a xenobiotic compound was a G.R.Prod-uct of Fisher Scientific,India and was ud as received.All chemi-cals were ud in analytical reagent grade and supplied by Fisher Scientific India.
2.2.Biomass sources and basal medium
Mixed microbial culture with a MLVSS of2.5g LÀ1and SVI of 200mL gÀ1was taken as a source of aerobic sludge from condary clarifier of Okhla wastewater treatment plant,New Delhi,India.It was brown initially with a loo,fluffy and irregular morphology. Its color was then changed to white andfinally to yellow during the cour of experiment.
In order to get more specific microorganism for TCP removal, activated sludge was initially conditioned over a15-day period to allow the biomass to adapt to TCP before inoculating into SBR. The aerobic sludge was divided into two parts during acclimation pha and fed with5–10mg LÀ1of
TCP along with respective co-substrates gluco and acetate in mineral salt medium(basal med-ium)in batch mode.Later on,the acclimated sludge was inoculated into SBR.
The basal medium ud in this experiment contained(g LÀ1): (Ahmad et al.,2010)Macro nutrient:NH4Cl(0.20),K2HPO4 (1.65),MgSO4.7H2O(0.13),KH2PO4(1.35)Micro nutrient:H3BO3 (0.05),FeCl2Á4H2O(0.05),ZnCl2(0.05),MnCl2Á4H2O(0.05), CuCl2Á2H2O(0.03),NH4SeO3Á5H2O(0.05),AlCl3Á6H2O(0.05), NiClÁ6H2O(0.05),NaSeO3Á5H2O(0.1).
A mother solution of TCP was prepared with strength of 1000mg LÀ1in double distilled water.Required concentrations of TCP were obtained by proportionate dilution with double distilled water.Initially,the reactors were fed with TCP(10mg LÀ1)in basal medium.The pH of the system was maintained at around8–9 through out the study.otc什么意思啊
2.3.Experimental t up
Two column type cylindrical reactors(quencing batch reac-tor)R1and R2made of transparent Perspex glass(height150cm and diameter5cm)was ud with a working volume of1.4L as shown in Fig.1S(supporting information).Reactors were main-tained at room temperature and started up with1.
4L of aerobic sludge.Reactor R1was fed with gluco while R2was fed with so-dium acetate as co-substrate along with TCP(Fig.1S).However,all other conditions remain same for both the reactors.Fine bubble aerator wasfixed at the bottom of both the reactors for supplying air at a superficial gas velocity above1.2cm sÀ1.A port wasfitted at70cm along the height of the SBR and ud for collecting efflu-ent samples.All the experiments were performed at constant tem-perature(30±5°C)and the dissolved oxygen was maintained at 4–5mg LÀ1in both the reactors during the study.
2.4.SBRs operation
Both reactors were operated at three different HRTs of48,24 and16h with a50%volumetric exchange ratio.Each cycle of the reactors consist of:5min influent addition,30–5min ttling, 5min effluent withdrawal,5min idle(no stirring)and the aeration was performed during the remaining period.Five percent of the sludge was wasted everyday which gives a constant SRT(sludge retention time)of20days.For measuring abiotic loss,a con-trolled reactor was ud having same dimensions as experimental reactor but without aerobic sludge.It was obrved that abiotic loss due to volatilization and photo degradation by sunlight were less than3%.
2.5.Analytical methods
SVI,MLVSS,COD,ClÀion and alkalinity were taken in accor-dance with the standard methods(APHA,2002).Biomass loss in effluent were monitored in terms of optical density at600nm using UV–visible spectrophotometer(Shimadzu-1601,Japan) according to the method given by(Ziagova and Liakopoulou-Kyriakides,2007).
The TCP was measured by UV–visible spectrophotometer(Shi-madzu-1601,Japan)in accordance with method given by(Zheng et al.,2004)and a calibration plot(absorbance versus concentra-tion of TCP)was drawn with correlation coefficient(R2)of0.98 and ud for estimating the concentration of unknown TCP solu-tions.Effluent samples were collected andfiltered using0.22l m pore sizefilter before taking concentration.The UV Spectra of the influent and effluent samples were recorded in the wavelength range190–700nm.The test samples drawn from experiments with higher concentrations of TCP were adequately diluted prior to absorbance determination.
2.6.Biodegradation pathway studies
FTIR of the influent,effluent and EPS(for both the reactors) were taken using Interspec2020Spectrolab(UK).The reaction end products were performed on a QP5050A GC system(Buck Sci-entific910Model,Shimadzu,Columbia,Md.)equipped with a DB-5 (30m by0.25mm)c
apillary column(RESTEK,USA).GC/MS studies were done using PERKIN ELMER CLARUS500Gas chromatograph-mass spectrometer.Samples for GC and GC/MS were extracted and derivatized by a previously described method(Louie et al.,2002). Briefly,the reaction mixture was acidified to pH4(10l l of concen-trated HCl per ml of the reaction mixture),and aromatic com-pounds were extracted into ethyl acetate(1:1v/v of ethyl acetate and effluent samples).
2.7.Batch kinetics
The ability of granules to degrade TCP was evaluated by study-ing the batch kinetics of biodegradation in250ml rum vials con-taining different concentrations of TCP(100,200,250,300,350, 400,450mg LÀ1)in mineral salt medium.100ml of granular sludge was added to each rum vials and the content was kept on a rotary shaker for8h and studied periodically(after every 30min till steady state).A kinetic study of the data was performed using the Haldane model for inhibitory substrate(Wang et al., 2007).
2.8.Genotoxicity by plasmid nicking assay
Escherichia coli K-12strain was ud to check the toxicity of degradation products by aerobic granules.This plasmid nicking as-say was carried out as described by(Rahman et al.,1990).0.5l g of
covalently clod circular pBR322DNA was treated with the con-trol as well as treated(effluent)and untreated(influent)TCP water samples in a total volume of20l L for1h.After the treatment,8l L of5x tracking dye[40mM EDTA,0.05%bromophenol blue and50% (v/v)glycerol]was added and loaded on1%agaro gel.The gel was run at50mA for2h and stained with ethidium bromide (0.5l g LÀ1)for30min at room temperature.After washing,the gel was visualized on fotodyne UV300transilluminator(USA) and photographed.
M.Z.khan et al./Bioresource Technology102(2011)7016–70217017
3.Results and discussion
3.1.Reactor performance in terms of COD removal
The plots show the variation of influent COD,effluent COD and %COD removal efficiency during the study(Fig.1).The COD values in the influent were1200mg LÀ1(R1)and850mg LÀ1(R2)in the beginning thereafter the values were stepwi incread as shown in Fig.1.Influent COD include the COD for TCP and co-substrate(gluco or acetate).Influent COD was higher in ca of R1due to higher COD value of gluco(1067mg LÀ1gÀ1gluco) as compared to COD of acetate(780mg LÀ1gÀ1acetate).Initially, low removal efficiencies(around70%)were obrved but as
soon as the system got stable(after day20),removal efficiency(reactor performance)increas and good results were obtained.
Both reactors R1and R2show good removal efficiency with a slight higher efficiency in ca of R1.This is due to the irregular morphology(folds,crevices and depressions)of gluco fed gran-ules which enables the higher mass transfer within the granule. The irregularities shorten the diffusion distances;improve the penetration of nutrients and substrates into the granule interior compared to more spherical granules fed with acetate as co-sub-strate.The obrvations were also supported by kinetic data. 3.2.Variation of influent TCP,SVI,MLVSS and OD
The variation of influent TCP(g LÀ1),SVI(mL gÀ1),MLVSS(g LÀ1) and OD in R1and R2during the experimental study are shown in Fig.2.Influent TCP was varied from10to360g LÀ1in R1and10–300g LÀ1in R2.Initially,same concentrations of influent TCP were fed into R1and R2,but,as the operation proceeds,the amount of biomass in R1increas giving high removal efficiency.Therefore, the influent TCP concentrations were incread at a faster rate in R1as compared to R2.
Settleability is an important parameter that enables the com-pact biomass(granules)to be retained in the reactor.The source
7018M.Z.khan et al./Bioresource Technology102(2011)7016–7021
aerobic sludge after acclimation had an SVI value of200ml gÀ1,it was then fed into R1and R2.After one week,the SVI value de-creas to140ml gÀ1(R1)and130ml gÀ1(R2)showing improve-ment in ttling properties of the sludge(Fig.2).In subquent weeks,SVI shows a decreasing trend,meanwhile,the aerobic sludge start changing into granular biomass.SVI values of35and 30ml gÀ1were obrved at the end of the operation in R1and R2,respectively(Fig.2).
项目管理制度
Biomass concentration(MLVSS)in the reactors shows variable behavior in between4.5–6.5g LÀ1.MLVSS values in R1and R2 were stabilized at6.5and6.2g LÀ1at the end of the study.Optical density of effluent(OD)shows the biomass loss during effluent withdrawal.Initially,OD of the effluent from R1and R2was1.85 and1.82.Since,granules have good ttling properties(as reflected by low SVI),biomass loss in the effluent decreas as the granu-lation proceeds,leading to a similar decrea in OD of effluent to stabilize at1.45(R1)and1.24(R2)at the end of the SBRs operation (Fig.2).The loss in ca of R2were less as compared to R1,due to more regular and compact structure of acetate-fed granules as mentioned in Section3.1.
However,the percent COD removal efficiency and specific deg-radation rate shows better substrate
degradation capability of glu-co fed granules.This is becau the choice of substrate has a great influence on granule microstructure.The den,regular and compact acetate fed granules failed to tolerate higher organic load-ing rate resulting in low biodegradation rate due to mass transfer limitations(Moy et al.,2002).
Prent study was performed at three different HRTs of48,24 and16h with a50%volumetric exchange ratio giving a cycle time of24,12and8h,respectively(Table1S and2S,supporting infor-mation).HRT has a profound effect on hydraulic conditions and the contact time among different reactants within the reactor. For optimizing the reactor performance,a proper HRT should be judiciously lected and carefully maintained.A HRT of48h was maintained forfirst25days,thereafter HRT was reduced to24h, meanwhile the sludge changes into granular biomass since short HRT favors granulation(Beun et al.,1999).Finally,the HRT was re-duced to16h(after day45)and kept constant for the rest of the study.It was found that;better granulation was occurred at a shorter HRT of16h.
Relea of ClÀion during TCP biodegradation was also moni-tored during the study(APHA2002)and shown in Table1S and 2S(supporting information).It is believed that biodegradation of chlorophenols involves a dehalogenation step which allow relea of ClÀion in the reactor.ClÀproduction was stoichiometrically re-lated to the TCP biodegradation and was low initially due to low biodegradation
rates.But as soon as the granules became the dom-inant part of biomass,degradation rate increas leading to a cor-responding ri in ClÀproduction in the effluent samples.However, ClÀrelea in R1is fairly greater than in R2due to high efficiency and degradation rate of gluco fed granules in R1(becau of greater mass transfer within the granule)(Table1S and2S,sup-porting information).
3.3.Characterization of the biodegradation products
aprilfool3.3.1.UV–visible spectral studies
The UV–visible studies were performed in the range190–700nm in order to compare the influent and effluent spectra dur-ing biodegradation of TCP in SBR.Spectra were taken at the end of the study(at16h HRT).UV spectra of TCP influent with gluco and acetate is shown in Fig.2S(supporting information).Influent and effluent spectra in both the cas(R1and R2)are similar with no significant peak of gluco or acetate within the wavelength re-gion studied.The influent spectrum is showing a prominent absorption peak at around320.0nm which confirm the prence of2,4,6-TCP.However,no such peak(at320.0nm)is obrved in ca of effluent coming out of the reactor,showing biodegradation of TCP in SBR.
3.3.2.Gas chromatographic analysischillax
GC was performed in order to determine the number of prod-ucts formed during biodegradation along with their retention time. Four peaks were obrved in the GC spectra of effluents samples of both reactors(Fig.2S int)extracted using ethyl acetate as de-scribed above.Largest peak corresponds to the hyl acetate at a retention time of11.325min.In addition to ethyl ace-tate,three more peaks(at1.366,6.275and12.45min)were ob-rved showing the formation of three biodegradation products which may be catechol,chlorocatechol or CO2.
3.3.3.FTIR spectral analysis
The FTIR spectra of pure TCP,influent TCP with gluco(R1), effluent and EPS during biodegradation of TCP in a quencing batch reactor are given in Fig.3S(supporting information).FTIR spectrum(1)of R1shows typical peaks corresponding to pure TCP.C A H and O A H stretching is obrved at2900and 3400cmÀ1.C@C stretching of benzenoid and quinoid moiety at 1450–1550cmÀ1.The bands at1249,1154,1125and1027cmÀ1 correspond to ring C A H in plane bending vibrations.Bands at 1337and1179cmÀ1are attributed to O A H deformation and bend-ing vibrations.A strong C A Cl peak is obrved at1090cmÀ1.Peaks visible at700–800cmÀ1show the substituted aromatic ring.Spec-trum(2)of R1(Fig.3S)shows influent containing TCP with gluco in mineral salt medium.Several small peaks are visible in the spec-trum with a prominent one at1300c
mÀ1due to C@C stretching of aromatic ring.Bands between1100and1000cmÀ1demonstrate hydroxyl group of aromatic ring.This absorption peak is obrved at a lower value than its normal absorption frequency becau of the interaction of aromatic ring with metal ions(region500–600cmÀ1)prent in tap water.
FTIR spectrum(3)of R1shows no prominent peak in the region 1300–1400cmÀ1(abnce of aromatic ring)confirming the bio-degradation of TCP in SBR.A prominent peak at2300cmÀ1shows the prence of CO2in the effluent samples.FTIR spectrum(4)R1 reprents the important functional groups prent in the EPS -creted by bacteria during TCP biodegradation.EPS shows a peak in the region1250–1300cmÀ1showing the prence of aromatic ring.A strong peak at1100cmÀ1shows the prence of O A H group of aromatic ring.The peak at686.74–763cmÀ1refers to out of plane C A H bending in a substituted aromatic system.The peaks provided evidence regarding the possibility of the formation of any of the following product-catechol,2-chlorophenol or chlorocate-chol etc.during TCP biodegradation.GC spectra(Section3.3.2.)also confirmed the formation of three different compounds.The pos-sibilities were further investigated by GC/MS studies.Similar FTIR spectra were obrved in ca of biodegradation of TCP in prence of acetate(R2)as additional supplement(Fig.3S).
3.3.
4.Gas chromatographic-mass spectrometric studies
梦想之旅There may be veral potential biodegradation products formed during biodegradation of TCP by mixed microbial culture depend-ing upon the pathway involved.The may be the products of dehalogenation after ring cleavage(chlorocatechols)and dehalo-genation before ring cleavage(2,4or2,6-dichlorophenol,2-chloro-phenol,catechol etc.)(Snyder et al.,2006).Mostly chlorinated aromatics degraded via ortho-cleavage,however,in some cas, successful degradation of chloroaromatics via3-chlorocatechol using the meta-cleavage has also been reported(Farrell and Quilty, 1999).Prent study showed the formation of3,5-dichlorocate-chol,a probable product of TCP biodegradation,which then trans-formed to6-chloro-hydroxy-quinol andfinally undergo an ortho-
M.Z.khan et al./Bioresource Technology102(2011)7016–70217019
degradation to give 2-chloro-4-oxo-hex-2-ene-1,6-dioic acid or 2-chloro-maleylacetic acid (M +1m /z 193)as shown in GC/MS spec-tra Fig.3S (supporting information).The propod pathway show-ing ortho -cleavage via 3,5-dichlorocatechol formation has been shown in Fig.4S (supporting information).A similar pathway has been reported by Louie et al.(2002)with a single difference in the
formation of 2,6-dichlor-p-hydroxyquinone instead of 3,5-dichlorcatechol.3,5-Dichlorocatechol was also detected as an intermediate in the biodegradation of TCP by Snyder et al.(2006).
3.4.Batch biodegradation kinetics
Biodegradation kinetics of TCP is an important parameter for prediction of its fate and design of the wastewater treatment plant (Tomei et al.,2008).The biodegradation rate of TCP by aerobic granules is often described by Haldane’s model (Fig.3a)for inhib-itory substrate which can be depicted as (Eq.(1)):
V ¼
V max S K s þS þS
K
i
ð1Þ
where V and V max are the specific and the maximum theoretical specific substrate degradation rates (mg TCP gVSS À1d À1),respec-tively,and S ,K s and K i are the substrate concentration,half-satura-tion constant,and inhibition constant (mg L À1),respectively.
The calculated values of kinetic parameters using non-linear regression model are:V max (G)=1344mg TCP gVSS À1d À1,K s (G)=1911.65mg L À1and K i (G)=29.85mg L À1for R1and V max (A)=1200mg TCP gVSS À1d À1,K s (A)=1828.68mg L À1and K i (A)=33.86mg L À1for R2with a correlation coefficient (R 2)of 0.9892and 0.9569,respectively (Fig.3a).On the basis of obrved values of kinetic parameters from fittings,the critical substrate concentration at which maximum reaction rate obrved
(C ÃTCP ¼ffiffiffiffiffiffiffiffiffiffiffiffiffi
K s 0K i p )is found to be 238.8and 248.78mg L À1for R1and R2,respectively.However,the inhibitory parameter b (b ¼ffiffiffiffiffiffiffiffiffiffiffiffi
K i =K s p )accounting for the extent of inhibitory effects (a smaller value of b gives a larger removal rate reduction at high sub-strate concentration)is 0.1249for R1and 0.1360for R2.The batch kinetic data in Fig.3a reveal that gluco fed granules (R1)have better degradation rate and removal efficien
cy than acetate fed granules (R2).
3.5.Genotoxicity by plasmid nicking assay
The genotoxicity by plasmid nicking assay experiment (Fig.3b)was conducted on influent TCP as well as treated TCP effluent sam-ple.The results show reduction in genotoxicity of TCP by SBR.Twenty microliters of TCP resulted in the conversion of the super coiled pBR322DNA to the relaxed form (lane B).However,20l L of treated effluent TCP resulted in a maximum protection of plas-mid from damage (lane C).The results showed that aerobic gran-ules were capable of reducing the genotoxicity of TCP in SBR in prence of gluco or acetate as co-substrate.4.Conclusion
Granules sustained an organic load of 4067(R1)and 3780mg COD L À1(R2)with percent removal efficiency of 97and 95.Spectral studies confirmed that the biodegradation of TCP oc-curred via formation of 3,5-dichlorocatechol pathway followed by an ortho-cleavage.Plasmid nicking assay for genotoxicity shows reduction in toxicity of TCP in SBR.Kinetic data showed the biodeg-radation of TCP followed Haldane model with V max of 1344and 1200mg TCP gVSS À1d À1for R1and R2,respectively.Prent study shows an attractive option for removing toxic TCP containing wastewater by aerobic granules in SBR.Acknowledgements
Authors are thankful to Aligarh Muslim University,Aligarh for providing necessary rearch facilities.UGC is to be thanked for providing rearch fellowship.Appendix A.Supplementary data
Supplementary data associated with this article can be found,in the online version,at doi:10.1016/j.biortech.2011.04.057.References
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