《水污染控制原理》课程教学资源（文献资料）Anaerobic biodegradation of ethylthionocarbamate by the mixed bacteria

Bioresource Technology102 (2011)10772-10775Contents lists available at SciVerse ScienceDirectRIORROUOCBioresource TechnologyAELSEVITERjournalhomepage:www.elsevier.com/locate/biortechShortCommunicationAnaerobic biodegradation of ethylthionocarbamate by the mixed bacteriaunder various electron acceptor conditionsShaohua Chen, Wenqi Gong*, Guangjun Mei, Weiyi HanSchool of Resources and Environmental Engineering. Wuhan University of Technology.Wuhan 430070, PR ChindARTICLEINFOABSTRACTArticle history:Biodegradation behavior and kinetics of ethylthionocarbamate under nitrate, sulfate and ferric reducingReceived 9 August 2011conditions by mixed cultures enriched from the anaerobic digester sludge was investigated. The resultsReceived in revised form 8 September 2011showed that ethylthionocarbamate could be degraded independently by the mixed cultures coupled toAccepted 8September 2011nitrate, sulfate, and ferric reduction, and meanwhile, nitrite, sulfide, and ferrous were accumulated asAvailable online 16September 2011a result of nitrate, sulfate and ferric reduction, respectively.Ferric was a more favorable terminal electronacceptor compared to nitrate and sulfate. The order of the electron acceptors with decreasing biodegra-Keywords:dation rates of the ethylthionocarbamate was: ferric> nitrate>sulfate,and the corresponding maximumEthylthionocarbamatebiodegradation rate was 7.240, 6.267, and 4.602 mg/(L-d), respectively. The anaerobic biodegradation ofAnaerobic biodegradationethylthionocarbamate under various electron acceptor conditions can be accurately described by firstNitrate reducingorder exponential decay kinetics.Sulfate reducing 2011 Elsevier Ltd. All rights reserved.Ferric reducingTo develop anaerobic bioremediation technologies, systematic1.Introductionstudies are required for understanding the biodegradation of ethyl-Ethylthionocarbamate has been widely used as flotationthionocarbamate under anaerobic conditions. The aim of this studyreagent in sulfide mineral processing for many decades (Chenwas to investigate the biodegradation of ethylthionocarbamateetal.,2011).Seriousenvironmentalproblemsassociatedwithflo-under various electron acceptor reducing conditions in the pres-tation reagents in mineral processing plant wastewater have beenenceof mixed cultures enriched from the anaerobic digester sludgewell documented (Chockalingam et al.,2003: Hissner et al.,1999).of a typical sewage treatment plant. And the utility of biodegrada-It is known that even small concentration of these reagents intion kinetics process is also illustrated.water streams is toxic to water life, besides their deleterious influ-ence on the end stream processes during recycling (Okibe and2. MethodsJohnson, 2002). However, investigations on the biodegradation ofethylthionocarbamateunderanaerobicconditionshavenotbeen2.1.Microbial sourcereported.Many compounds have been shown to be degraded by microor-The anaerobic digested sludge used as inoculum for the presentganisms using various electron acceptors. With few exceptions, allresearch was obtained from the anaerobic digester of EzhouBTEX compounds (Dou et al.,2008:Szykowny and Keasling.1997).Sewage Treatment Plant.naphthalene (Dou et al., 2009) and dimethyl phthalate (Wu et al.,2007) have been shown to be degraded by microorganisms under2.2.Culturemediumandenrichmentnitrate reducing conditions. Studies also showed that several com-pounds including BTEX (Hu et al., 2007; Dou et al., 2008), nonyl-The electron acceptors used were nitrate (2.55g/L NaNO3).phenol polyethoxylates and alkanes (Lu et al., 2008) could besulfate (4.26 g/L Na2SO4) and ferric (402.78 mg/L Fe3*). Relativelydegraded under sulfate-reducing conditions. Furthermore, biodeg-high concentration of sulfate,nitrateand ferric were used to insureradation of BTEX (Dou et al., 2008), nonylphenol polyethoxylatesthedevelopmentofsulfate,nitrateandferricreducingconditions,(Lu et al., 2007) and other organic compounds with ferric as therespectively (Lu et al., 2007. 2008). In addition, all mediumelectronacceptorhas been elucidatedcontained the following constituents: NH.CI (1.5 g/L). KH2PO4(0.6 g/L), MgCl2-6H20 (0.1 g/L), CaCl2 (0.1 g/L), yeast extract(1.0 g/L), vitamin (1%, v/v) and trace solutions (1%, v/v). Further-more, the sulfate and ferric reducing medium were supplemented*Corresponding author.Tel.: +86 18986286480; fax: +86 27 87882128.with NaHCO3 (2.52 g/L) and Na2S.9H2O (0.5 g/L). Syringes andE-mail address: gongwenqi@yahoo.com.cn (W.Gong).0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2011.09.030

Short Communication Anaerobic biodegradation of ethylthionocarbamate by the mixed bacteria under various electron acceptor conditions Shaohua Chen, Wenqi Gong ⇑ , Guangjun Mei, Weiyi Han School of Resources and Environmental Engineering, Wuhan University of Technology, Wuhan 430070, PR China article info Article history: Received 9 August 2011 Received in revised form 8 September 2011 Accepted 8 September 2011 Available online 16 September 2011 Keywords: Ethylthionocarbamate Anaerobic biodegradation Nitrate reducing Sulfate reducing Ferric reducing abstract Biodegradation behavior and kinetics of ethylthionocarbamate under nitrate, sulfate and ferric reducing conditions by mixed cultures enriched from the anaerobic digester sludge was investigated. The results showed that ethylthionocarbamate could be degraded independently by the mixed cultures coupled to nitrate, sulfate, and ferric reduction, and meanwhile, nitrite, sulfide, and ferrous were accumulated as a result of nitrate, sulfate and ferric reduction, respectively. Ferric was a more favorable terminal electron acceptor compared to nitrate and sulfate. The order of the electron acceptors with decreasing biodegra￾dation rates of the ethylthionocarbamate was: ferric > nitrate > sulfate, and the corresponding maximum biodegradation rate was 7.240, 6.267, and 4.602 mg/(Ld), respectively. The anaerobic biodegradation of ethylthionocarbamate under various electron acceptor conditions can be accurately described by first order exponential decay kinetics. 2011 Elsevier Ltd. All rights reserved. 1. Introduction Ethylthionocarbamate has been widely used as flotation reagent in sulfide mineral processing for many decades (Chen et al., 2011). Serious environmental problems associated with flo￾tation reagents in mineral processing plant wastewater have been well documented (Chockalingam et al., 2003; Hissner et al., 1999). It is known that even small concentration of these reagents in water streams is toxic to water life, besides their deleterious influ￾ence on the end stream processes during recycling (Okibe and Johnson, 2002). However, investigations on the biodegradation of ethylthionocarbamate under anaerobic conditions have not been reported. Many compounds have been shown to be degraded by microor￾ganisms using various electron acceptors. With few exceptions, all BTEX compounds (Dou et al., 2008; Szykowny and Keasling, 1997), naphthalene (Dou et al., 2009) and dimethyl phthalate (Wu et al., 2007) have been shown to be degraded by microorganisms under nitrate reducing conditions. Studies also showed that several com￾pounds including BTEX (Hu et al., 2007; Dou et al., 2008), nonyl￾phenol polyethoxylates and alkanes (Lu et al., 2008) could be degraded under sulfate-reducing conditions. Furthermore, biodeg￾radation of BTEX (Dou et al., 2008), nonylphenol polyethoxylates (Lu et al., 2007) and other organic compounds with ferric as the electron acceptor has been elucidated. To develop anaerobic bioremediation technologies, systematic studies are required for understanding the biodegradation of ethyl￾thionocarbamate under anaerobic conditions. The aim of this study was to investigate the biodegradation of ethylthionocarbamate under various electron acceptor reducing conditions in the pres￾ence of mixed cultures enriched from the anaerobic digester sludge of a typical sewage treatment plant. And the utility of biodegrada￾tion kinetics process is also illustrated. 2. Methods 2.1. Microbial source The anaerobic digested sludge used as inoculum for the present research was obtained from the anaerobic digester of Ezhou Sewage Treatment Plant. 2.2. Culture medium and enrichment The electron acceptors used were nitrate (2.55 g/L NaNO3), sulfate (4.26 g/L Na2SO4) and ferric (402.78 mg/L Fe3+). Relatively high concentration of sulfate, nitrate and ferric were used to insure the development of sulfate, nitrate and ferric reducing conditions, respectively (Lu et al., 2007, 2008). In addition, all medium contained the following constituents: NH4Cl (1.5 g/L), KH2PO4 (0.6 g/L), MgCl26H2O (0.1 g/L), CaCl2 (0.1 g/L), yeast extract (1.0 g/L), vitamin (1%, v/v) and trace solutions (1%, v/v). Further￾more, the sulfate and ferric reducing medium were supplemented with NaHCO3 (2.52 g/L) and Na2S9H2O (0.5 g/L). Syringes and 0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.09.030 ⇑ Corresponding author. Tel.: +86 18986286480; fax: +86 27 87882128. E-mail address: gongwenqi@yahoo.com.cn (W. Gong). Bioresource Technology 102 (2011) 10772–10775 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

10773S.Chen etal./BioresourceTechnology102(2011)10772-10775needlesusedforsubstrateandsamplecollectionwereflushedwith30→ghigh purity nitrogen gas passed over hot reduced copper fillings toremove trace of oxygen. Serum bottles used in the experiments27were sealed with butyl rubber stoppers and aluminum caps.Enrich-mentwasconductedanaerobicallyina5o0mLserumbottlecon-24sterilecontroltaining 100 mL various-medium mixture and 100 mL sludgewithout electronacceptorsample.During enrichmentethylthionocarbamateconcentrationsulfate-reducing conditionwas gradually increased from 10 to 50 mg/Lat 12-day intervals un-nitrate-reduction conditionder incubation at 30°Cand 130 rpm.After2monthsthemixedbac-18under Fe(III)-reducing conditionterial consortia capable of biodegrading ethylthionocarbamatewere obtained.152.3.Experimental procedure12-VTheenriched bacteria was transferred into500mL serumbottleF49flled with 200 mL medium and yeast extract was omitted from the151824300391221276medium. And the enriched bacteria were added to get the initialtime(d)concentration of 1.0 g MLSS/L The various electron acceptors andethylthionocarbamate (30 mg/L) were added to each medium.Fig, 1. Anaerobic biodegradation of ethylthionocarbamate under various electronacceptor reducing conditions.Also, the initial solution pH was adjusted to 6.8 with 0.5 M HCI.All of thebottles wereflushed withhighpuritynitrogengasforing the period of the first 12 days,accounting for 93.43%,91.83%30min to maintain the anaerobic integrity of the system (Jonesand 97.79% of the total biodegradation, respectively. And moreand Ingle, 2005).The bottles were sealed with butyl rubber stop-than 66.43% of ethylthionocarbamatewere removed withinpers and aluminum caps. In order to account for abiotic ethylthio-12daysundertheconditionofferricreduction.nocarbamate degradation, control experiments containing noUnder these terminal electron acceptor conditions, the order ofelectron acceptor and no microorganisms were run in parallel.the electron acceptors with decreasing biodegradation rate of eth-All experiments were carried out in a thermostated water bathylthionocarbamate was: ferric > nitrate> sulfate, and the corre-with 130 rpm for 30 days at 30C.Thereafter,samples were with-sponding maximum biodegradation rate was 7.240, 6.267 anddrawn periodically collected to measure the concentration of eth-4.602 mg/(L-d), respectively. The degradation rate decreased shar-ylthionocarbamate, nitrate, nitrite, sulfate, sulfide, ferric andplywhenmostethylthionocarbamatewasremovedfromthesolu-ferrous iron. All the experiments were conducted in triplicate. Eachtion.Therefore,the results demonstrated that ferric was a moredatawasthemeanofthreemeasurements.favorable terminal electron acceptor compared to nitrate andsulfate.2.4. Analytical methods3.2.Variation of concentration ofnitrate,nitrite,sulfate,sulfide,ferricA pH meter (ORZON818, USA) was employed for measuring pHand ferrous during biodegradationvalues.Ethylthionocarbamate concentration was analyzed using aUV-vis spectrophotometer (Shimadzu,Japan).Ferrous and ferricThe variation of concentration of sulfate and sulfide, nitrate andironwereanalyzedbythe1.10-phenanthrolinemethodatawave-nitrite, ferric and ferrous concentrations is shown in Figs. 2 and 4,length of 510nm(Hu et al.,2007).Sulfateconcentration wasrespectively.A comparison between Figs.1 and 2 indicated that ni-measured through barium chromate spectrophotometry at a wave-tratereduction went hand-in-hand with ethylthionocarbamatelength of 420 nm.Sulfide concentration was determined by thedegradation, which indicated that ethylthionocarbamate degrada-methylene blue formation reaction in a spectrophotometer attion was coupled to nitrate reduction and was attributable to the665nm(Douetal.,2008).Nitrateandnitritewereanalyzedthroughactivity of the enriched bacteria. The accumulation of nitrite wasultraviolet spectrophotometricmethod usinga spectrophotometerfound during the reduction of nitrate, but the inhibitory effect on(Shimadzu, Japan) (ling et al., 2010).thedegradationofethylthionocarbamatewasnotobvious3. Results and discussion7322550283.1.Anaerobic degradation of ethylthionocarbamate under various2540electron acceptorreducingconditionsRFrom Fig. 1, in the absence of any terminal electron acceptornitrate2520and intheabioticcontrol experiments,thedecreaseofethylthioA-nitritenocarbamate was negligible over a period of30 days, showing that2510F12abioticprocesswas notplayingan important rolein ethylthionoc-osarbamate abatement from the solution. Under the condition with-82500-eout any terminal electron acceptor, there was no significant change-4in ethylthionocarbamate concentration. It is demonstrated that the2490disappearance of ethylthionocarbamate under various electronF0acceptorreducingconditionswasduetobiologicalprocess.How248001215182430392127ever, the enriched mixed bacteria could rapidly degrade ethylthio-ttime(d)nocarbamate without lag period under various electron acceptorreduction conditions.Underthe conditions of nitrate,sulfateandFig 2. Variation of concentration of nitrate and nitrite during anaerobic biodegferric reduction,most ethylthionocarbamate was biodegraded dur-radation of ethylthionocarbamate

needles used for substrate and sample collection were flushed with high purity nitrogen gas passed over hot reduced copper fillings to remove trace of oxygen. Serum bottles used in the experiments were sealed with butyl rubber stoppers and aluminum caps. Enrich￾ment was conducted anaerobically in a 500 mL serum bottle con￾taining 100 mL various-medium mixture and 100 mL sludge sample. During enrichment ethylthionocarbamate concentration was gradually increased from 10 to 50 mg/L at 12-day intervals un￾der incubation at 30 C and 130 rpm. After 2 months the mixed bac￾terial consortia capable of biodegrading ethylthionocarbamate were obtained. 2.3. Experimental procedure The enriched bacteria was transferred into 500 mL serum bottle filled with 200 mL medium and yeast extract was omitted from the medium. And the enriched bacteria were added to get the initial concentration of 1.0 g MLSS/L. The various electron acceptors and ethylthionocarbamate (30 mg/L) were added to each medium. Also, the initial solution pH was adjusted to 6.8 with 0.5 M HCl. All of the bottles were flushed with high purity nitrogen gas for 30 min to maintain the anaerobic integrity of the system (Jones and Ingle, 2005). The bottles were sealed with butyl rubber stop￾pers and aluminum caps. In order to account for abiotic ethylthio￾nocarbamate degradation, control experiments containing no electron acceptor and no microorganisms were run in parallel. All experiments were carried out in a thermostated water bath with 130 rpm for 30 days at 30 C. Thereafter, samples were with￾drawn periodically collected to measure the concentration of eth￾ylthionocarbamate, nitrate, nitrite, sulfate, sulfide, ferric and ferrous iron. All the experiments were conducted in triplicate. Each data was the mean of three measurements. 2.4. Analytical methods A pH meter (ORZ0N818, USA) was employed for measuring pH values. Ethylthionocarbamate concentration was analyzed using a UV–vis spectrophotometer (Shimadzu, Japan). Ferrous and ferric iron were analyzed by the 1,10-phenanthroline method at a wave￾length of 510 nm (Hu et al., 2007). Sulfate concentration was measured through barium chromate spectrophotometry at a wave￾length of 420 nm. Sulfide concentration was determined by the methylene blue formation reaction in a spectrophotometer at 665 nm (Dou et al., 2008). Nitrate and nitrite were analyzed through ultraviolet spectrophotometric method using a spectrophotometer (Shimadzu, Japan) (Jing et al., 2010). 3. Results and discussion 3.1. Anaerobic degradation of ethylthionocarbamate under various electron acceptor reducing conditions From Fig. 1, in the absence of any terminal electron acceptor and in the abiotic control experiments, the decrease of ethylthio￾nocarbamate was negligible over a period of 30 days, showing that abiotic process was not playing an important role in ethylthionoc￾arbamate abatement from the solution. Under the condition with￾out any terminal electron acceptor, there was no significant change in ethylthionocarbamate concentration. It is demonstrated that the disappearance of ethylthionocarbamate under various electron acceptor reducing conditions was due to biological process. How￾ever, the enriched mixed bacteria could rapidly degrade ethylthio￾nocarbamate without lag period under various electron acceptor reduction conditions. Under the conditions of nitrate, sulfate and ferric reduction, most ethylthionocarbamate was biodegraded dur￾ing the period of the first 12 days, accounting for 93.43%, 91.83% and 97.79% of the total biodegradation, respectively. And more than 66.43% of ethylthionocarbamate were removed within 12 days under the condition of ferric reduction. Under these terminal electron acceptor conditions, the order of the electron acceptors with decreasing biodegradation rate of eth￾ylthionocarbamate was: ferric > nitrate > sulfate, and the corre￾sponding maximum biodegradation rate was 7.240, 6.267 and 4.602 mg/(Ld), respectively. The degradation rate decreased shar￾ply when most ethylthionocarbamate was removed from the solu￾tion. Therefore, the results demonstrated that ferric was a more favorable terminal electron acceptor compared to nitrate and sulfate. 3.2. Variation of concentration of nitrate, nitrite, sulfate, sulfide, ferric and ferrous during biodegradation The variation of concentration of sulfate and sulfide, nitrate and nitrite, ferric and ferrous concentrations is shown in Figs. 2 and 4, respectively. A comparison between Figs. 1 and 2 indicated that ni￾trate reduction went hand-in-hand with ethylthionocarbamate degradation, which indicated that ethylthionocarbamate degrada￾tion was coupled to nitrate reduction and was attributable to the activity of the enriched bacteria. The accumulation of nitrite was found during the reduction of nitrate, but the inhibitory effect on the degradation of ethylthionocarbamate was not obvious Fig. 1. Anaerobic biodegradation of ethylthionocarbamate under various electron acceptor reducing conditions. Fig. 2. Variation of concentration of nitrate and nitrite during anaerobic biodeg￾radation of ethylthionocarbamate. S. Chen et al. / Bioresource Technology 102 (2011) 10772–10775 10773

10774S.Chenetal./BioresourceTechnology102(2011)10772-10775322504004260+2820024sulfideconcentration(mg/L)二F2016sulfateferric ironsulfideferrous iron12-8504Fo4210-F0150-301215182124270369036912151821242730time(d)time(d)Fig.3. Variation of concentration of sulfate and sulfide during anaerobic biodeg-Fig. 4.Variation of concentration of ferric and ferrous during anaerobic biodegra-radation ofethylthionocarbamatedation ofethylthionocarbamatethroughout the degradation even if the concentration of nitrite wasup to 30.67 mg/L.Table 1 indicated that when nitrate was a terminal electronAs can be seen from Figs.1 and 3, the samephenomena wereacceptor,the measured mass ratio between nitrate and ethylthio-observed under the condition of sulfate reduction.From Fig.3,itnocarbamate consumption was higher than the theoretical ratio,couldbe concluded that sulfideproduced was much lessthan sul-thereasonfor which is that the theoretical massratiowas calcu-fate consumed, the important reason for which may be that somelated by assuming nitrate was ultimately reduced to nitrogenof the sulfide was in the form of H,S, which could not be detectedgas. In fact nitrate was not completely transferred to nitrogenin the solution (Dou et al, 2008). Meanwhile, the accumulation ofgas, but part of them was accumulated as nitrite (Dou et al.,hydrogensulfidewasfoundduringthereductionofsulfate,andthe2009,2008).and the corresponding stoichiometric equation canresultisinagreementwith theassumption inprevious studies.be stated as follows:From Figs.1 and 3 it could be found that there was a good relation-shipbetweenethylthionocarbamatedegradationandthereductionof sulfate, and it could also be concluded that the accumulation of2(CHs);CHOCSNHCH,CH+ +33NO 12CO2+N2+33NO+11H,0+2H++252sulfide in the medium was not toxic to the enriched bacteria. The(4)reason may be that the mixed bacteria were enriched from theBased on the Eq. (4), the theoretical mass ratio between nitratemedium that contained sulfide about 66.62 mg/L (Dou et al., 2008)and ethylthionocarbamate consumption was 6.959,which wasAs shown in Fig. 4, ferrous iron also accumulated during thehigher than the corresponding measured value. From Table 1 itreduction of ferric iron. Because the presence of ferrous iron cou-couldbeobservedthatthemeasuredmassratiowas3.557.whichpled with the loss of ferric iron, and the theoretically expectedwashigherthanthetheoreticalvalueof2.784thatwasexpectedamount of ferrous iron produced in biotransformation processassumingthecompletereduction of nitrateto nitrogengas withcanbecalculatedaccordingtotheamountofferricironconsumed.complete ethylthionocarbamate mineralization,but was lowerThe amount of ferrous iron was 93.15% of that theoreticallythan thetheoretical valueof 6.959 thatwas calculated accordingexpected, which suggested the fact that the biodegradation of eth-to the assumption that nitrate was only reduced to nitrite. Fromylthionocarbamatewascoupledtoferricironreduction.Fig. 2 it could also be observed that the accumulation of nitritewas occurred,atthesametimetheproductionofnitritewaslower3.3.Stoichiometrybetween the consumption of electron acceptors andthan the consumption of nitrate,which further supported the factethylthionocarbamatedegradationthat only part of the nitrite was reduced to nitrogen gas.However, it could be observed that under sulfate reducing andGiventhattherewasno cellgrowth,thetheoretical stoichiomferric reducingcondition,themeasured massratiowasslightlyetric equations for anaerobic degradation ofethylthionocarbamatelower thanthe theoretical mass ratios,and the relationshipsto carbon dioxide with nitrate, sulfate and ferric as the terminalbetweenthemeasuredandtheoreticalratioforwas92.60%andelectronacceptorswereasfollows:90.78%, respectively. The reason for which may be that ethylthio-5(CH3)2CHOCSNHCH,CH3 + 33NO; + 23H*nocarbamatewasnotcompletelytransformedtocarbondioxideduring the whole degradations. Furthermore, another possible rea-→ 30C02 + 19N2 + 44H20 + 5s2-(1)son in part is that during this experiment the anaerobic oxidationof ethylthionocarbamate was also coupled to cultures growth and2(CH3), CHOCSNHCH2CHs + 11SO2converted into cell materials. 6C02 + N2 + 11H20 + 13s2- + 4H*(2)2(CH3)2CHOCSNHCH2CH3 + 68Fe3+ + 34H20Table 1Theoretical and measured mass ratios between electron acceptors and ethylthionoc-→ 12HCO + N2 + 68Fe2++ 82H++ S2(3)arbamateconsumption.Fe3NO5SoFBased on Eqs. ((1)-(3). the theoretical mass ratio of electronacceptor consumption and ethylthionocarbamate degradationTheoretical MeasuredTheoreticalMeasuredTheoretical Measuredcould beobtained,and themeasuredmassratio could bealso cal-2.7843.5573.5923.26812.95211.758culated, and is listed in Table 1

throughout the degradation even if the concentration of nitrite was up to 30.67 mg/L. As can be seen from Figs. 1 and 3, the same phenomena were observed under the condition of sulfate reduction. From Fig. 3, it could be concluded that sulfide produced was much less than sul￾fate consumed, the important reason for which may be that some of the sulfide was in the form of H2S, which could not be detected in the solution (Dou et al., 2008). Meanwhile, the accumulation of hydrogen sulfide was found during the reduction of sulfate, and the result is in agreement with the assumption in previous studies. From Figs. 1 and 3 it could be found that there was a good relation￾ship between ethylthionocarbamate degradation and the reduction of sulfate, and it could also be concluded that the accumulation of sulfide in the medium was not toxic to the enriched bacteria. The reason may be that the mixed bacteria were enriched from the medium that contained sulfide about 66.62 mg/L (Dou et al., 2008). As shown in Fig. 4, ferrous iron also accumulated during the reduction of ferric iron. Because the presence of ferrous iron cou￾pled with the loss of ferric iron, and the theoretically expected amount of ferrous iron produced in biotransformation process can be calculated according to the amount of ferric iron consumed. The amount of ferrous iron was 93.15% of that theoretically expected, which suggested the fact that the biodegradation of eth￾ylthionocarbamate was coupled to ferric iron reduction. 3.3. Stoichiometry between the consumption of electron acceptors and ethylthionocarbamate degradation Given that there was no cell growth, the theoretical stoichiom￾etric equations for anaerobic degradation of ethylthionocarbamate to carbon dioxide with nitrate, sulfate and ferric as the terminal electron acceptors were as follows: 5ðCH3Þ2CHOCSNHCH2CH3 þ 33NO 3 þ 23Hþ ! 30CO2 þ 19N2 þ 44H2O þ 5S2 ð1Þ 2ðCH3Þ2CHOCSNHCH2CH3 þ 11SO2 4 ! 6CO2 þ N2 þ 11H2O þ 13S2 þ 4Hþ ð2Þ 2ðCH3Þ2CHOCSNHCH2CH3 þ 68Fe3þ þ 34H2O ! 12HCO 3 þ N2 þ 68Fe2þ þ 82Hþ þ S2 ð3Þ Based on Eqs. ((1)–(3)), the theoretical mass ratio of electron acceptor consumption and ethylthionocarbamate degradation could be obtained, and the measured mass ratio could be also cal￾culated, and is listed in Table 1. Table 1 indicated that when nitrate was a terminal electron acceptor, the measured mass ratio between nitrate and ethylthio￾nocarbamate consumption was higher than the theoretical ratio, the reason for which is that the theoretical mass ratio was calcu￾lated by assuming nitrate was ultimately reduced to nitrogen gas. In fact nitrate was not completely transferred to nitrogen gas, but part of them was accumulated as nitrite (Dou et al., 2009, 2008), and the corresponding stoichiometric equation can be stated as follows: 2ðCH3Þ2CHOCSNHCH2CH3 þ33NO 3 ! 12CO2 þN2 þ33NO 2 þ11H2Oþ2Hþ þ2S2 ð4Þ Based on the Eq. (4), the theoretical mass ratio between nitrate and ethylthionocarbamate consumption was 6.959, which was higher than the corresponding measured value. From Table 1 it could be observed that the measured mass ratio was 3.557, which was higher than the theoretical value of 2.784 that was expected assuming the complete reduction of nitrate to nitrogen gas with complete ethylthionocarbamate mineralization, but was lower than the theoretical value of 6.959 that was calculated according to the assumption that nitrate was only reduced to nitrite. From Fig. 2 it could also be observed that the accumulation of nitrite was occurred, at the same time the production of nitrite was lower than the consumption of nitrate, which further supported the fact that only part of the nitrite was reduced to nitrogen gas. However, it could be observed that under sulfate reducing and ferric reducing condition, the measured mass ratio was slightly lower than the theoretical mass ratios, and the relationships between the measured and theoretical ratio for was 92.60% and 90.78%, respectively. The reason for which may be that ethylthio￾nocarbamate was not completely transformed to carbon dioxide during the whole degradations. Furthermore, another possible rea￾son in part is that during this experiment the anaerobic oxidation of ethylthionocarbamate was also coupled to cultures growth and converted into cell materials. Fig. 4. Variation of concentration of ferric and ferrous during anaerobic biodegra￾dation of ethylthionocarbamate. Fig. 3. Variation of concentration of sulfate and sulfide during anaerobic biodeg￾radation of ethylthionocarbamate. Table 1 Theoretical and measured mass ratios between electron acceptors and ethylthionoc￾arbamate consumption. NO 3 SO2 4 Fe3+ Theoretical Measured Theoretical Measured Theoretical Measured 2.784 3.557 3.592 3.268 12.952 11.758 10774 S. Chen et al. / Bioresource Technology 102 (2011) 10772–10775

10775S.Chen et al./Bioresource Technology 102 (2011) 10772-10775Table 2nocarbamate degradation was between the theoretical ratio thatKinetic parameters of biodegradation under various electron acceptor conditions.was calculated by assuming that nitrate was reduced to nitrogenR2DBoand nitrite.AConditionsSO,2-16.35096.871114.12460.9875AcknowledgementsN019.62316.212711.16990.99085.61278.77760.980221.8680Theauthorsaregratefulto thefinancial supportsof National"863"Plan Research Project (2007AA06Z123).IndependentInnovation Research Funds of Wuhan University of Technology3.4.Biodegradation kinetics model(2010-YB-16) and Hubei Key Laboratory of Pollutant Analysis andReuseTechnologyOpenFund Project (KY2010G19)The first order exponential decay equation was employed tocompare the biodegradation of ethylthionocarbamate under vari-Referencesouselectronacceptorconditionsandcanbeexpressedasfollows:Chen, S.H., Gong.W.Q., Mei, GJ. Zhou, Q. Bai, C.P., Xu, N., 2011.Primary) + BoC = A × exp(-Dbiodegradation of suifide mineral flotation collectors. Miner. Eng, 24 (8), 953-955.where, A is the decay intensity constant, D is decay index, t is theChockalingam, E.Subramanian, S.,Natarajan, K.A,2003. Studies on biodegradationof organic flotation collectors using Bacillus polymyxa. Hydrometallurgy 71 (1-reaction time and Bo is a constant. The calculated kinetic parame-2),249256.tersaresummarizedinTable2.Dou, J.F., Liu, X, Ding, AZ., 2009. Anaerobic degradation of naphthalene by theTable 2 shows that the first order exponential decay equationmixed bacteria under nitrate reducing conditions. J. Hazard.Mater.165,325331can accurately describe the biodegradation of ethylthionocarba-Dou, J.F., Liu, X, Hu, Z.F., Deng, D., 2008. Anaerobic BTEX biodegradation linked tomate under various electron acceptor conditions. The ordernitrate and sulfate reduction.J.Hazard,Mater.151 (2-3),720-729of the decay intensity constants of ethylthionocarbamate is:Hissner, F., Daus, B., Heinig. K, 1999. Determination of flotation reagents used intin-mining by capillary electrophoresis.J.Chromatogr.A 853 (1-2), 497-502.Are+ > Ano > Aso- these results are consistent with the discus-Hu,ZF.Dou.j.F.,Liu, X_, Zheng.XL.Deng. D_2007.Anaerobic biodegradation ofsion in the previous section. According to thermodynamicbenzene series compounds by mixed cultures based on optional electronicprinciples, the reaction could be released higher energy foracceptors. J. Environ. Sci. China 19 (9). 1049-1054.Jing. C., Ping. Z, Mahmood, Q., 2010. Influence of various nitrogenous electronoxidizing ethylthionocarbamate under ferric reducing condition,acceptors on the anaerobic sulfide oxidation.Bioresour.Technol. 101 (9),2931-followed by NO3- and SO.2-2937.Jones, B.D_ Ingle,J.D.,2005.Evaluation of redox indicators for determining sulfatereducing and dechlorinating conditions. Water Res. 39 (18),4343-43544.ConclusionsLu. J. Jin, Q, He, Y.L, 2007. Biodegradation of nonylphenol polyethoxylates underFe(l)-reducing conditions. Chemosphere 69 (7), 1047-1054.The enriched bacteria were capable of biodegrading ethylthio-Lu, J. Jin, Q. He, Y.L., Wu, J. Zhao, J. 2008. Biodegradation of nonylphenolpolyethoxylates under sulfate-reducing conditions.Sci. Total Environ, 399nocarbamate without a lag phase under nitrate, sulfate and ferric(13), 121127reducing conditions, and theirbiodegradationprocesses fitted wellOkibe, N_ Johnson, D.B., 2002. Toxicity of flotation reagents to moderatelywith the first order exponential decay kinetics equation. Ferric wasthermophilic bioleaching microorganisms. Biotechnol. Lett.24 (23),2011-2016.Szykowny, D., Keasling. J.D., 1997. Kinetics of BTEX degradation by a nitrate-a more favorable terminal electron acceptor compared to nitratereducing mixed culture. Ann. NY Acad. Sci. 829, 135-141.and sulfate. The order of the electron acceptors with decreasingWu, D.L, Hu, B,L,Zheng, P.Mahmood, Q.,2007.Anoxic biodegradation of dimethylbiodegradation rate of ethylthionocarbamate was ferric>nitrate>phthalate (DMP)byactivatedsludgecultures under nitrate-reducingsulfate.Themeasured massratio of nitratereduction toethylthio-conditions.J.Environ.Sci.19,1252-1256

3.4. Biodegradation kinetics model The first order exponential decay equation was employed to compare the biodegradation of ethylthionocarbamate under vari￾ous electron acceptor conditions and can be expressed as follows: Ct ¼ A  expð t DÞ þ B0 where, A is the decay intensity constant, D is decay index, t is the reaction time and B0 is a constant. The calculated kinetic parame￾ters are summarized in Table 2. Table 2 shows that the first order exponential decay equation can accurately describe the biodegradation of ethylthionocarba￾mate under various electron acceptor conditions. The order of the decay intensity constants of ethylthionocarbamate is: AFe3þ > ANO 3 > ASO2 4 , these results are consistent with the discus￾sion in the previous section. According to thermodynamic principles, the reaction could be released higher energy for oxidizing ethylthionocarbamate under ferric reducing condition, followed by NO3 and SO4 2. 4. Conclusions The enriched bacteria were capable of biodegrading ethylthio￾nocarbamate without a lag phase under nitrate, sulfate and ferric reducing conditions, and their biodegradation processes fitted well with the first order exponential decay kinetics equation. Ferric was a more favorable terminal electron acceptor compared to nitrate and sulfate. The order of the electron acceptors with decreasing biodegradation rate of ethylthionocarbamate was ferric > nitrate > sulfate. The measured mass ratio of nitrate reduction to ethylthio￾nocarbamate degradation was between the theoretical ratio that was calculated by assuming that nitrate was reduced to nitrogen and nitrite. Acknowledgements The authors are grateful to the financial supports of National ‘‘863’’ Plan Research Project (2007AA06Z123), Independent Innovation Research Funds of Wuhan University of Technology (2010-YB-16) and Hubei Key Laboratory of Pollutant Analysis and Reuse Technology Open Fund Project (KY 2010G19). References Chen, S.H., Gong, W.Q., Mei, G.J., Zhou, Q., Bai, C.P., Xu, N., 2011. Primary biodegradation of sulfide mineral flotation collectors. Miner. Eng. 24 (8), 953– 955. Chockalingam, E., Subramanian, S., Natarajan, K.A., 2003. Studies on biodegradation of organic flotation collectors using Bacillus polymyxa. Hydrometallurgy 71 (1– 2), 249–256. Dou, J.F., Liu, X., Ding, A.Z., 2009. Anaerobic degradation of naphthalene by the mixed bacteria under nitrate reducing conditions. J. Hazard. Mater. 165, 325– 331. Dou, J.F., Liu, X., Hu, Z.F., Deng, D., 2008. Anaerobic BTEX biodegradation linked to nitrate and sulfate reduction. J. Hazard. Mater. 151 (2–3), 720–729. Hissner, F., Daus, B., Heinig, K., 1999. Determination of flotation reagents used in tin-mining by capillary electrophoresis. J. Chromatogr. A 853 (1–2), 497–502. Hu, Z.F., Dou, J.F., Liu, X., Zheng, X.L., Deng, D., 2007. Anaerobic biodegradation of benzene series compounds by mixed cultures based on optional electronic acceptors. J. Environ. Sci. China 19 (9), 1049–1054. Jing, C., Ping, Z., Mahmood, Q., 2010. Influence of various nitrogenous electron acceptors on the anaerobic sulfide oxidation. Bioresour. Technol. 101 (9), 2931– 2937. Jones, B.D., Ingle, J.D., 2005. Evaluation of redox indicators for determining sulfate￾reducing and dechlorinating conditions. Water Res. 39 (18), 4343–4354. Lu, J., Jin, Q., He, Y.L., 2007. Biodegradation of nonylphenol polyethoxylates under Fe(III)-reducing conditions. Chemosphere 69 (7), 1047–1054. Lu, J., Jin, Q., He, Y.L., Wu, J., Zhao, J., 2008. Biodegradation of nonylphenol polyethoxylates under sulfate-reducing conditions. Sci. Total Environ. 399 (1–3), 121–127. Okibe, N., Johnson, D.B., 2002. Toxicity of flotation reagents to moderately thermophilic bioleaching microorganisms. Biotechnol. Lett. 24 (23), 2011–2016. Szykowny, D., Keasling, J.D., 1997. Kinetics of BTEX degradation by a nitrate￾reducing mixed culture. Ann. NY Acad. Sci. 829, 135–141. Wu, D.L., Hu, B.L., Zheng, P., Mahmood, Q., 2007. Anoxic biodegradation of dimethyl phthalate (DMP) by activated sludge cultures under nitrate-reducing conditions. J. Environ. Sci. 19, 1252–1256. Table 2 Kinetic parameters of biodegradation under various electron acceptor conditions. Conditions A DB0 R2 SO4 2 16.3509 6.8711 14.1246 0.9875 NO3 19.6231 6.2127 11.1699 0.9908 Fe3+ 21.8680 5.6127 8.7776 0.9802 S. Chen et al. / Bioresource Technology 102 (2011) 10772–10775 10775