Biodegradation of Tetrachlorothylene Using Methanol as Co-metabolic Substrate

BIOMEDICAL AND ENVIRONMENTAL SCIENCES 21, 98-102 (2008)www.besjoumal.comBiodegradation of Tetrachlorothylene Using Methanol asCo-metabolic Substrate'QI YANG**2, HAI-TAO SHANG", Hur-DILI , HONG-Bo xI*, AND JIAN-LONG WANG*"School of Water Resources and Environment, China University of Geoscience, Bejing 100083, China;"Laboratory of Geomicrobiology, China University of Geoscience, Beijing 100083, China; *Laboratoryof Environmental Technology INET, Tsinghua University, Beijing 10084, ChinaObjective To investigate the biodegradation of tetracbloroethylene (PCE) using methanol as electron donor byacclimated anaerobic sludge. Methods HP- 6890 gas chromatograph (GC), together with HP-7694 autosampler, waused to analyze the concentration of PCE and intermediates. Results PCE could be decholrinated reductively to DCEvia TCE, and probably further to VC and ethylene. The degradation of PCE and TCE conformed to first-order reactionkinetics. The reaction rate constants were 0.8991 d' and 0.068 d', respectively, and the corresponding half.life were 0.77dand 10.19 d, respectively. TCE production rate constant was 0.1333 d', sbowing that PCE was degraded more rapidly thanTCE. Conclusion Methanol is an electron donor suitable for PCE degradation and the cometabolic electron donors arenot limiting factors for PCE degradation.Key words: Tetrachloroethylene; Anaerobic cometabolism; BiodegradationINTRODUCTIONsubstrate. Furthermore under some conditions, PCEcan be degraded into non-chlorinated end productsPCE (tetrachloroethylene), a priority. pollutantsuch as ethylenebsl and ethane that are hamless tolisted by US EPA, is a chlorinated solvent'", which isenvironment.The objective of the study was to investigate thewidely used as degreasing agent and dry cleaningdegradation of PCE under anaerobic condition usingsolvent, and it is also a suspected carcinogen'2. Duemethanol as co-metabolic substrate.to unreasonable disposal and leakage, PCE hasbecome one. of the most common organiccontaminants'. The methods used for PCE removalMATERIALS AND METHODSinclude physical, chemical, and biological procedures.PCE can transfer from a place into another place ofChemicalspolluted zone through physical process, but it cannotPCE was analytical grade purchased frombe removed ultimately from environment5!. AlthoughBeijing Chemical Company, Ltd. Other chemicalsPCE removal efficiency is high by chemical method,were chemical grade and used as received.the cost is too high'*. Biological method is of interestbecause of its simple operation and low cost. ManyMediumstudies have shown that PCE is recalcitrant to aerobiccondition'5S, but can be degraded via reductiveInorganic salt medium consisting of (in g:Ll)dechlorination under anaerobic condition. When PCENH4C: 0.32, KH2PO: 0.063, CaCl2: 0.063, Na2CO3:is an only carbon source, it cannot be degraded"or is0.1316, 1 mL trace element stock solution. The stockdegraded slowlyl8. Therefore many studies have beensolution of trace element containing (in mgL)carried out under co-metabolic conditions. ManyMgSO4 7H2O: 5000, FeCl2:4H2O: 6000, CoCl2:6H2O: .researchersl-1]1 reported that PCE degradation is1036.88，H;BO3:100， ZnSO47H2O: 100,much more rapid using methanol as co-metabolicCuSO45H2O: 50, NiSO46H2O: 879.47, MnCl24H2O:'This work was supported from the National Natural Science Foundation of China (No. 40102027; 50578151) and the Natural ScienceFoundation of Beijing (No. 8052017). The School-enterprise cooperation project of Beijing Municipal Education Commission (No.5190065005).Correspondence should be adressed to Qi YANG Te: 86- 10-82320921. Fax: 86 10-82321081. E-mail: voa@cub.edu.cnBiographical note of the first author. Qi YANG male, bom in 1966, profesor中国煤化工LhinUnversiyof Geosciences (eiing), majoring in eaviromental bioremediation and wastewaYHCNMHGCopynght日2008 by China CDC9BIODEGRADATION OF TETRACHLOROTHYLENE995000, NH4)sMo)O24H2O: 640.Gas btleAir pipe nExperimental ProceduresSludge cultivationAnacrobic sludge wascollected from the UASB reactor in FengtaiWastewater Treatment Plant in Beijing, After- Sampling portanaerobic sludge was taken, it was incubated in 10L冬r -Water bathglass bottle at constant temperature of 35C, usingglucose as carbon source. Two g:L" glucose and 0.5t Anacrobic sludge 冬mL trace element were put into 1L inorganic saltmedium. Hydraulic retention time (HRT) was 5777777?7????7707?0?77???days.Fa 1. Schematic diagram of experimental apparatus.Sludge acclimationIntermittent mode wasused in sludge acclimation. Four hundred mMethods. ss and Vss analysis were performed byincubated anaerobic sludge and 250 mL inorganic saltweighing after oven drying. Methane and other gasesmedium were added into 700 mL glass botle withwere collected with fermentation tube.n HP-6890 gas chromatograph (GC) withsampling site in side and Teflon rubber stopperHP-7694 autosampler was used. The concentration ofequipped with gas-conducting pipe. PCE wasPCE was determined by the head-space method usingdissolved in methanol and added into reactor, whichwas stoppered quickly. Acclimation plan is shown ingas chromatography. The inlet temperature was set atTable 1. Acclimation was performed at the160C; chromatographic column with a flow rate of1.0 mL:min"; the initial temperature in GC oven attemperature of 35"C in a water bath (Fig.1).70"C was maintained for 10 min; the ECDTABLE 1temperature was controlled at 300C; in headspace,glass temperature was 50'C and loop 60"C; thePlan of Sludge Acclimation Using Methanol as Co-metabolic Substrateequilibrium time was 10 min; injection time wasAcclimation time(d) 1 5 12 20 30 37 441.00 min. Nitrogen was used as carrier gas at a rate ofPCE (mgL)about 30 mL:min' (GB11890-89, 1996). The limit ofdetection for PCE was 0.05 μg:L'.PCE degradationInorganic salt medium andPCE stock solution were put into the reactor inRESULTSsequence. Chlorinated hydrocarbons were measuredAcclimation of Anaerobic Sludgeafter being stabilized 20 min. PCE and its degradativeproducts were measured.The sample of anaerobic sludge was ollctedAnalytical Methodsand incubated for about 20 days; then the biologicalactivities were measured after glucose, inorganic salts,COD analysis was performed in accordance withand trace elements were added. The results are shownthe dichromate reflux method described in Standardin Table 2.TABLE2Biological Activity of Anaerobic SludgeCODSS(g:L) VSS(g:L) VSs/SS (%)Methane ProducedInfueat (mgL ) Effluent (mgL) Removal Eficiency (%)(mL CHv(g VSSd)y')69.553.4542.83182288.78During the acclimation period the PCE wasaclimated anaerobic sludge possessed the capacityadded according to Table 1. The concentrations ofof PCE degradation. Figure 3 reveals the possiblePCE and its degradative products, such as TCE andintermediates accumulation during PCE degradation.DCE, were analyzed. The results are depicted in Figs.The variation of TCE and DCE concentrations with2 and 3.time showed that TCE began to accumulate at theFigure 2 shows that PCE removal efficiency12th dreached more than 92% after 10 days' acclimation.PCEL中国煤化工After 30 days, the PCE removal efficiency waalmost 100% and kept stable, which revealed that theTYHCN M H Ge dgadaio0oYANG ETAL.simultaneously with the consumption of methanol.10Furthermore, biogas was generated through all the0fexperiment. The relationship betweenPCEdegradation, methanol consumption, and gas昌60production using methanol as the sole substrate and40co-substrate is ilustrated in Fig. 5.120000PCE(ug/L)Methanol (sole substrate, mg/L)1 300Methanol (co-substrate, mgL)800Sole substrate (mL)Acclimation time (d)Co-substrate (mL)600FIG 2. PCE removal eficiency in acclimation.4001000.20003of曾+ TCETime (d)E 0.十DCEPIG 5. Relationship between PCE removal,methanol consumption, and gas volumeg 0.(methanol as the sole substrate andco-substrate).50 60Degradation kineticsThrough regressionanalysis, the relationship between ln(c/co) and timeon PCE is shown in Fig. 6. As seen from Fig. 6, theFIa 3. Degradation products of PCE inPCE degradation eguation was as follows:acclimation.c=1395.6eD-8991 with R2 equal to 0.9833 and n equalwas investigated by using acclimated anaerobic sludge.to 7, which conformed to the first- order kinetics.The concentration of PCE and its degradative productsReaction rate constant is 0.8991 d"', half life 0.77 d.were measured and the results are depicted in Fig.4.TCE was accumulated at the beginning of thexperiment. TCE reached maximum at the fifth day,which was 600 mg:L"， then decreased. Therelationship between ln (c/cmax) and time for TCE-+PCE--TCE士-DCEs00 Fproduction during PCE degradation and TCEdegradation is depicted in Figs. 7 and 8. The equationof TCE production was c=338.6.1333 (n=S,R'=0.9219), which accorded with first- order kinetics.The rate constant was 0.1333 d'. The equation ofTCE degradation was c=607.1ex. (n=3,R2=0.9791). The rate constant was 0.068 d', half life10.19 d. According to these results, it can beFIa 4. Relation between liquid PCE, degrationconcluded that degradation of PCE and TCEproducts, and intial PCE concentration.accumulation conform to the first-order kinetics. Therate constant of PCE degradation was much higherFigure 4 shows that TCE accumulation occurredthan that of TCE production. The result wasas soon as PCE degradation started. There was no lagconsistent with that of Valter experiment2. TCEphase for PCE degradation. The PCE removalaccumulation rate was almost two times higher thaneficiency reached over 95% at the third day and 900TCE degradation rate, which indicated that TCEμg:L' PCE could be completely degraded after 6production amount was much greater than that ofdays. The accumulation of TCE increased at theTCE degradation, so TCE appeared through all thebeginning, but decreased slightly from the sixth day.experiment. The half life of PCE degradation wasDCEs were detected at the third day, but there was aless than 1 day, but that of TCE was 10.19 day.relatively slow increase.Furth中国煤化工hase for TCERelationshipbetweenaccu-rate ofPCE wasmethanol consumption, and gas production In6.20iYHCNMHGnovalofPCEinthis study, anaerobic removal of PCE occurredthe anaerobic sludge was 143 μg (gVSS-L)-.BIODEGRADATION OF TETRACHLOROTHYLENE100大Others might be transformned into VC, ethylene, CH4,and CO2. In this study, the other two degradativec=1395. 6e-09.intermediate products, VC and ethylene, were not。-2R=0.9833detected simultaneously. Much information indicatedthat VC transformation rate was slower than that ofgother chlorinated etbylene. Sometimes VC was 3orders of magnitude lower in transformation rate thanPCEl2?! vC transformation was limiting.ratese/l612131.Most of PCE was removed at the thrd day, whileTime ()the volume of gas production and methanolFIG 6. Regression curve of In (CIC.) andconsumption also reached maximum. From then on,time on PCE.gas was produced slowly, and methanol was almostconsumed completely. The rate of sole methanol04rdegradation was higher and the volume of the CH4was larger by using methanol as co -substrate. Fromgas production and methanol consumption it can be0.0 texplained that most of PCE removal appeared at theinitial experiment. In following days PCE removalwas stable, which might be due to methanol-0.4-c-338.96el3exhaustion. That is to say, with methanolR=0.9291consumption, most PCE had been removed. InThomas' experiment"3), degradation of 3.5 μmol!L;1-0.8PCE needed 320 μmolL1 methanol, but in ourTime (d)experiment, degradation of 8.4 umol:L' PCE neededFIG7. Regression curve of In (C/Cmx) and19.08 mmolL' methanol. In his study, the quantitytime during TCE production.of methanol needed was also much larger than that inFederico's studys'. It might be due to the presence of0.00microorganisms that readily consumed methanol inthe sludge.-0.05c= 607.1-08R20.9791CONCLUSIONS三-0.10-The following conclusions can be drawn fromthe present laboratory study:(1) Acclimation of anaerobic sludge showed that-0.15.12anaerobic sludge can accommodate PCE quickly anddegrade it.Time(d)(2) PCE is degraded via reductive dechlorination,FiG 8. Regression curve of In (CICmx) andand the degradative products may be DCEs, VC ortime during TCE degration.ethylene when methanol is supplied as co-metabolicsubstrate.DISCUSSION(3) The degradation of PCE and theaccumulation of TCE can be described by first-orderBioremediation is becoming an increasinglykinetic equation and the reaction rate constants arepopular technology for treatment of contaminated0.8991 dI and 0.068 d', respectively.groundwater. Much published work has so farfocused largely on kinetics of anaerobic sludge. PCEREFERENCESdegradation may be atributed to the reductivedechlorination under methanogenic conditions, i.e. a1. Babu Z F Stephen A B (1988). Dependence ofchlorine atom in PCE was replaced by a hydrogentetrachloroethyleae dechlorination on methanogenic substrateatom. Our results were consistent with those reportedcop中国煤化工-CM. App Environby other researchers3sl. In our experiment, initial2. 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