- Email: [email protected]
Biological removal of the xenobiotic trichloroethylene (TCE) through cometabolism in nitrifying systems
Bioresource Technology 101 (2010) 430–433
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biological removal of the xenobiotic trichloroethylene (TCE) through cometabolism in nitrifying systems B. Alpaslan Kocamemi a,*, F. Çeçen b a b
Department of Environmental Engineering, Faculty of Engineering, University of Marmara, Kuyubasi, Istanbul, Turkey Institute of Environmental Sciences, University of Bogazici, Bebek, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 31 March 2009 Received in revised form 13 July 2009 Accepted 17 July 2009 Available online 2 September 2009 Keywords: Activated sludge Cometabolism Nitrification Trichloroethylene Xenobiotics
a b s t r a c t In the present study, cometabolic TCE degradation was evaluated using NH4–N as the growth-substrate. At initial TCE concentrations up to 845 lg/L, TCE degradation followed first-order kinetics. The increase in ammonium utilization rate favored the degradation of TCE. This ensured that biological transformation of TCE in nitrifying systems is accomplished through a cometabolic pathway by the catalysis of non-specific ammonia oxygenase enzyme of nitrifiers. The transformation yield (Ty) of TCE, the amount of TCE degraded per unit mass of NH4–N, strongly depended on the initial NH4–N and TCE concentrations. In order to allow a rough estimation of TCE removal and nitrification at different influent TCE and NH4–N concentrations, a linear relationship was developed between 1/Ty and the initial NH4–N/TCE ratio. The estimated Ty values lead to the conclusion that nitrifying systems are promising candidates for biological removal of TCE through cometabolism. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Trichloroethylene (TCE) is a xenobiotic compound that is mainly used as degreaser and solvent in automotive, metal, electroplating, textile and paper industries. The large-scale use of TCE in various industries over the past years has led to widespread distribution of this toxic and carcinogenic compound in the environment, especially in groundwater media. TCE can also be monitored in surface media. But, the highly volatile characteristics usually results in quick evaporation in open surface water bodies. Considerable effort has been focused on removal of TCE by biological processes. To date, no isolates or enrichment cultures utilizing TCE as growth-substrate under aerobic conditions have yet been obtained (Bradley, 2003; Field and Sierra-Alvarez, 2004). But, some aerobic bacterial cultures containing non-specific oxygenase enzymes (e.g., methane, toluene, phenol, propane and ammonia oxidizers) were identified capable to degrade TCE by cometabolism (Arciero et al., 1989; Chang and Alvarez-Cohen, 1995; Chu and Alvarez-Cohen, 2000; Han et al., 2007; Shukla et al., 2009). In this process, biotransformation of a non-growth compound (e.g., TCE) is accomplished concurrently with metabolism of a growth-supporting substrate through the catalysis of non-specific oxygenase enzymes. The organism obtains no benefit from cometabolism of the non-growth-supporting substrate. * Corresponding author. Tel.: +90 216 3480292x270; fax: +90 216 3481369. E-mail addresses: [email protected], [email protected] (B.A. Kocamemi). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.07.079
Moreover, the presence of the non-growth-supporting substrate may inhibit the metabolism of the growth-supporting substrate, thereby decreasing or preventing bacterial growth (Ely et al., 1997). In addition, cometabolism of the non-growth-supporting substrate consumes reductant NAD(P)H, which can only be regenerated from growth-supporting substrate degradation. Therefore, in this process, a growth-supporting substrate must be available (at least periodically) to grow new cells, provide reductant and induce production of non-specific enzymes (Alvarez-Cohen and Speitel, 2001). Among the organisms capable to degrade TCE cometabolically, this study focused on ammonia oxidizers (nitrifiers). In literature, the studies evaluating the cometabolism of TCE by nitrifying species (Arciero et al., 1989; Rasche et al., 1991; Ely et al., 1995, 1997; Hyman et al., 1995; Yang et al., 1999) are very limited compared to those performed with other organisms (e.g., methanotrophs). Nitrification studies up to date were mainly performed with pure Nitrosomonas europaea cultures. In our previous studies, using a mixed culture enriched in terms of nitrifying bacteria, we first evaluated the inhibitory effect of TCE on nitrification during cometabolic degradation for a broad TCE range and at a fixed growth-supporting substrate (NH4–N) concentration (Alpaslan Kocamemi and Çeçen, 2005). Later (Alpaslan Kocamemi and Çeçen, 2007), we focused on the kinetics of the inhibitory effect of TCE on nitrification, which directly influences the sustainability of TCE degradation in nitrifying systems. The inhibition type and inhibition coefficient of TCE were determined for a broad range of NH4–N and TCE.
431
B.A. Kocamemi, F. Çeçen / Bioresource Technology 101 (2010) 430–433
In the present study, which is complementary to our previous study (Alpaslan Kocamemi and Çeçen, 2007), cometabolic TCE degradation was evaluated by a mixed culture enriched for nitrifiers. A batch suspended-growth system was fed with NH4–N as the growth-supporting substrate. The dependence of the process on various factors, such as the NH4–N utilization rate and the relative initial ratio of NH4–N and TCE, was quantitatively analyzed. 2. Methods
Table 1 The estimated first-order TCE degradation constant at various NH4–N levels. Initial NH4–N concentration (mg/L)
First-order TCE degradation rate constant (L/g VSS h)
Goodness-of-fit of linear regression (R2)
25 50 100 200 400
0.26 0.31 0.48 0.73 1.03
0.98 0.94 0.95 0.95 0.93
2.1. Mixed culture enriched for nitrifiers
2.2. Experimental procedure Four sets of experiments were performed in 200 mL capped glass bottles at a constant wastewater temperature of 25 °C and pH range of 7–8. In each set, five runs were performed at constant TCE, but at varying initial NH4–N (25–400 mg/L). The initial TCE was kept at 40, 110, 325, 845 lg/L in the first, second, third and fourth set of experiments. In all experiments, the stock synthetic NH4–N solution [37.75 g/L (NH4)2SO4 and 95 g/L NaHCO3] and the stock TCE solution [500 mg/L TCE] were used in diluted form to adjust the desired NH4–N and TCE concentrations. The stock mineral solution [2 g/L MgSO47H2O, 0.1 g/L CaCO3, 0.4 g/L FeSO47H2O, 0.2 g/L MnSO4H2O and 0.3 g/L K2HPO4] was diluted 40 folds. In each experiment, the stock culture was rinsed thoroughly with water and diluted to MLVSS ranging between 210 and 530 mg/L. Experiments were started under oxygen-supersaturated conditions (initial DO of 35–40 mg/L) and ended before the DO in the test bottle dropped below 4 mg/L. Continuous diffused aeration was avoided to prevent the possible risk of TCE volatilization. Complete mixing in the test bottles was maintained by magnetic stirring. Blank experiments were performed to evaluate the abiotic loss of TCE resulting from stirring action. These experiments demonstrated a negligible TCE exchange rate of 0.0028 lg/L/min per initial TCE (lg/L) (data not shown, Alpaslan Kocamemi, 2005). In all runs, samples were analyzed with respect to time for NHþ 4 —N with the Nessler Method (APHA, 1998) using Hach DR/2000 spectrophotometer and for TCE with HP 5890 Gas Chromatograph equipped with an electron capture detector. The measured TCE concentrations were corrected by the abiotic TCE loss rate. The specific ammonium utilization rate (qNH4 —N ) and the specific cometabolic TCE degradation rates (qTCE) were then calculated from the slopes of NH4–N/VSS versus time and TCE/VSS versus time plottings through linear regression analysis, respectively.
monas species. Photomicrographs of these analyses were shown in a former paper (Alpaslan Kocamemi and Çeçen, 2007). Further, as shown by DGGE analyses (Alpaslan Kocamemi and Çeçen, 2007), no changes had occurred in the diversity of amoA gene sequences during the period of about 7 months after enrichment. Since a shift in microbial population was not the case, the rates in the present study are not expected to change during the experimental period. Evaluation of the cometabolic degradation rate of TCE (qTCE) with respect to initial TCE concentrations (Alpaslan Kocamemi, 2005, data not shown) showed that at each initial NH4–N concentration studied, qTCE increased linearly with TCE concentration. The first-order cometabolic TCE degradation rate constants obtained from the linearization of the graphs are summarized in Table 1. The cometabolic TCE degradation followed first-order kinetics because the bulk TCE concentrations (0–825 lg/L) were very small. If higher bulk TCE concentrations were present, TCE degradation rate would most probably reach the saturation level. However, it is very likely that also in real remediation systems, such as groundwater, TCE degradation follows first-order kinetics in the concentration ranges below 1000 lg/L. The first-order TCE degradation rate constants in the present study are considerably lower in comparison to those reported for pure N. europaea species as 30.8 L/g VSS h and 42.5 L/gVSS h for the initial TCE concentrations of 0–3300 and 2100 lg/L, respectively (Alvarez-Cohen and Speitel, 2001). However, in those studies performed with pure cultures, the culture consisted of pure nitrifier species only and the reported values based on the assumption that the dry cell mass consisted of 50% protein (Alvarez-Cohen and Speitel, 2001). On the other hand, in the present study, the mixed culture still contains cells other than nitrifiers although it was enriched for nitrifiers. Therefore, the values expressed on VSS basis are quite lower compared to those reported in pure culture studies.
1050 900
Cometabolic degradation rate of TCE (µg TCE /gVSS.h)
The mixed culture was initially taken from the Pasakoy Advanced Biological Sewage Treatment Plant in Istanbul. The culture was then enriched for nitrifiers as described previously (Alpaslan Kocamemi and Çeçen, 2005). After the enrichment period, this mixed culture was continuously fed with NH4–N and mineral solutions only based on the fill-and-draw principle. The sludge samples taken from the stock enriched nitrifier culture were analyzed by Florescent In-Situ Hybridization (FISH) to ensure the enrichment of the mixed culture for nitrifiers. Denaturing gradient gel electrophoresis (DGGE) analyses were also performed to observe whether any changes occurred in the diversity of amoA gene sequences 7 months after the enrichment period. The procedures of FISH and DGGE analyses were as described previously (Alpaslan Kocamemi and Çeçen, 2009).
750
40 ppb TCE 110 ppb TCE
600
325 ppb TCE 450 845 ppb TCE 300 150 0 0
3. Results and discussions The FISH analysis of the stock culture revealed that the dominant members of the microbial community consisted of Nitroso-
10
20
30
40
50
60
NH4-N utilization rate (mg NH 4-N/gVSS.h) Fig. 1. Cometabolic degradation rates of TCE (qTCE) with respect to the NH4–N utilization rates (qNH4 —N ).
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B.A. Kocamemi, F. Çeçen / Bioresource Technology 101 (2010) 430–433
Table 2 Estimated transformation yields (Ty) for TCE. Culture
Initial TCE concentration (lg/L)
Ty
Reference
lgcometabolic substrateðTCEÞ mg growth-substrate ðNH4 —NÞ
Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed
culture enriched for nitrifiers culture enriched for nitrifiers culture enriched for nitrifiers culture enriched for nitrifiers methanotrophic culture culture of toluene degraders culture of phenol degraders culture of propane degraders
40 110 325 845 1000 30,000 20,000 16,000
1.4 2.3 3.3 24.4 16 2.5 1.9 11.9
The degradation rate of TCE (qTCE) was further expressed in dependence of ammonium utilization rates (qNH4 —N ) as shown in Fig. 1. At each initial TCE concentration, this rate exhibited an increase with ammonium utilization rates, which ensured the cometabolic removal of TCE by nitrifier species. The increases in qTCE at increased qNH4 —N values may be most probably related with the regeneration of the NAD(P)H (reductant), the potentially limiting reactant of cometabolic processes, by the growth-supporting substrate NH4–N. In our previous study (Alpaslan Kocamemi and Çeçen, 2007), TCE was found to be a competitive inhibitor of ammonia oxidation, which means that both TCE and NH4–N compete for the same active site of the enzyme ammonia monooxygenase (AMO). Therefore, a decrease in TCE degradation rate may be expected at higher ammonium utilization rates or vice versa. In consistence with this hypothesis, in our previous study (Alpaslan Kocamemi and Çeçen, 2007), it was shown that increase in TCE concentration resulted in a relative decrease in qNH4 —N (i.e., inhibition of nitrification). But, in the present study, NH4–N exhibited a positive effect on TCE degradation at even higher NH4–N concentrations. This may be related to the considerably greater affinity of TCE for the enzyme AMO compared to ammonia as reported before (Alpaslan Kocamemi and Çeçen, 2007). In order to assess the cometabolic TCE degradation potential of the culture, the transformation yields (Ty) of TCE, which is the mass of TCE degraded per unit mass of NH4–N consumed, were estimated by dividing the maximum qTCE values shown in Fig. 1 by the corresponding qNH4 —N . Those values are reported in Table 2 and compared with other cultures in various studies. As seen from Table 2, Ty values determined in the present study were variable depending on the TCE concentration. The minimum amount of NH4–N required for the degradation of 1 lg TCE at the maximum rate was found as 0.7 mg NH4–N at 40 lg/L TCE, 0.4 mg NH4–N at 110 lg/L TCE, 0.3 mg NH4–N at 325 lg/L TCE and 0.04 mg NH4–N at 845 lg/L TCE. Ty values for other organisms reported in Table 2 are not directly comparable with those determined in the present study because they were determined at higher initial TCE concentrations. However, the order of magnitude of Ty values in the present study are quite comparable to those reported in other studies using other cultures. This indicates that nitrifying systems using NH4–N as the growth-supporting substrate have a quite high TCE degradation capacity. The evaluation of qNH4 —N /qTCE (1/Ty) values against the initial NH4–N/TCE values (data not shown, Alpaslan Kocamemi, 2005) indicated that in the studied NH4–N (25–400 mg/L) and TCE (40– 845 lg/L) concentration ranges, up to an NH4–N/TCE ratio of 4800 a strong linear relationship existed between qNH4 —N /qTCE (1/ Ty) and the initial NH4–N/TCE ratio. As shown in Eq. (1) the constant belonging to this relationship was found as 0.2651:
1=T y ¼ 0:2651ðNH4 —N=TCEÞ
ð1Þ
Eq. (1) can be a useful tool in the prediction of Ty that can be achieved at different NH4–N and TCE concentrations.
This study This study This study This study Alvarez-Cohen Alvarez-Cohen Alvarez-Cohen Alvarez-Cohen
and and and and
Speitel Speitel Speitel Speitel
(2001) (2001) (2001) (2001)
4. Conclusions The cometabolic removal of TCE was successfully demonstrated for a mixed nitrifying culture. In the studied TCE range (0–845 lg/ L), the cometabolic TCE degradation followed first-order kinetics. Cometabolic degradation of TCE exhibited an increase with NH4– N utilization. The transformation yield of TCE strongly correlated with the initial levels of TCE and NH4–N. The transformation yield of TCE in this mixed nitrifying culture was quite comparable to other cultures capable of cometabolic degradation. This suggests that in addition to other aerobic systems containing non-specific oxygenase enzymes also nitrifying systems are promising candidates for biological removal of TCE through cometabolism.
Acknowledgements The financial supports of this study by TUBITAK (Project No.: IÇTAG A038) and Research Fund of Bogazici University (Project No.: B.A.P. 02Y101D) is gratefully acknowledged. We are grateful to Ercument Aktuz for his assistance in GC analyses.
References Alpaslan Kocamemi, B., 2005. Cometabolic Degradation of Trichloroethylene (TCE) and 1,2-Dichloroethane (1,2-DCA) in Nitrification Systems. Ph.D. dissertation, Bogazici University, Istanbul, Turkey. Alpaslan Kocamemi, B., Çeçen, F., 2005. Cometabolic degradation of TCE in enriched nitrifying batch systems. J. Hazard. Mater. B125, 260–265. Alpaslan Kocamemi, B., Çeçen, F., 2007. Kinetic analysis of the inhibitory effect of trichloroethylene (TCE) on nitrification in cometabolic degradation. Biodegradation 18, 71–81. Alpaslan Kocamemi, B., Çeçen, F., 2009. Biodegradation of 1,2-dichloroethane (1,2DCA) by cometabolism in a nitrifying biofilm reactor. Int. Biodeterior. Biodegrad. 63, 717–726. Alvarez-Cohen, L., Speitel, G.E., 2001. Kinetics of aerobic cometabolism of chlorinated solvents. Biodegradation 12, 105–126. APHA, AWWA, WEF, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Heath Association, Washington, DC, USA. Arciero, D., Vannelli, T., Logan, M., Hooper, A.B., 1989. Degradation of trichloroethylene by the ammonia-oxidizing bacterium Nitrosomonas europaea. Biochem. Biophys. Res. Commun. 159 (2), 640–643. Bradley, P.M., 2003. History and ecology of chloroethene biodegradation: a review. Bioremed. J. 7 (2), 81–109. Chang, H.L., Alvarez-Cohen, L., 1995. Transformation capacities of chlorinated organics by mixed cultures enriched on methane, propane, toluene, or phenol. Biotechnol. Bioeng. 45, 440–449. Chu, K.H., Alvarez-Cohen, L., 2000. Treatment of chlorinated solvents by nitrogenfixing and nitrate-supplied methane oxidizers in columns packed with unsaturated porous media. Environ. Sci. Technol. 34 (9), 1784–1793. Ely, R.L., Hyman, M.R., Arp, D.J., Guenther, R.B., Williamson, K.J., 1995. A cometabolic kinetics model incorporating enzyme inhibition, inactivation, and recovery: II. Trichloroethylene degradation experiments. Biotechnol. Bioeng. 46 (3), 232– 245. Ely, R.L., Williamson, K.J., Hyman, M.R., Arp, D.J., 1997. Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response. Biotechnol. Bioeng. 54 (6), 520–534. Field, J.A., Sierra-Alvarez, R., 2004. Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds. Rev. Environ. Sci. Bio/Technol. 3, 185– 254.
B.A. Kocamemi, F. Çeçen / Bioresource Technology 101 (2010) 430–433 Han, Y.L., Tom Kuo, M.C., Tseng, I.C., Lu, C.J., 2007. Semicontinuous microcosm study of aerobic cometabolism of trichloroethylene using toluene. J. Hazard. Mater. 148, 583–591. Hyman, M.R., Russell, S.A., Ely, R.L., Williamson, K.J., Arp, D.J., 1995. Inhibition, inactivation, and recovery of ammonia-oxidizing activity in cometabolism of trichloroethylene by Nitrosomonas europaea. Appl. Environ. Microbiol. 61 (4), 1480–1487. Rasche, M.E., Hyman, M.R., Arp, D.J., 1991. Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia
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monooxygenase and substrate specificity. Appl. Environ. Microbiol. 57 (10), 2986–2994. Shukla, A.K., Vishwakarma, P., Upadhyay, S.N., Tripathi, A.K., Prasana, H.C., Dubey, S.K., 2009. Biodegradation of trichloroethylene (TCE) by methanotrophic community. Bioresour. Technol. 100, 2469–2474. Yang, L., Chang, Y.-F., Chou, M.-S., 1999. Feasibility of bioremediation of trichloroethylene contaminated sites by nitrifying bacteria through cometabolism with ammonia. J. Hazard. Mater. B69, 111–126.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biological removal of the xenobiotic trichloroethylene (TCE) through cometabolism in nitrifying systems B. Alpaslan Kocamemi a,*, F. Çeçen b a b
Department of Environmental Engineering, Faculty of Engineering, University of Marmara, Kuyubasi, Istanbul, Turkey Institute of Environmental Sciences, University of Bogazici, Bebek, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 31 March 2009 Received in revised form 13 July 2009 Accepted 17 July 2009 Available online 2 September 2009 Keywords: Activated sludge Cometabolism Nitrification Trichloroethylene Xenobiotics
a b s t r a c t In the present study, cometabolic TCE degradation was evaluated using NH4–N as the growth-substrate. At initial TCE concentrations up to 845 lg/L, TCE degradation followed first-order kinetics. The increase in ammonium utilization rate favored the degradation of TCE. This ensured that biological transformation of TCE in nitrifying systems is accomplished through a cometabolic pathway by the catalysis of non-specific ammonia oxygenase enzyme of nitrifiers. The transformation yield (Ty) of TCE, the amount of TCE degraded per unit mass of NH4–N, strongly depended on the initial NH4–N and TCE concentrations. In order to allow a rough estimation of TCE removal and nitrification at different influent TCE and NH4–N concentrations, a linear relationship was developed between 1/Ty and the initial NH4–N/TCE ratio. The estimated Ty values lead to the conclusion that nitrifying systems are promising candidates for biological removal of TCE through cometabolism. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Trichloroethylene (TCE) is a xenobiotic compound that is mainly used as degreaser and solvent in automotive, metal, electroplating, textile and paper industries. The large-scale use of TCE in various industries over the past years has led to widespread distribution of this toxic and carcinogenic compound in the environment, especially in groundwater media. TCE can also be monitored in surface media. But, the highly volatile characteristics usually results in quick evaporation in open surface water bodies. Considerable effort has been focused on removal of TCE by biological processes. To date, no isolates or enrichment cultures utilizing TCE as growth-substrate under aerobic conditions have yet been obtained (Bradley, 2003; Field and Sierra-Alvarez, 2004). But, some aerobic bacterial cultures containing non-specific oxygenase enzymes (e.g., methane, toluene, phenol, propane and ammonia oxidizers) were identified capable to degrade TCE by cometabolism (Arciero et al., 1989; Chang and Alvarez-Cohen, 1995; Chu and Alvarez-Cohen, 2000; Han et al., 2007; Shukla et al., 2009). In this process, biotransformation of a non-growth compound (e.g., TCE) is accomplished concurrently with metabolism of a growth-supporting substrate through the catalysis of non-specific oxygenase enzymes. The organism obtains no benefit from cometabolism of the non-growth-supporting substrate. * Corresponding author. Tel.: +90 216 3480292x270; fax: +90 216 3481369. E-mail addresses: [email protected], [email protected] (B.A. Kocamemi). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.07.079
Moreover, the presence of the non-growth-supporting substrate may inhibit the metabolism of the growth-supporting substrate, thereby decreasing or preventing bacterial growth (Ely et al., 1997). In addition, cometabolism of the non-growth-supporting substrate consumes reductant NAD(P)H, which can only be regenerated from growth-supporting substrate degradation. Therefore, in this process, a growth-supporting substrate must be available (at least periodically) to grow new cells, provide reductant and induce production of non-specific enzymes (Alvarez-Cohen and Speitel, 2001). Among the organisms capable to degrade TCE cometabolically, this study focused on ammonia oxidizers (nitrifiers). In literature, the studies evaluating the cometabolism of TCE by nitrifying species (Arciero et al., 1989; Rasche et al., 1991; Ely et al., 1995, 1997; Hyman et al., 1995; Yang et al., 1999) are very limited compared to those performed with other organisms (e.g., methanotrophs). Nitrification studies up to date were mainly performed with pure Nitrosomonas europaea cultures. In our previous studies, using a mixed culture enriched in terms of nitrifying bacteria, we first evaluated the inhibitory effect of TCE on nitrification during cometabolic degradation for a broad TCE range and at a fixed growth-supporting substrate (NH4–N) concentration (Alpaslan Kocamemi and Çeçen, 2005). Later (Alpaslan Kocamemi and Çeçen, 2007), we focused on the kinetics of the inhibitory effect of TCE on nitrification, which directly influences the sustainability of TCE degradation in nitrifying systems. The inhibition type and inhibition coefficient of TCE were determined for a broad range of NH4–N and TCE.
431
B.A. Kocamemi, F. Çeçen / Bioresource Technology 101 (2010) 430–433
In the present study, which is complementary to our previous study (Alpaslan Kocamemi and Çeçen, 2007), cometabolic TCE degradation was evaluated by a mixed culture enriched for nitrifiers. A batch suspended-growth system was fed with NH4–N as the growth-supporting substrate. The dependence of the process on various factors, such as the NH4–N utilization rate and the relative initial ratio of NH4–N and TCE, was quantitatively analyzed. 2. Methods
Table 1 The estimated first-order TCE degradation constant at various NH4–N levels. Initial NH4–N concentration (mg/L)
First-order TCE degradation rate constant (L/g VSS h)
Goodness-of-fit of linear regression (R2)
25 50 100 200 400
0.26 0.31 0.48 0.73 1.03
0.98 0.94 0.95 0.95 0.93
2.1. Mixed culture enriched for nitrifiers
2.2. Experimental procedure Four sets of experiments were performed in 200 mL capped glass bottles at a constant wastewater temperature of 25 °C and pH range of 7–8. In each set, five runs were performed at constant TCE, but at varying initial NH4–N (25–400 mg/L). The initial TCE was kept at 40, 110, 325, 845 lg/L in the first, second, third and fourth set of experiments. In all experiments, the stock synthetic NH4–N solution [37.75 g/L (NH4)2SO4 and 95 g/L NaHCO3] and the stock TCE solution [500 mg/L TCE] were used in diluted form to adjust the desired NH4–N and TCE concentrations. The stock mineral solution [2 g/L MgSO47H2O, 0.1 g/L CaCO3, 0.4 g/L FeSO47H2O, 0.2 g/L MnSO4H2O and 0.3 g/L K2HPO4] was diluted 40 folds. In each experiment, the stock culture was rinsed thoroughly with water and diluted to MLVSS ranging between 210 and 530 mg/L. Experiments were started under oxygen-supersaturated conditions (initial DO of 35–40 mg/L) and ended before the DO in the test bottle dropped below 4 mg/L. Continuous diffused aeration was avoided to prevent the possible risk of TCE volatilization. Complete mixing in the test bottles was maintained by magnetic stirring. Blank experiments were performed to evaluate the abiotic loss of TCE resulting from stirring action. These experiments demonstrated a negligible TCE exchange rate of 0.0028 lg/L/min per initial TCE (lg/L) (data not shown, Alpaslan Kocamemi, 2005). In all runs, samples were analyzed with respect to time for NHþ 4 —N with the Nessler Method (APHA, 1998) using Hach DR/2000 spectrophotometer and for TCE with HP 5890 Gas Chromatograph equipped with an electron capture detector. The measured TCE concentrations were corrected by the abiotic TCE loss rate. The specific ammonium utilization rate (qNH4 —N ) and the specific cometabolic TCE degradation rates (qTCE) were then calculated from the slopes of NH4–N/VSS versus time and TCE/VSS versus time plottings through linear regression analysis, respectively.
monas species. Photomicrographs of these analyses were shown in a former paper (Alpaslan Kocamemi and Çeçen, 2007). Further, as shown by DGGE analyses (Alpaslan Kocamemi and Çeçen, 2007), no changes had occurred in the diversity of amoA gene sequences during the period of about 7 months after enrichment. Since a shift in microbial population was not the case, the rates in the present study are not expected to change during the experimental period. Evaluation of the cometabolic degradation rate of TCE (qTCE) with respect to initial TCE concentrations (Alpaslan Kocamemi, 2005, data not shown) showed that at each initial NH4–N concentration studied, qTCE increased linearly with TCE concentration. The first-order cometabolic TCE degradation rate constants obtained from the linearization of the graphs are summarized in Table 1. The cometabolic TCE degradation followed first-order kinetics because the bulk TCE concentrations (0–825 lg/L) were very small. If higher bulk TCE concentrations were present, TCE degradation rate would most probably reach the saturation level. However, it is very likely that also in real remediation systems, such as groundwater, TCE degradation follows first-order kinetics in the concentration ranges below 1000 lg/L. The first-order TCE degradation rate constants in the present study are considerably lower in comparison to those reported for pure N. europaea species as 30.8 L/g VSS h and 42.5 L/gVSS h for the initial TCE concentrations of 0–3300 and 2100 lg/L, respectively (Alvarez-Cohen and Speitel, 2001). However, in those studies performed with pure cultures, the culture consisted of pure nitrifier species only and the reported values based on the assumption that the dry cell mass consisted of 50% protein (Alvarez-Cohen and Speitel, 2001). On the other hand, in the present study, the mixed culture still contains cells other than nitrifiers although it was enriched for nitrifiers. Therefore, the values expressed on VSS basis are quite lower compared to those reported in pure culture studies.
1050 900
Cometabolic degradation rate of TCE (µg TCE /gVSS.h)
The mixed culture was initially taken from the Pasakoy Advanced Biological Sewage Treatment Plant in Istanbul. The culture was then enriched for nitrifiers as described previously (Alpaslan Kocamemi and Çeçen, 2005). After the enrichment period, this mixed culture was continuously fed with NH4–N and mineral solutions only based on the fill-and-draw principle. The sludge samples taken from the stock enriched nitrifier culture were analyzed by Florescent In-Situ Hybridization (FISH) to ensure the enrichment of the mixed culture for nitrifiers. Denaturing gradient gel electrophoresis (DGGE) analyses were also performed to observe whether any changes occurred in the diversity of amoA gene sequences 7 months after the enrichment period. The procedures of FISH and DGGE analyses were as described previously (Alpaslan Kocamemi and Çeçen, 2009).
750
40 ppb TCE 110 ppb TCE
600
325 ppb TCE 450 845 ppb TCE 300 150 0 0
3. Results and discussions The FISH analysis of the stock culture revealed that the dominant members of the microbial community consisted of Nitroso-
10
20
30
40
50
60
NH4-N utilization rate (mg NH 4-N/gVSS.h) Fig. 1. Cometabolic degradation rates of TCE (qTCE) with respect to the NH4–N utilization rates (qNH4 —N ).
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B.A. Kocamemi, F. Çeçen / Bioresource Technology 101 (2010) 430–433
Table 2 Estimated transformation yields (Ty) for TCE. Culture
Initial TCE concentration (lg/L)
Ty
Reference
lgcometabolic substrateðTCEÞ mg growth-substrate ðNH4 —NÞ
Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed
culture enriched for nitrifiers culture enriched for nitrifiers culture enriched for nitrifiers culture enriched for nitrifiers methanotrophic culture culture of toluene degraders culture of phenol degraders culture of propane degraders
40 110 325 845 1000 30,000 20,000 16,000
1.4 2.3 3.3 24.4 16 2.5 1.9 11.9
The degradation rate of TCE (qTCE) was further expressed in dependence of ammonium utilization rates (qNH4 —N ) as shown in Fig. 1. At each initial TCE concentration, this rate exhibited an increase with ammonium utilization rates, which ensured the cometabolic removal of TCE by nitrifier species. The increases in qTCE at increased qNH4 —N values may be most probably related with the regeneration of the NAD(P)H (reductant), the potentially limiting reactant of cometabolic processes, by the growth-supporting substrate NH4–N. In our previous study (Alpaslan Kocamemi and Çeçen, 2007), TCE was found to be a competitive inhibitor of ammonia oxidation, which means that both TCE and NH4–N compete for the same active site of the enzyme ammonia monooxygenase (AMO). Therefore, a decrease in TCE degradation rate may be expected at higher ammonium utilization rates or vice versa. In consistence with this hypothesis, in our previous study (Alpaslan Kocamemi and Çeçen, 2007), it was shown that increase in TCE concentration resulted in a relative decrease in qNH4 —N (i.e., inhibition of nitrification). But, in the present study, NH4–N exhibited a positive effect on TCE degradation at even higher NH4–N concentrations. This may be related to the considerably greater affinity of TCE for the enzyme AMO compared to ammonia as reported before (Alpaslan Kocamemi and Çeçen, 2007). In order to assess the cometabolic TCE degradation potential of the culture, the transformation yields (Ty) of TCE, which is the mass of TCE degraded per unit mass of NH4–N consumed, were estimated by dividing the maximum qTCE values shown in Fig. 1 by the corresponding qNH4 —N . Those values are reported in Table 2 and compared with other cultures in various studies. As seen from Table 2, Ty values determined in the present study were variable depending on the TCE concentration. The minimum amount of NH4–N required for the degradation of 1 lg TCE at the maximum rate was found as 0.7 mg NH4–N at 40 lg/L TCE, 0.4 mg NH4–N at 110 lg/L TCE, 0.3 mg NH4–N at 325 lg/L TCE and 0.04 mg NH4–N at 845 lg/L TCE. Ty values for other organisms reported in Table 2 are not directly comparable with those determined in the present study because they were determined at higher initial TCE concentrations. However, the order of magnitude of Ty values in the present study are quite comparable to those reported in other studies using other cultures. This indicates that nitrifying systems using NH4–N as the growth-supporting substrate have a quite high TCE degradation capacity. The evaluation of qNH4 —N /qTCE (1/Ty) values against the initial NH4–N/TCE values (data not shown, Alpaslan Kocamemi, 2005) indicated that in the studied NH4–N (25–400 mg/L) and TCE (40– 845 lg/L) concentration ranges, up to an NH4–N/TCE ratio of 4800 a strong linear relationship existed between qNH4 —N /qTCE (1/ Ty) and the initial NH4–N/TCE ratio. As shown in Eq. (1) the constant belonging to this relationship was found as 0.2651:
1=T y ¼ 0:2651ðNH4 —N=TCEÞ
ð1Þ
Eq. (1) can be a useful tool in the prediction of Ty that can be achieved at different NH4–N and TCE concentrations.
This study This study This study This study Alvarez-Cohen Alvarez-Cohen Alvarez-Cohen Alvarez-Cohen
and and and and
Speitel Speitel Speitel Speitel
(2001) (2001) (2001) (2001)
4. Conclusions The cometabolic removal of TCE was successfully demonstrated for a mixed nitrifying culture. In the studied TCE range (0–845 lg/ L), the cometabolic TCE degradation followed first-order kinetics. Cometabolic degradation of TCE exhibited an increase with NH4– N utilization. The transformation yield of TCE strongly correlated with the initial levels of TCE and NH4–N. The transformation yield of TCE in this mixed nitrifying culture was quite comparable to other cultures capable of cometabolic degradation. This suggests that in addition to other aerobic systems containing non-specific oxygenase enzymes also nitrifying systems are promising candidates for biological removal of TCE through cometabolism.
Acknowledgements The financial supports of this study by TUBITAK (Project No.: IÇTAG A038) and Research Fund of Bogazici University (Project No.: B.A.P. 02Y101D) is gratefully acknowledged. We are grateful to Ercument Aktuz for his assistance in GC analyses.
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