Correspondence/Rebuttal pubs.acs.org/est
Response to Comment on “Anaerobic Mercury Methylation and Demethylation by Geobacter Bemidjiensis Bem”
W
e offer our response to comments by Regnell1 regarding the calculations of methylation/demethylation rates reported in our paper “Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem”.2 We disagree with the proposed approach, resulting in the calculated demethylation rate being nearly 2 orders of magnitude slower for methylmercury (MeHg) added externally than for MeHg produced internally. The kinetics of bacterial mercury (Hg) methylation or demethylation is the result of many coupled processes (i.e., Hg sorption, reduction, oxidation, methylation, and demethylation) and thus is far more complex than described by the rate equations in the comment by Regnell. Furthermore, environmental factors, such as presence of small thiols and culture conditions, affect Hg(II) bioavailability and its uptake, thus impacting the observed methylation and demethylation rates. Without a full understanding of all these processes and mechanisms, any presented kinetic model for calculating methylation or demethylation rates and comparing them with others may prove to be a fruitless exercise in data fitting. We now address these points in detail below. First, Regnell noted that “the observation that more Hg(0) than MeHg was produced from HgCl2 suggests that the rate of demethylation (Kdemeth) was higher than the rate of methylation (Kmeth), unless Hg0 was a result mainly of direct reduction of the added Hg”. However, his subsequent analysis assumed that Hg(0) is a product of demethylation only. We clearly attributed formation of Hg(0) primarily to biological reduction of Hg(II) in the methylation assay (see our original manuscript p. 4368),2 since G. bemidjiensis is a known metal-reducing bacterium. In fact, when MeHg was added as the sole source of Hg, we observed lower amounts of Hg(0) produced than MeHg degraded, due to oxidation of Hg(0) (see Figure 2 in Lu et al.).2 Second, Regnell’s model assumed that in the Hg(II) methylation assay all added Hg is available for bacterial methylation, and that demethylation competes with methylation with Kmeth decreasing linearly from 0.03 to 0.015 h−1 over time during demethylation.1 This assumption is incorrect because ∼38% of the added Hg(II) was rapidly reduced to Hg(0), as described above. More importantly, studies have shown that in pure cultures Hg methylation often exhibits a plateau or a maximum, usually within a few hours or a day, despite the presence of a large quantity of inorganic Hg in the system.3−7 Although the mechanism of this stalled methylation is not fully understood, and is a subject of future investigations related to Hg bioavailability, we can reasonably assume a near zero MeHg production after 8 h Hg(II) incubation. Given these considerations, we calculated Kmeth by fitting the data as a pseudo-first-order reaction for the first 8 h,2 assuming that methylation is limited by the amount of bioavailable Hg, which is subsequently converted to MeHg (∼1.2 nM). As a result, our estimated methylation rate was about 30 times higher than the value obtained by Regnell. After 8 h, MeHg concentration decreased linearly with time. The data thus fitted well with a © 2016 American Chemical Society
zero-order kinetics. This linear relationship also holds true when MeHg is added as the sole source of Hg in demethylation assays (see Figure 2 in Lu et al.).2 Lastly, demethylation rates calculated by Regnell differed by nearly 2 orders of magnitude for MeHg added externally and for MeHg produced internally by the cells. These results seem quite unrealistic. They would imply that indigenously produced MeHg and exogenously added MeHg would end up in different pools or have different availabilities to cells for demethylation. However, our results indicate that the exogenously added MeHg is rapidly absorbed and degraded by the cells (see Figure 3b and Figure 2a in Lu et al.).2 Similarly, in a study of Hg methylation and demethylation by the sulfate-reducing bacterium Desulfobulbus propionicus (DSM6523) with isotope labeled Hg(II) and MeHg, Bridou et al.8 observed only a 2-fold difference in demethylation rates between MeHg added externally and MeHg produced internally. The discrepancy here again illustrates that, while different assumptions could be made and different kinetic rate expressions could be used, the calculated methylation or demethylation rates may be only meaningful and comparable if the mechanisms of these processes are well understood, and all the experimental and environmental conditions are fully considered.
Xia Lu*,†,‡ Yurong Liu‡,§ Alexander Johs‡ Linduo Zhao‡ Tieshan Wang† Ziming Yang‡ Hui Lin‡ Dwayne A. Elias∥ Eric M. Pierce‡ Liyuan Liang‡,⊥ Tamar Barkay# Baohua Gu*,‡ †
School of Nuclear Science and Technology, Lanzhou University, Lanzhou, China ‡ Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States § State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China ∥ Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States ⊥ Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States # Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey 08901, United States Published: August 12, 2016 9800
DOI: 10.1021/acs.est.6b03687 Environ. Sci. Technol. 2016, 50, 9800−9801
Environmental Science & Technology
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Correspondence/Rebuttal
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Regnell, O. Comment on ″Anaerobic Mercury Methylation and 1 Demethylation by Geobacter bemidjiensis Bem″. Environ. Sci. Technol. 2016, this issue. (2) Lu, X.; Liu, Y.; Johs, A.; Zhao, L.; Wang, T.; Yang, Z.; Lin, H.; Elias, D. A.; Pierce, E. M.; Liang, L.; Barkay, T.; Gu, B. Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem. Environ. Sci. Technol. 2016, 50 (8), 4366−4373. (3) Graham, A. M.; Bullock, A. L.; Maizel, A. C.; Elias, D. A.; Gilmour, C. C. Detailed assessment of the kinetics of Hg-cell association, Hg methylation, and methylmercury degradation in several Desulfovibrio species. Appl. Environ. Microbiol. 2012, 78 (20), 7337− 7346. (4) Hu, H.; Lin, H.; Zheng, W.; Tomanicek, S. J.; Johs, A.; Feng, X.; Elias, D. A.; Liang, L.; Gu, B. Oxidation and methylation of dissolved elemental mercury by anaerobic bacteria. Nat. Geosci. 2013, 6 (9), 751−754. (5) Lin, H.; Lu, X.; Liang, L.; Gu, B. Cysteine inhibits mercury methylation by Geobacter Sulf urreducens PCA mutant ΔomcBESTZ. Environ. Sci. Technol. Lett. 2015, 2, 144−148. (6) Lin, H.; Morrell-Falvey, J. L.; Rao, B.; Liang, L.; Gu, B. Coupled mercury-cell sorption, reduction, and oxidation affecting methylmercury production by Geobacter sulfurreducens PCA. Environ. Sci. Technol. 2014, 48 (20), 11969−11976. (7) Schaefer, J. K.; Rocks, S. S.; Zheng, W.; Liang, L.; Gu, B.; Morel, F. M. Active transport, substrate specificity, and methylation of Hg (II) in anaerobic bacteria. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (21), 8714−8719. (8) Bridou, R.; Monperrus, M.; Gonzalez, P. R.; Guyoneaud, R.; Amouroux, D. Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using species-specific isotopic tracers. Environ. Toxicol. Chem. 2011, 30 (2), 337−344.
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DOI: 10.1021/acs.est.6b03687 Environ. Sci. Technol. 2016, 50, 9800−9801
