Correspondence/Rebuttal pubs.acs.org/est
Response to Comment on Microbial Community Composition Is Unaffected by Anode Potential
W
e thank Commault et al.1 for their interest in our publication.2 Their comment correctly points out that we stated that anode potential did not affect community composition, and that we did not classify our microbial communities below the genus. We did not attempt to classify our community below the genus level as we did not feel that our sequences were long enough (∼400 bp fully processed) and we did not have enough sequence coverage (only ∼1000 sequences per sample) to confidently classify these microorganisms down to the species level. Classification to this level was not our intention as this study was aimed at further scrutinizing the results presented in the study by Torres et al.,3 which suggested that the anodic community varied at the Class level based on set anode potential. Whenever a species was referred to in our paper, we were careful to state that the sequence was “most similar to” a certain species, which implied that we had not performed the requisite analyses to confidently classify our sequences at the species or strain level, but instead recognized it as most similar to a published species. Our intention in this study was to identify all members of the biofilm community acclimated to different set potentials using universal primers. The use of a universal primer set in our study,2 instead of a Geobacter specific primer set,4 further hindered our ability to confidently distinguish between different species and strains within the Geobacter genus. We do not discount the possibility of changes in communities at the species or strain level. We also used the phrase “most similar to” in our community analysis because we know that both physiological adaptations and genetic shifts can occur within anodic biofilms. For example, in a previous publication we demonstrated physiological adaptation to set potential by showing that a pure culture of G. sulfurreducens strain PCA could preferentially express various exocellular electron transfer components at different levels to gain energy from a wide range of anode potentials.5 We also recognize that there can be genetic shifts in anodic biofilms, and that classification using even full length sequences may not correctly identify species on the basis of 16S rRNA sequences. For example, in a recent study, Sun et al. isolated a Geobacter strain from an anode biofilm in a microbial electrolysis cell,6 and classified it as a new species,7 Geobacter anodireducens, even though it was “most similar to G. sulf urreducens” (98% similarity) according to the NCBI database on the basis of an approximately full length 16S rRNA gene (1457 bp). This level of classification required an in-depth analysis of the molecular and physiological properties of the newly adapted microorganism to confidently identify and differentiate this new species from other sequenced organisms published in the NCBI database. As more of these novel species are identified and as sequencing technology improves, it will likely become easier to classify sequences to a more specific level with higher confidence in community analysis studies. It is difficult for us to compare the results of our study with previous results obtained by Commault et al.4 because their © 2014 American Chemical Society
operating conditions, which were quite different from ours, could have an effect on the community development independent of the set anode potential. The operating temperature in the study by Commault et al. (18 °C) was much lower than that used in our study (30 °C), and they added a methanogenic inhibitor (BES; 2-bromoethanosulfonate),4 which could have altered bacterial community composition.8 We are also unable to comment on Figure 1 presented by Commault et al.1 because the methods for constructing the phylogenetic tree were unclear. However, if we assume the sequence length was ∼300 bp, based on the Geobacter specific primers used in their previous study, and the number of sequences provided in the analysis to be ∼50,4 then the two major groups are only different by ∼1%, based on the scale bar of 0.3%. A sequencing error of only one or two base pairs could result in the classification of a new strain when using short sequences and these small errors could prohibit accurate identification of strains. Furthermore, no bootstrap values were provided to assign a confidence level for the placement of the sequences in the tree and the published strains were not included in the tree along with the samples. A more detailed electrochemical analysis of the systems presented in the original work by Commault et al.4 may also have relevance to this discussion. For example, nonturnover cyclic voltammograms showing clear differences in redox peak positions combined with a detailed genetic analysis of the system would further support their claim of strain selection rather than only a physiological shift. We recognize that as technology develops and techniques are refined, that new results may show our conclusions to be true only to the genus level and that a strain selection process does occur. However, based on the intent of this study, as outlined in this comment and the original published manuscript, and the available techniques and information at the time of the experiment, we are confident in asserting that the overall community composition is unaffected by the set anode potential at the given levels of taxonomic classification.2
Xiuping Zhu† Matthew D. Yates† Marta C. Hatzell† Hari Ananda Rao‡ Pascal E. Saikaly‡ Bruce E. Logan*,† †
Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡ Water Desalination and Reuse Center, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia Published: December 5, 2014 14853
dx.doi.org/10.1021/es503791t | Environ. Sci. Technol. 2014, 48, 14853−14854
Environmental Science & Technology
■
Correspondence/Rebuttal
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 814 863 7908; fax: +1 814 863 7304; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Commault, A. S.; Lear, G.; Weld, R. J. Comment on microbial community composition is unaffected by anode potential. Environ. Sci. Technol. 2014, DOI: 10.1021/es501982m. (2) Zhu, X. P.; Yates, M. D.; Hatzell, M. C.; Rao, H. A.; Saikaly, P. E.; Logan, B. E. Microbial community composition is unaffected by anode potential. Environ. Sci. Technol. 2014, 48, 1352−1358. (3) Torres, C. I.; Krajmalnik-Brown, R.; Parameswaran, P.; Marcus, A. K.; Wanger, G.; Gorby, Y. A.; Rittmann, B. E. Selecting anoderespiring bacteria based on anode potential: Phylogenetic, electrochemical, and microscopic characterization. Environ. Sci. Technol. 2009, 43, 9519−9524. (4) Commault, A. S.; Lear, G.; Packer, M. A.; Weld, R. J. Influence of anode potentials on selection of Geobacter strains in microbial electrolysis cells. Bioresour. Technol. 2013, 139, 226−234. (5) Zhu, X. P.; Yates, M. D.; Logan, B. E. Set potential regulation reveals additional oxidation peaks of Geobacter sulfurreducens anodic biofilms. Electrochem. Commun. 2012, 22, 116−119. (6) Sun, D.; Call, D.; Wang, A.; Cheng, S.; Logan, B. E. Geobacter sp. SD-1 with enhanced electrochemical activity in high-salt concentration solutions. Environ. Microbiol. Report 2014, 6, 723−729. (7) Sun, D.; Wang, A.; Cheng, S.; Yates, M. D.; Logan, B. E. Geobacter anodireducens sp. nov., a novel exoelectrogenic microbe in bioelectrochemical systems. Int. J. Syst. Evol. Microbiol. 2014, 64, 3485−3491. (8) Chiu, P. C.; Lee, M. 2-bromoethanesulfonate affects bacteria in a trichloroethene-dechlorinating culture. Appl. Environ. Microbiol. 2001, 67, 2371−2374.
14854
dx.doi.org/10.1021/es503791t | Environ. Sci. Technol. 2014, 48, 14853−14854
Response to Comment on Microbial Community Composition Is Unaffected by Anode Potential
W
e thank Commault et al.1 for their interest in our publication.2 Their comment correctly points out that we stated that anode potential did not affect community composition, and that we did not classify our microbial communities below the genus. We did not attempt to classify our community below the genus level as we did not feel that our sequences were long enough (∼400 bp fully processed) and we did not have enough sequence coverage (only ∼1000 sequences per sample) to confidently classify these microorganisms down to the species level. Classification to this level was not our intention as this study was aimed at further scrutinizing the results presented in the study by Torres et al.,3 which suggested that the anodic community varied at the Class level based on set anode potential. Whenever a species was referred to in our paper, we were careful to state that the sequence was “most similar to” a certain species, which implied that we had not performed the requisite analyses to confidently classify our sequences at the species or strain level, but instead recognized it as most similar to a published species. Our intention in this study was to identify all members of the biofilm community acclimated to different set potentials using universal primers. The use of a universal primer set in our study,2 instead of a Geobacter specific primer set,4 further hindered our ability to confidently distinguish between different species and strains within the Geobacter genus. We do not discount the possibility of changes in communities at the species or strain level. We also used the phrase “most similar to” in our community analysis because we know that both physiological adaptations and genetic shifts can occur within anodic biofilms. For example, in a previous publication we demonstrated physiological adaptation to set potential by showing that a pure culture of G. sulfurreducens strain PCA could preferentially express various exocellular electron transfer components at different levels to gain energy from a wide range of anode potentials.5 We also recognize that there can be genetic shifts in anodic biofilms, and that classification using even full length sequences may not correctly identify species on the basis of 16S rRNA sequences. For example, in a recent study, Sun et al. isolated a Geobacter strain from an anode biofilm in a microbial electrolysis cell,6 and classified it as a new species,7 Geobacter anodireducens, even though it was “most similar to G. sulf urreducens” (98% similarity) according to the NCBI database on the basis of an approximately full length 16S rRNA gene (1457 bp). This level of classification required an in-depth analysis of the molecular and physiological properties of the newly adapted microorganism to confidently identify and differentiate this new species from other sequenced organisms published in the NCBI database. As more of these novel species are identified and as sequencing technology improves, it will likely become easier to classify sequences to a more specific level with higher confidence in community analysis studies. It is difficult for us to compare the results of our study with previous results obtained by Commault et al.4 because their © 2014 American Chemical Society
operating conditions, which were quite different from ours, could have an effect on the community development independent of the set anode potential. The operating temperature in the study by Commault et al. (18 °C) was much lower than that used in our study (30 °C), and they added a methanogenic inhibitor (BES; 2-bromoethanosulfonate),4 which could have altered bacterial community composition.8 We are also unable to comment on Figure 1 presented by Commault et al.1 because the methods for constructing the phylogenetic tree were unclear. However, if we assume the sequence length was ∼300 bp, based on the Geobacter specific primers used in their previous study, and the number of sequences provided in the analysis to be ∼50,4 then the two major groups are only different by ∼1%, based on the scale bar of 0.3%. A sequencing error of only one or two base pairs could result in the classification of a new strain when using short sequences and these small errors could prohibit accurate identification of strains. Furthermore, no bootstrap values were provided to assign a confidence level for the placement of the sequences in the tree and the published strains were not included in the tree along with the samples. A more detailed electrochemical analysis of the systems presented in the original work by Commault et al.4 may also have relevance to this discussion. For example, nonturnover cyclic voltammograms showing clear differences in redox peak positions combined with a detailed genetic analysis of the system would further support their claim of strain selection rather than only a physiological shift. We recognize that as technology develops and techniques are refined, that new results may show our conclusions to be true only to the genus level and that a strain selection process does occur. However, based on the intent of this study, as outlined in this comment and the original published manuscript, and the available techniques and information at the time of the experiment, we are confident in asserting that the overall community composition is unaffected by the set anode potential at the given levels of taxonomic classification.2
Xiuping Zhu† Matthew D. Yates† Marta C. Hatzell† Hari Ananda Rao‡ Pascal E. Saikaly‡ Bruce E. Logan*,† †
Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡ Water Desalination and Reuse Center, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia Published: December 5, 2014 14853
dx.doi.org/10.1021/es503791t | Environ. Sci. Technol. 2014, 48, 14853−14854
Environmental Science & Technology
■
Correspondence/Rebuttal
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 814 863 7908; fax: +1 814 863 7304; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Commault, A. S.; Lear, G.; Weld, R. J. Comment on microbial community composition is unaffected by anode potential. Environ. Sci. Technol. 2014, DOI: 10.1021/es501982m. (2) Zhu, X. P.; Yates, M. D.; Hatzell, M. C.; Rao, H. A.; Saikaly, P. E.; Logan, B. E. Microbial community composition is unaffected by anode potential. Environ. Sci. Technol. 2014, 48, 1352−1358. (3) Torres, C. I.; Krajmalnik-Brown, R.; Parameswaran, P.; Marcus, A. K.; Wanger, G.; Gorby, Y. A.; Rittmann, B. E. Selecting anoderespiring bacteria based on anode potential: Phylogenetic, electrochemical, and microscopic characterization. Environ. Sci. Technol. 2009, 43, 9519−9524. (4) Commault, A. S.; Lear, G.; Packer, M. A.; Weld, R. J. Influence of anode potentials on selection of Geobacter strains in microbial electrolysis cells. Bioresour. Technol. 2013, 139, 226−234. (5) Zhu, X. P.; Yates, M. D.; Logan, B. E. Set potential regulation reveals additional oxidation peaks of Geobacter sulfurreducens anodic biofilms. Electrochem. Commun. 2012, 22, 116−119. (6) Sun, D.; Call, D.; Wang, A.; Cheng, S.; Logan, B. E. Geobacter sp. SD-1 with enhanced electrochemical activity in high-salt concentration solutions. Environ. Microbiol. Report 2014, 6, 723−729. (7) Sun, D.; Wang, A.; Cheng, S.; Yates, M. D.; Logan, B. E. Geobacter anodireducens sp. nov., a novel exoelectrogenic microbe in bioelectrochemical systems. Int. J. Syst. Evol. Microbiol. 2014, 64, 3485−3491. (8) Chiu, P. C.; Lee, M. 2-bromoethanesulfonate affects bacteria in a trichloroethene-dechlorinating culture. Appl. Environ. Microbiol. 2001, 67, 2371−2374.
14854
dx.doi.org/10.1021/es503791t | Environ. Sci. Technol. 2014, 48, 14853−14854
