the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 30 Sep 2024
Cr(VI) reduction, electricity production, and microbial resistance variation in paddy soil under microbial fuel cell operation
Abstract. Microbial fuel cell (MFC) is an efficient in-situ approach to combat pollutants and generate electricity. This study constructed a soil MFC (SMFC) to reduce Cr(VI) in paddy soil and investigate its influence on microbial community and microbial resistance characteristics. Fe3O4 nanoparticle as the cathodic catalyst effectively boosted power generation (0.97 V, 102.0 mW/m2), whose porous structure and reducibility also contributed to Cr reduction and immobilization. After 30 days, 93.67 % of Cr(VI) was eliminated. The bioavailable Cr decreased by 97.44 % while the residual form increased by 88.89 %. SMFC operation greatly changed soil enzymatic activity and microbial structure, with exoelectrogens like Desulfotomaculum (3.32 % in anode) and Cr(VI)-reducing bacteria like Hydrogenophaga (2.07 % in cathode) more than 1000 folds of soil. In particular, SMFC operation significantly enhanced the abundance of heavy metal resistance genes (HRGs). Among them, chrA, chrB, and chrR increased by 99.54~3314.34 % in SMFC anode than control, probably attributed to the enrichment of potential tolerators like Acinetobacter, Limnohabitans, and Desulfotomaculum. These key taxa were positively correlated with HRGs but negatively correlated with pH, EC, and Cr(VI), which could have driven Cr(VI) reduction. This study provided novel evidence for bioelectrochemical system application in contaminated paddy soil, which could be a potential approach for environmental remediation and detoxification.
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RC1: 'Comment on egusphere-2024-2771', Anonymous Referee #1, 16 Oct 2024
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The manuscript titled " Cr(VI) reduction, electricity production, and microbial resistance variation in paddy soil under microbial fuel cell operation " (egusphere-2024-2771) focused on SMFC for Cr(VI) contaminated paddy soil remediation and soil microbial ecology restoration, which presented systematic research about the effect of MFC on soil microbial community and metal resistance. In this study, an SMFC was constructed to remove Cr(VI) in paddy soil and investigate its influence on microbial ecology. The mechanism of Cr(VI) reduction and the change of microbial community structure were comprehensively studied, and the variation of HRGs was also discussed. This research provides a good reference for the microbial remediation of polluted soil and improves the practical application of MFC. In conclusion, the work presented some interesting ideas and new knowledge, which met the journal's scope. However, some shortcomings need to be improved before acceptance for publication. Specific revision suggestions are as follows:
- In the introduction, the author should provide more relevant research on HRGs as appropriate to highlight the innovation of the article.
- some writing details need attention. For example, the subtitle of a paper is sorted incorrectly. Such as section 2.4.
- Lines 77-78, please explain why the author chose to add 118.8 mg/kg chromium to the soil.
- In section 2.5.3, it is hoped that the author can provide specific operations on " The abundance of HRGs in the surface soil of SMFC and OMFC anode after the operation was analyzed using an SYBR Green real-time fluorescence quantitative PCR system ".
- Lines 127-137, for the determination method of Microbial community structure, it is suggested that the author supplement the relevant references.
- In lines 181-183, The authors need a more complete explanation for why the voltage of SMFC drops rapidly in the first week and then rises again.
- The Cr shown in both Figure 1e and Figure 1k is easy to be confused. It is recommended to mark them in the appropriate position in the diagram.
- Figure 2 shows that the cathode is GF while the anode is not represented by the material name. Please uniformly use the electrode or material noun to provide a good reading experience.
- In section 3.4.2, it is suggested that the author briefly explain why the soil biochemical indicators should be determined and what contribution these indicators have to the subject.
- In Part 4, give necessary description of Figure 6 ”Cr(VI)reduction Mechanism of during SMFC operation”.
Citation: https://doi.org/10.5194/egusphere-2024-2771-RC1 -
AC1: 'Reply on RC1', Can Wang, 19 Oct 2024
reply
Review of ction, electricity production, and microbial resistance variation in paddy soil under microbial fuel cell operation” by Niu et al. for consideration in EGUsphere.
The manuscript titled " Cr(VI) reduction, electricity production, and microbial resistance variation in paddy soil under microbial fuel cell operation " (egusphere-2024-2771) focused on SMFC for Cr(VI) contaminated paddy soil remediation and soil microbial ecology restoration, which presented systematic research about the effect of MFC on soil microbial community and metal resistance. In this study, an SMFC was constructed to remove Cr(VI) in paddy soil and investigate its influence on microbial ecology. The mechanism of Cr(VI) reduction and the change of microbial community structure were comprehensively studied, and the variation of HRGs was also discussed. This research provides a good reference for the microbial remediation of polluted soil and improves the practical application of MFC. In conclusion, the work presented some interesting ideas and new knowledge, which met the journal's scope. However, some shortcomings need to be improved before acceptance for publication. Specific revision suggestions are as follows:
We would like to thank the reviewers for their time and feedback on this manuscript. Please find our point-to-point responses below.
- In the introduction, the author should provide more relevant research on HRGs as appropriate to highlight the innovation of the article.
Response: Thanks for the suggestion.
The change of HRGs in paddy soils is the focus of our discussion and an important innovation point in our research. Widespread antibiotic resistance poses a serious threat to human health. The observed increase in antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in natural environments has been attributed to the selective pressure generated by overuse and misuse of veterinary animal feeding and aquaculture [1]. Microorganisms have deployed various strategies to counteract the toxic effects of antibiotics. These include active efflux of the antibiotic from the microbial cell; modification of antibiotic targets; and enzymatic modification of the antibiotic [2]. Heavy metals (HMs) accumulate in the environment, and since HMs do not degrade, they create permanent and selective stress on environmental microbes. Even a sub-dose of Cr (especially the Cr(VI) state) can promote plasmid-mediated horizontal gene transfer (HGT) [3], causing enrichment of heavy metal resistance genes (HRGs), threatening environmental safety [4-7]. Meanwhile, due to the co-selection effect, the long-term existence of HMs also causes the enrichment of antibiotic-resistant bacteria, further increasing the resistance gene spreading risk in the environment [8]. Although the research on ARGs in soil is extensive, the study of MFC operation on HRGs in soil is still very scarce.
In the revised manuscript, we will further supplement the relevant content in the introduction.
Some of the newly added references are as follows:
[1] Ashbolt Nicholas, J., Amézquita, A., Backhaus, T., Borriello, P., Brandt Kristian, K., Collignon, P., Coors, A., Finley, R., Gaze William, H., Heberer, T., Lawrence John, R., Larsson, D.G.J., McEwen Scott, A., Ryan James, J., Schönfeld, J., Silley, P., Snape Jason, R., Van den Eede, C., Topp, E., (2013). Human Health Risk Assessment (HHRA) for Environmental Development and Transfer of Antibiotic Resistance. Environmental Health Perspectives 121(9), 993-1001. https://doi.org/10.1289/ehp.1206316.
[2] van Hoek, A.H., Mevius, D., Guerra, B., Mullany, P., Roberts, A.P., Aarts, H.J., (2011). Acquired Antibiotic Resistance Genes: An Overview. Frontiers in Microbiology 2. https://doi.org/10.3389/fmicb.2011.00203.
[3] Zhang, Y., Gu, A.Z., Cen, T., Li, X., He, M., Li, D., Chen, J., (2018). Sub-inhibitory concentrations of heavy metals facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes in water environment. Environmental Pollution 237, 74-82. https://doi.org/10.1016/j.envpol.2018.01.032.
[4] Guo, S., Xiao, C., Zhou, N., Chi, R., (2021). Speciation, toxicity, microbial remediation and phytoremediation of soil chromium contamination. Environmental Chemistry Letters 19(2), 1413-1431. https://doi.org/10.1007/s10311-020-01114-6.
[5] Wang, C., Jia, Y., Li, J., Li, P., Wang, Y., Yan, F., Wu, M., Fang, W., Xu, F., Qiu, Z., (2023). Influence of microbial augmentation on contaminated manure composting: metal immobilization, matter transformation, and bacterial response. J. Hazard. Mater. 441, 129762. https://doi.org/https://doi.org/10.1016/j.jhazmat.2022.129762.
[6] Wang, C., Jia, Y., Li, J., Wang, Y., Niu, H., Qiu, H., Li, X., Fang, W., Qiu, Z., (2023). Effect of bioaugmentation on tetracyclines influenced chicken manure composting and antibiotics resistance. Science of The Total Environment 867. https://doi.org/10.1016/j.scitotenv.2023.161457.
[7] Wang, C., Tan, H., Li, H., Xie, Y., Liu, H., Xu, F., Xu, H., (2020). Mechanism study of Chromium influenced soil remediated by an uptake-detoxification system using hyperaccumulator, resistant microbe consortium, and nano iron complex. Environ. Pollut. 257, 113558. https://doi.org/10.1016/j.envpol.2019.113558.
[8] Men, C., Liu, R., Xu, F., Wang, Q., Guo, L., Shen, Z., (2018). Pollution characteristics, risk assessment, and source apportionment of heavy metals in road dust in Beijing, China. Science of The Total Environment 612, 138-147. https://doi.org/https://doi.org/10.1016/j.scitotenv.2017.08.123.
- Some writing details need attention. For example, the subtitle of a paper is sorted incorrectly. Such as section 2.4.
Response: Thank you for your careful examination. During revision, we will carefully review and revise the chapter order to ensure its accuracy.
The original title "2.6 Microbial response during operation " will be amended to "2.5 Microbial response during operation "; The original title "2.6.1 Soil biochemical response " will be amended to "2.5.1 Soil biochemical response "; The original title "2.6.2 Microbial community structure " will be amended to "2.5.2 Microbial community structure "; The original title "2.6.3 HRG Fluctuation " will be amended to "2.5.3 HRG Fluctuation "; The original title "2.7 Data analysis " will be amended to "2.6 Data analysis ";
- Lines 77-78, please explain why the author chose to add 118.8 mg/kg chromium to the soil.
Response: Thank you for your kind suggestion. In the current study, the Cr concentration in the soil was set referring the Chinese Soil Environmental Quality Standard (GB15618-2018). The standard stimulated that the first-level risk value of heavy metal risk control in agricultural land soil (if it exceeds the value, there will be soil ecological environment pollution risk) is between 90-150 mg/kg for Cr. At the same time, we refer to other similar soil studies to determine the final chromium concentration in the soil is about 130 mg/kg, and the final value was 118.8mg/kg [1-3]. We will elaborate on this in the revised document.
Some of the newly added references are as follows:
[1] Li, Y., Lin, J., Wu, Y., Jiang, S., Huo, C., Liu, T., Yang, Y., Ma, Y., (2024). Transformation of exogenous hexavalent chromium in soil: Factors and modelling. Journal of Hazardous Materials 480, 135799. https://doi.org/https://doi.org/10.1016/j.jhazmat.2024.135799.
[2] Liu, S., Pu, S., Deng, D., Huang, H., Yan, C., Ma, H., Razavi, B.S., (2020). Comparable effects of manure and its biochar on reducing soil Cr bioavailability and narrowing the rhizosphere extent of enzyme activities. Environment International 134, 105277. https://doi.org/https://doi.org/10.1016/j.envint.2019.105277.
[3] Mandal, S., Sarkar, B., Bolan, N., Ok, Y.S., Naidu, R., (2017). Enhancement of chromate reduction in soils by surface modified biochar. Journal of Environmental Management 186, 277-284. https://doi.org/https://doi.org/10.1016/j.jenvman.2016.05.034.
- In section 2.5.3, it is hoped that the author can provide specific operations on " The abundance of HRGs in the surface soil of SMFC and OMFC anode after the operation was analyzed using an SYBR Green real-time fluorescence quantitative PCR system ".
Response: Thank you for your professional advice. In the revised manuscript, the related paragraph was revised as:
Microbial DNA Rapid extraction kit (Shenggong Bioengineering Co., LTD., Shanghai, China) was used to extract total DNA from fresh samples. The abundance of HRGs in the surface soil of SMFC and OMFC anode after operation was analyzed using a SYBR Green real-time fluorescence quantitative PCR system (7500, Thermo Fisher, USA) [1]. The soil of OMFC was used for comparison. The detected genes included HRGs (chrA, chrB, chrR, recG, nfsA, zupT, fpvA) and MGEs (intI, tnpA02, tnpA04, tnpA05). The primer sequences are provided in Table S2. The specific detection steps were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s. Forty cycles were performed to make three replicates, and 16S rRNA was used as the internal reference gene. The relative gene expression results were analyzed using the 2^(-ΔΔCt) method, which is commonly used for relative quantification, where ΔΔCt = (Ct target gene - Ct internal reference gene) experimental group - (Ct target gene - Ct internal reference gene) control group.
Some of the newly added references are as follows:
[1] Wang, C., Jia, Y., Li, J., Li, P., Wang, Y., Yan, F., Wu, M., Fang, W., Xu, F., Qiu, Z., (2023). Influence of microbial augmentation on contaminated manure composting: metal immobilization, matter transformation, and bacterial response. J. Hazard. Mater. 441, 129762. https://doi.org/https://doi.org/10.1016/j.jhazmat.2022.129762.
- Lines 127-137, for the determination method of Microbial community structure, it is suggested that the author supplement the relevant references.
Response: Thank you for your helpful advice. We will supplement the methods for determining microbial community structure in Section 2.5.2 with references. Some of the newly added references are as follows:
[1] Bokulich, N. A. , Kaehler, B. D. , Ram, R. J. , Matthew, D. , Evan, B. , & Rob, K. , et al. (2018). Optimizing taxonomic classification of marker-gene amplicon sequences with qiime 2’s q2-feature-classifier plugin. Microbiome, 6(1), 90-.
[2] Deng, Y., Jiang, Y. H., Yang, Y., He, Z., Luo, F., and Zhou, J. (2012). Molecular ecological network analyses. BMC Bioinformatics 13(1), 113-.
[3] Faust, K., and Raes, J. (2012). Microbial interactions: from networks to models. Nat Rev Microbiol 10, 538-550.
- In lines 181-183, The authors need a more complete explanation for why the voltage of SMFC drops rapidly in the first week and then rises again.
Response: Thank you for your professional advice. In the revised manuscript, the related paragraph was revised as:
Initially, CMFC showed a working circuit voltage (WCV) of 0.55 V and an open circuit voltage (OCV) of 0.68 V (Fig. 2g). In the first week, WCV dropped quickly to 0.45 V but bounced back and stabilized at 0.75 V on day 25, implying the adaptation process of the anode microbes in the soil. We reasonably concluded that in the complex heterogeneous environment of soil, the anodic electrochemical active bacteria need some time to adapt to fluctuating environmental conditions facing environmental disturbances. During SMFC operation, the anode microbial community could be gradually selected and enriched, and a stable adaptive community is formed, so the SMFC voltage tends to be stable. This conjecture is also reflected in subsequent results.
- The Cr shown in both Figure 1e and Figure 1k is easy to be confused. It is recommended to mark them in the appropriate position in the diagram.
Response: Thank you for your kind suggestion. We will attach clear and eye-catching ICONS to the relevant images in the revised manuscript.
The corresponding picture modification can be seen in the attachment.
Fig. 1 Characterization of electrode materials before and after operation by EDS and SEM. (a-c) EDS and SEM images of cathode loaded with Fe3O4; (d-f) EDS and SEM images of cathode after the SMFC operation; (g-h) EDS and SEM images of anode microorganisms; (j-l) EDS and SEM images of the anode after SMFC operation.
- Figure 2 shows that the cathode is GF while the anode is not represented by the material name. Please uniformly use the electrode or material noun to provide a good reading experience.
Response: Thanks for the suggestion. We will use uniform material nouns in the revised manuscript. And Figure is replotted as follows:
The corresponding picture modification can be seen in the attachment.
Fig. 2 Characterization of electrode materials. (a-b) Fe2p spectra of cathode/Fe3O4 composite cathode, (c) cyclic voltammetry (CV) curve of cathode/Fe3O4, (d-e) Cr2p spectra of GF composite cathode and Anodic Aluminum foam after operation, (f) XRD spectrum of the cathode-Fe3O4, Power generation performance of SMFC during long-term operation. (g)output voltage distribution, (h) polarization curves and power density curves (15-day vs. 30-day).
- In section 3.4.2, it is suggested that the author briefly explain why the soil biochemical indicators should be determined and what contribution these indicators have to the subject.
Response: Thanks for the suggestion.
Soil enzyme activity is an important index to evaluate soil environmental change[1-2]. Soil enzymes, as biocatalysts involved in biochemical reactions, play an important role in nutrient mineralization, decomposition of organic matter, and nutrient cycling. The dynamic measurement of soil enzyme activity in SMFC helps us to understand and analyze the change state of soil microecology. The interaction analysis of soil enzymes with other indicators will also help us to understand the specific action mechanism of microecology that contributes to Cr reduction. The results showed that the changes in soil enzyme activity were closely related to the changes in soil microbial community structure during SMFC operation, and the changes in the abundance of resistance genes were also reflected.
Some of the newly added references are as follows:
[1] Chen, Y., Zuo, M., Yang, D., He, Y., Wang, H., Liu, X., Zhao, M., Xu, L., Ji, J., Liu, Y., Gao, T., (2024). Synergistically Effect of Heavy Metal Resistant Bacteria and Plants on Remediation of Soil Heavy Metal Pollution. Water, Air, & Soil Pollution 235(5), 296. https://doi.org/10.1007/s11270-024-07100-w.
[2] Liu, H., Xu, F., Xie, Y., Wang, C., Zhang, A., Li, L., Xu, H., (2018). Effect of modified coconut shell biochar on availability of heavy metals and biochemical characteristics of soil in multiple heavy metals contaminated soil. Science of The Total Environment 645, 702-709. https://doi.org/https://doi.org/10.1016/j.scitotenv.2018.07.115.
- In Part 4, give the necessary description of Figure 6 “Cr(VI)reduction Mechanism of during SMFC operation”.
Response: Thank you for your kind advice. In the revised manuscript, the related paragraph was revised as:
Microorganisms have developed efficient detoxification strategies to counteract the toxic effects of heavy metal stress[1]. In SMFC system, Cr(VI) reduction is the synergistic result of multiple approaches. Adsorption/biological adsorption: including the adsorption of anode and cathode materials, surface catalyst adsorption, and microbial membrane adsorption; Bioelectrochemistry reduction and microbial reduction: including intracellular sequestration, export, reduced permeability, extracellular sequestration, and extracellular detoxification (Figure 6).
Some of the newly added references are as follows:
[1] Rouch, D.A., Lee, B.T.O., Morby, A.P., (1995). Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance. Journal of Industrial Microbiology 14(2), 132-141. https://doi.org/10.1007/BF01569895.
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