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Master of Science Thesis course 25 – 2021 Can Tho University Figure 4. 8 COD removal efficiency for MFCs using (A) different substrate types at 10mM and (B) mixtures of acetate and lactate (1:1 ratio) at different concentrations Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 39

Master of Science Thesis course 25 – 2021 Can Tho University In comparison, mixed acetate and lactate (at ratio 1:1 and 10 mM concentration) produced a lower result at 84  4% (Figure 4.8A). This result implies that the dominant bacterial species grown with acetate performed well in oxidizing acetate but not necessarily generating electricity; hence, it can be concluded that these bacteria are not electroactive. Comparing MFCs fed with both acetate and lactate (1:1 ratio) at different concentrations (Figure 4.8B), it was observed that the highest COD removal was obtained at 30 mM substrate concentration, which also showed the best electricity generation performance among the MFCs. This result suggests that at 30 mM mixed substrate, organic decomposition and electricity generation are enhanced, which implies that EAB growth was favored. However, COD removal decreased at 40 mM substrate concentration. This result is consistent with several studies where high substrate concentrations were found to inhibit power production in MFCs [151]. As expected, the initial COD values were proportional to the substrate concentration (i.e., higher initial COD at higher substrate concentration) (Figure 4.8). However, there is no trend found on the COD removal efficiency determined by the final COD reading. The final COD value reflects the organic compounds that are present after the oxidation process in the anode. These compounds may have remained un-decomposed together with byproducts of the bacterial culture present in the anode. The COD changes indicate the localized metabolism of the bacteria, but these do not differentiate consumption by EAB against non-electrogenic microorganisms [154]. The final COD values only show that the MFCs differed in the developed microbial mix culture, which further implies that the byproducts also vary according to the bacterial species that dominate the mixed culture. Since identifying bacterial species present was out of the scope of this study, predicting the type and amount of byproducts was not attempted. As a result, no trend on the final COD readings could be expected. HPLC data (Table 4.2) confirmed the presence of different organic compounds in varying amounts after the wastewater treatment process, which was initially either low in amount or not present in the anolyte samples. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 40

Master of Science Thesis course 25 – 2021 Can Tho University Table 4. 2 The substrate consumption analysis by HPLC Acetate 10mM Lactatate (mg/L) st nd rd th Lactate 10mM Acetatate (mg/L) Acetate and Lactate, 10mM Lactate (mg/L) 1 day 2 day 3 day 4 day Acetate and Lactate, 20mM Acetate (mg/L) - - - - Acetate and Lactate, 30mM Lactatate (mg/L) - - Acetate and Lactate, 40mM Acetatate (mg/L) 792.97 110.17 - - Lactate (mg/L) 1091.98 - - - Acetate (mg/L) - - - - - - - Lactatate (mg/L) 539.25 - - - Acetatate (mg/L) 385.17 - - - Lactate (mg/L) 1085.80 Acetate (mg/L) 805.58 183.10 - - 184.20 - 1665.25 - - 1220.44 619.41 - 138.94 2718.40 560.74 1634.02 - 1440.45 Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 41

Master of Science Thesis course 25 – 2021 Can Tho University Chapter 5: CONCLUSION AND SUGGESTIONS 5.1 CONCLUSION This study attempted to investigate the effect of substrates on biofilm formation and the efficiency of HCMFCs operating on recirculation mode using a mixed bacterial culture inherent in domestic wastewater as biological catalysts. This study further affirms that MFCs that treat domestic wastewater require a robust mixed substrate. The performance of the MFCs was found to be affected by the concentration of substrates. For systems supplied with different concentrations of mixture acetate and lactate, electricity production increased with COD value. The MFCs with the mixture of acetate and lactate at 30 mM concentration had the best power generation producing maximum power density up to 956.75 mW/m2, COD removal, and internal resistance among the four MFCs with different substrate concentrations. 80–91% COD removal efficiency was attained for different concentrations. Furthermore, it was found that internal resistance decreased with an increase in COD value. Additionally, among all the MFCs tested, effective substrate degradation having COD removal at 91.4% was observed with acetate. This research also showed that high substrate concentrations could inhibit the development and formation of biofilm systems. The results suggest that there is an optimal value for substrate concentration. These findings provide useful and progressive insights into future applications of MFCs in wastewater treatment. 5.2 SUGGESTIONS Based on the results of this study, the author recommends the following: The biome community in wastewater contains many species, their operation greatly affects the performance of MFC. They can support each other leading to enhanced performance, or they may inhibit species that generate power in MFC. Therefore, there is a need for further research on bacteria species present in wastewater. Further understanding of the bacterial species is comparable to other studies and explains the processes in this study. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 42

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Master of Science Thesis course 25 – 2021 Can Tho University 42. Liu, H., R. Ramnarayanan, and B.E. Logan, Production of electricity during wastewater treatment using a single chamber microbial fuel cell. Environmental science & technology, 2004. 38(7): p. 2281-2285. 43. Rabaey, K., et al., Tubular microbial fuel cells for efficient electricity generation. Environmental science & technology, 2005. 39(20): p. 8077-8082. 44. He, Z., S.D. Minteer, and L.T. Angenent, Electricity generation from artificial wastewater using an upflow microbial fuel cell. Environmental science & technology, 2005. 39(14): p. 5262-5267. 45. Min, B. and B.E. Logan, Continuous electricity generation from domestic wastewater and organic substrates in a flat plate microbial fuel cell. Environmental science & technology, 2004. 38(21): p. 5809-5814. 46. Tender, L.M., et al., Harnessing microbially generated power on the seafloor. Nature biotechnology, 2002. 20(8): p. 821-825. 47. Hasvold, Ø., et al., Sea-water battery for subsea control systems. Journal of Power Sources, 1997. 65(1-2): p. 253-261. 48. Hutchinson, A.J., J.C. Tokash, and B.E. Logan, Analysis of carbon fiber brush loading in anodes on startup and performance of microbial fuel cells. Journal of Power Sources, 2011. 196(22): p. 9213-9219. 49. Rinaldi, A., et al., Engineering materials and biology to boost performance of microbial fuel cells: a critical review. Energy & Environmental Science, 2008. 1(4): p. 417-429. 50. Kalathil, S., S. Patil, and D. Pant, Microbial fuel cells: electrode materials. 2017, Elsevier, Amsterdam, The Netherlands. p. 1-10. 51. Zhou, M., et al., An overview of electrode materials in microbial fuel cells. Journal of Power Sources, 2011. 196(10): p. 4427-4435. 52. Rimboud, M., et al., Electroanalysis of microbial anodes for bioelectrochemical systems: basics, progress and perspectives. Physical Chemistry Chemical Physics, 2014. 16(31): p. 16349-16366. 53. Santoro, C., et al., Current generation in membraneless single chamber microbial fuel cells (MFCs) treating urine. Journal of Power Sources, 2013. 238: p. 190-196. 54. Lanas, V., Y. Ahn, and B.E. Logan, Effects of carbon brush anode size and loading on microbial fuel cell performance in batch and continuous mode. Journal of Power Sources, 2014. 247: p. 228-234. 55. Jiang, D. and B. Li, Granular activated carbon single-chamber microbial fuel cells (GAC-SCMFCs): a design suitable for large-scale wastewater treatment processes. Biochemical Engineering Journal, 2009. 47(1-3): p. 31-37. 56. Wu, S., et al., Combined carbon mesh and small graphite fiber brush anodes to enhance and stabilize power generation in microbial fuel cells treating domestic wastewater. Journal of Power Sources, 2017. 356: p. 348-355. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 46

Master of Science Thesis course 25 – 2021 Can Tho University 57. You, J., et al., Electricity generation and struvite recovery from human urine using microbial fuel cells. Journal of Chemical Technology & Biotechnology, 2016. 91(3): p. 647-654. 58. Ieropoulos, I., J. Greenman, and C. Melhuish, Microbial fuel cells based on carbon veil electrodes: stack configuration and scalability. International Journal of Energy Research, 2008. 32(13): p. 1228-1240. 59. Guerrini, E., et al., Performance explorations of single chamber microbial fuel cells by using various microelectrodes applied to biocathodes. International journal of hydrogen energy, 2014. 39(36): p. 21837-21846. 60. Zhao, F., et al., Activated carbon cloth as anode for sulfate removal in a microbial fuel cell. Environmental science & technology, 2008. 42(13): p. 4971-4976. 61. Santoro, C., et al., The effects of carbon electrode surface properties on bacteria attachment and start up time of microbial fuel cells. Carbon, 2014. 67: p. 128-139. 62. Seviour, T., et al., Voltammetric profiling of redox-active metabolites expressed by Pseudomonas aeruginosa for diagnostic purposes. Chemical Communications, 2015. 51(18): p. 3789-3792. 63. Jiang, D., et al., A pilot-scale study on utilizing multi-anode/cathode microbial fuel cells (MAC MFCs) to enhance the power production in wastewater treatment. International Journal of Hydrogen Energy, 2011. 36(1): p. 876-884. 64. Baudler, A., S. Riedl, and U. Schröder, Long-term performance of primary and secondary electroactive biofilms using layered corrugated carbon electrodes. Frontiers in Energy Research, 2014. 2: p. 30. 65. Ter Heijne, A., et al., Performance of non-porous graphite and titanium- based anodes in microbial fuel cells. Electrochimica Acta, 2008. 53(18): p. 5697-5703. 66. He, G., et al., Effect of fiber diameter on the behavior of biofilm and anodic performance of fiber electrodes in microbial fuel cells. Bioresource technology, 2011. 102(22): p. 10763-10766. 67. Karra, U., et al., Power generation and organics removal from wastewater using activated carbon nanofiber (ACNF) microbial fuel cells (MFCs). International journal of hydrogen energy, 2013. 38(3): p. 1588-1597. 68. Chen, S., et al., A three‐dimensionally ordered macroporous carbon derived from a natural resource as anode for microbial bioelectrochemical systems. ChemSusChem, 2012. 5(6): p. 1059-1063. 69. Zheng, S., et al., Binder-free carbon black/stainless steel mesh composite electrode for high-performance anode in microbial fuel cells. Journal of Power Sources, 2015. 284: p. 252-257. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 47

Master of Science Thesis course 25 – 2021 Can Tho University 70. Baudler, A., et al., Does it have to be carbon? Metal anodes in microbial fuel cells and related bioelectrochemical systems. Energy & Environmental Science, 2015. 8(7): p. 2048-2055. 71. Santoro, C., et al., Influence of anode surface chemistry on microbial fuel cell operation. Bioelectrochemistry, 2015. 106: p. 141-149. 72. Li, B., et al., Surface modification of microbial fuel cells anodes: approaches to practical design. Electrochimica Acta, 2014. 134: p. 116-126. 73. Pan, K. and P. Zhou, Performance enhancement with a hydrophilic self- immobilized redox mediator modified anode in Chlorella vulgaris-based microbial solar cell. ACS Sustainable Chemistry & Engineering, 2015. 3(9): p. 1974-1981. 74. Zhang, C., et al., Enhanced power generation of microbial fuel cell using manganese dioxide-coated anode in flow-through mode. Journal of Power Sources, 2015. 273: p. 580-583. 75. Wei, J., P. Liang, and X. Huang, Recent progress in electrodes for microbial fuel cells. Bioresource technology, 2011. 102(20): p. 9335-9344. 76. Zhou, M., et al., Anode modification by electrochemical oxidation: A new practical method to improve the performance of microbial fuel cells. Biochemical Engineering Journal, 2012. 60: p. 151-155. 77. Guo, K., et al., Engineering electrodes for microbial electrocatalysis. Current opinion in biotechnology, 2015. 33: p. 149-156. 78. Epifanio, M., et al., Effects of atmospheric air plasma treatment of graphite and carbon felt electrodes on the anodic current from Shewanella attached cells. Bioelectrochemistry, 2015. 106: p. 186-193. 79. Blanchet, E., et al., Two-dimensional carbon cloth and three-dimensional carbon felt perform similarly to form bioanode fed with food waste. Electrochemistry Communications, 2016. 66: p. 38-41. 80. Babauta, J.T., et al., Microscale gradients of oxygen, hydrogen peroxide, and pH in freshwater cathodic biofilms. ChemSusChem, 2013. 6(7): p. 1252. 81. Rahimnejad, M., et al., A review on the effect of proton exchange membranes in microbial fuel cells. Biofuel Research Journal, 2014. 1(1): p. 7-15. 82. Leong, J.X., et al., Ion exchange membranes as separators in microbial fuel cells for bioenergy conversion: a comprehensive review. Renewable and Sustainable Energy Reviews, 2013. 28: p. 575-587. 83. Winfield, J., et al., Towards disposable microbial fuel cells: natural rubber glove membranes. International journal of hydrogen energy, 2014. 39(36): p. 21803-21810. 84. Zhuang, L., et al., Membrane-less cloth cathode assembly (CCA) for scalable microbial fuel cells. Biosensors and Bioelectronics, 2009. 24(12): p. 3652-3656. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 48

Master of Science Thesis course 25 – 2021 Can Tho University 85. Ieropoulos, I., et al. EcoBot-III-A Robot with Guts. in ALIFE. 2010. 86. Philamore, H., et al., Cast and 3D printed ion exchange membranes for monolithic microbial fuel cell fabrication. Journal of Power Sources, 2015. 289: p. 91-99. 87. Winfield, J., et al., Urine-activated origami microbial fuel cells to signal proof of life. Journal of Materials Chemistry A, 2015. 3(13): p. 7058-7065. 88. Zuo, Y., et al., Tubular membrane cathodes for scalable power generation in microbial fuel cells. Environmental science & technology, 2007. 41(9): p. 3347-3353. 89. Hernández-Fernández, F., et al., New application of supported ionic liquids membranes as proton exchange membranes in microbial fuel cell for waste water treatment. Chemical Engineering Journal, 2015. 279: p. 115-119. 90. Bergel, A., D. Féron, and A. Mollica, Catalysis of oxygen reduction in PEM fuel cell by seawater biofilm. Electrochemistry Communications, 2005. 7(9): p. 900-904. 91. Reimers, C.E., et al., Harvesting energy from the marine sediment− water interface. Environmental science & technology, 2001. 35(1): p. 192-195. 92. Kundu, P.P. and K. Dutta, Progress and recent trends in microbial fuel cells. 2018: Elsevier. 93. Cheng, S., H. Liu, and B.E. Logan, Power densities using different cathode catalysts (Pt and CoTMPP) and polymer binders (Nafion and PTFE) in single chamber microbial fuel cells. Environmental science & technology, 2006. 40(1): p. 364-369. 94. Lan, T.-H., et al., 2D Numerical physical model settings for three electron transfer pathways in microbial fuel cells. Sensors and Materials, 2017. 29(7): p. 1055-1060. 95. Pankratova, G., L. Hederstedt, and L. Gorton, Extracellular electron transfer features of Gram-positive bacteria. Analytica chimica acta, 2019. 1076: p. 32-47. 96. Sydow, A., et al., Electroactive bacteria—molecular mechanisms and genetic tools. Applied microbiology and biotechnology, 2014. 98(20): p. 8481-8495. 97. Torres, C.I., et al., A kinetic perspective on extracellular electron transfer by anode-respiring bacteria. FEMS microbiology reviews, 2010. 34(1): p. 3-17. 98. Logan, B.E., Exoelectrogenic bacteria that power microbial fuel cells. Nature Reviews Microbiology, 2009. 7(5): p. 375-381. 99. Carlson, H.K., et al., Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria. Proceedings of the National Academy of Sciences, 2012. 109(5): p. 1702-1707. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 49

Master of Science Thesis course 25 – 2021 Can Tho University 100. Light, S.H., et al., A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria. Nature, 2018. 562(7725): p. 140-144. 101. Holmes, D.E., et al., Genes for two multicopper proteins required for Fe (III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes. Microbiology, 2008. 154(5): p. 1422-1435. 102. Lovley, D.R., Syntrophy goes electric: direct interspecies electron transfer. Annual review of microbiology, 2017. 71: p. 643-664. 103. Rotaru, A.-E., et al., A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energy & Environmental Science, 2014. 7(1): p. 408-415. 104. Ha, P.T., et al., Syntrophic anaerobic photosynthesis via direct interspecies electron transfer. Nature communications, 2017. 8(1): p. 1-7. 105. Tang, J., et al., Secondary mineralization of ferrihydrite affects microbial methanogenesis in Geobacter-Methanosarcina cocultures. Applied and environmental microbiology, 2016. 82(19): p. 5869-5877. 106. Hasan, K., et al., Electrochemical communication between heterotrophically grown Rhodobacter capsulatus with electrodes mediated by an osmium redox polymer. Bioelectrochemistry, 2013. 93: p. 30-36. 107. Lee, Y., et al., Flavin mononucleotide mediated microbial fuel cell in the presence of Shewanella putrefaciens CN32 and iron-bearing mineral. Biotechnology and bioprocess engineering, 2015. 20(5): p. 894-900. 108. Harrington, T.D., et al., The mechanism of neutral red-mediated microbial electrosynthesis in Escherichia coli: menaquinone reduction. Bioresource technology, 2015. 192: p. 689-695. 109. Koochana, P.K., et al., Phenothiazines and phenoxazines: as electron transfer mediators for ferritin iron release. Dalton transactions, 2019. 48(10): p. 3314-3326. 110. Sund, C.J., et al., Effect of electron mediators on current generation and fermentation in a microbial fuel cell. Applied Microbiology and Biotechnology, 2007. 76(3): p. 561-568. 111. Schröder, U., Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Physical Chemistry Chemical Physics, 2007. 9(21): p. 2619-2629. 112. Ren, Z., et al., Characterization of microbial fuel cells at microbially and electrochemically meaningful time scales. Environmental science & technology, 2011. 45(6): p. 2435-2441. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 50

Master of Science Thesis course 25 – 2021 Can Tho University 113. Stone, H.A. and C. Duprat, Low-Reynolds-number flows, in Fluid- Structure Interactions in Low-Reynolds-Number Flows. 2015, Royal Society of Chemistry. p. 25-77. 114. Schlichting, H. and K. Gersten, Boundary-layer theory. 2016: Springer. 115. Pham, H.T., et al., High shear enrichment improves the performance of the anodophilic microbial consortium in a microbial fuel cell. Microbial biotechnology, 2008. 1(6): p. 487-496. 116. Mohsen, Q., S.A. Fadl-Allah, and N.S. El-Shenawy, Electrochemical impedance spectroscopy study of the adsorption behavior of bovine serum albumin at biomimetic calcium–phosphate coating. Int. J. Electrochem. Sci, 2012. 7(5): p. 4510-4527. 117. He, Z. and F. Mansfeld, Exploring the use of electrochemical impedance spectroscopy (EIS) in microbial fuel cell studies. Energy & Environmental Science, 2009. 2(2): p. 215-219. 118. da Silva, A.M.V., R.J.B. da Silva, and M.F.G. Camões, Optimization of the determination of chemical oxygen demand in wastewaters. Analytica chimica acta, 2011. 699(2): p. 161-169. 119. Kishimoto, N. and M. Okumura, Feasibility of Mercury-free Chemical Oxygen Demand (COD) Test with Excessive Addition of Silver Sulfate. Journal of Water and Environment Technology, 2018. 16(6): p. 221-232. 120. GANCEDO, M.C. and B. Luh, HPLC analysis of organic acids and sugars in tomato juice. Journal of Food Science, 1986. 51(3): p. 571-573. 121. Sun, G., A. Thygesen, and A.S. Meyer, Acetate is a superior substrate for microbial fuel cell initiation preceding bioethanol effluent utilization. Applied microbiology and biotechnology, 2015. 99(11): p. 4905-4915. 122. Thangavel, S., et al., Protection efficiencies of surface‐active inhibitors in zinc‐air batteries. International Journal of Energy Research, 2020. 44(14): p. 11883-11893. 123. Kim, G., et al., Bacterial community structure, compartmentalization and activity in a microbial fuel cell. Journal of applied microbiology, 2006. 101(3): p. 698-710. 124. Liu, S., Chapter 11: How Cells Grow. Bioprocess Engineering. Elsevier, Amsterdam, 2013: p. 549. 125. Ge, Z., et al., Reducing effluent discharge and recovering bioenergy in an osmotic microbial fuel cell treating domestic wastewater. Desalination, 2013. 312: p. 52-59. 126. Chae, K.-J., et al., Effect of different substrates on the performance, bacterial diversity, and bacterial viability in microbial fuel cells. Bioresource technology, 2009. 100(14): p. 3518-3525. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 51

Master of Science Thesis course 25 – 2021 Can Tho University 127. Hibbing, M.E., et al., Bacterial competition: surviving and thriving in the microbial jungle. Nature reviews microbiology, 2010. 8(1): p. 15-25. 128. Jung, S. and J.M. Regan, Comparison of anode bacterial communities and performance in microbial fuel cells with different electron donors. Applied microbiology and biotechnology, 2007. 77(2): p. 393-402. 129. Das, B., et al., Crosslinked poly (vinyl alcohol) membrane as separator for domestic wastewater fed dual chambered microbial fuel cells. International Journal of Hydrogen Energy, 2020. 130. Choudhury, P., et al., Performance improvement of microbial fuel cell (MFC) using suitable electrode and Bioengineered organisms: A review. Bioengineered, 2017. 8(5): p. 471-487. 131. Wang, L., et al., Methane production in a bioelectrochemistry integrated anaerobic reactor with layered nickel foam electrodes. Bioresource technology, 2020. 313: p. 123657. 132. Artyushkova, K., et al., Relationship between surface chemistry, biofilm structure, and electron transfer in Shewanella anodes. Biointerphases, 2015. 10(1): p. 019013. 133. You, J., et al., Stability and reliability of anodic biofilms under different feedstock conditions: Towards microbial fuel cell sensors. Sensing and bio- sensing research, 2015. 6: p. 43-50. 134. Ghangrekar, M. and V. Shinde, Performance of membrane-less microbial fuel cell treating wastewater and effect of electrode distance and area on electricity production. Bioresource Technology, 2007. 98(15): p. 2879-2885. 135. Kondaveeti, S.K., J.S. Seelam, and G. Mohanakrishna, Anodic electron transfer mechanism in bioelectrochemical systems, in Microbial Fuel Cell. 2018, Springer. p. 87-100. 136. Sure, S., et al., Microbial nanowires: an electrifying tale. Microbiology, 2016. 162(12): p. 2017-2028. 137. Malvankar, N.S. and D.R. Lovley, Microbial nanowires: a new paradigm for biological electron transfer and bioelectronics. ChemSusChem, 2012. 5(6): p. 1039-1046. 138. He, L., et al., Advances in microbial fuel cells for wastewater treatment. Renewable and Sustainable Energy Reviews, 2017. 71: p. 388-403. 139. Amano, N., et al., Methylomusa anaerophila gen. nov., sp. nov., an anaerobic methanol-utilizing bacterium isolated from a microbial fuel cell. International journal of systematic and evolutionary microbiology, 2018. 68(4): p. 1118-1122. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 52

Master of Science Thesis course 25 – 2021 Can Tho University 140. Xiang, K., et al., GldA overexpressing-engineered E. coli as superior electrocatalyst for microbial fuel cells. Electrochemistry communications, 2009. 11(8): p. 1593-1595. 141. Qiao, Y., X.-S. Wu, and C.M. Li, Interfacial electron transfer of Shewanella putrefaciens enhanced by nanoflaky nickel oxide array in microbial fuel cells. Journal of Power Sources, 2014. 266: p. 226-231. 142. Ieropoulos, I., J. Winfield, and J. Greenman, Effects of flow-rate, inoculum and time on the internal resistance of microbial fuel cells. Bioresource technology, 2010. 101(10): p. 3520-3525. 143. Hong, Y., et al., Adaptation to high current using low external resistances eliminates power overshoot in microbial fuel cells. Biosensors and Bioelectronics, 2011. 28(1): p. 71-76. 144. Winfield, J., et al., The overshoot phenomenon as a function of internal resistance in microbial fuel cells. Bioelectrochemistry, 2011. 81(1): p. 22-27. 145. Pasternak, G., J. Greenman, and I. Ieropoulos, Dynamic evolution of anodic biofilm when maturing under different external resistive loads in microbial fuel cells. Electrochemical perspective. Journal of power sources, 2018. 400: p. 392-401. 146. Liang, P., et al., Composition and distribution of internal resistance in three types of microbial fuel cells. Applied Microbiology and Biotechnology, 2007. 77(3): p. 551-558. 147. Liu, P., et al., Stimulated electron transfer inside electroactive biofilm by magnetite for increased performance microbial fuel cell. Applied energy, 2018. 216: p. 382-388. 148. Zhang, F., et al., Mesh optimization for microbial fuel cell cathodes constructed around stainless steel mesh current collectors. Journal of Power Sources, 2011. 196(3): p. 1097-1102. 149. Wang, C.-T., et al., Exposing effect of comb-type cathode electrode on the performance of sediment microbial fuel cells. Applied Energy, 2017. 204: p. 620-625. 150. Harimawan, A., et al., Influence of Electrode Distance on Electrical Energy Production of Microbial Fuel Cell using Tapioca Wastewater. Journal of Engineering and Technological Sciences, 2018. 50(6): p. 841-855. 151. Khater, D.Z., K. El-Khatib, and H.M. Hassan, Microbial diversity structure in acetate single chamber microbial fuel cell for electricity generation. Journal of Genetic Engineering and Biotechnology, 2017. 15(1): p. 127-137. 152. Lal, D., Microbes to generate electricity. Indian journal of microbiology, 2013. 53(1): p. 120-122. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 53

Master of Science Thesis course 25 – 2021 Can Tho University 153. Oh, S. and B.E. Logan, Hydrogen and electricity production from a food processing wastewater using fermentation and microbial fuel cell technologies. Water research, 2005. 39(19): p. 4673-4682. 154. Asensio, Y., et al., Influence of the fuel and dosage on the performance of double-compartment microbial fuel cells. Water research, 2016. 99: p. 16-23. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 54

Received: 25 September 2020 Revised: 27 January 2021 Accepted: 29 January 2021 DOI: 10.1002/er.6550 RESEARCH ARTICLE Lactate and acetate applied in dual-chamber microbial fuel cells with domestic wastewater Kha Lil Dinh1 | Chin-Tsan Wang2 | Hue Ngan Dai3 | Van Man Tran3 | My Loan Phung Le3 | Imee A. Saladaga4 | Yu-An Lin5 1Department of Chemistry, College of Summary Natural Sciences, Can Tho University, For about 20 years, microbial fuel cells (MFCs) are an emerging technology Can Tho City, Vietnam that has gained attention for its new wastewater treatment and energy genera- 2Department of Mechanical and Electro- tion, especially its ability to convert chemical energy from a broad range of Mechanical Engineering, National Ilan substances into electricity. However, MFC has not been widely commercial- University, Yilan City, Taiwan ized due to low efficiency. Studies have shown that substrate loading is an 3Department of Physical Chemistry, important factor in scaling up. Therefore, this study investigates the effect of Faculty of Chemistry, VNUHCM– substrate type and concentration on honeycomb MFCs (HCMFCs). The effect University of Science, Ho Chi Minh City, of different concentrations ranging from 10 to 40 mM of lactate and acetate Vietnam (1:1 ratio) substrates was investigated. Power efficiency was analyzed using 4Department of Chemical Engineering, polarization and power density curves. Results showed that the performance College of Engineering, Eastern Visayas of MFCs and biofilm formation is affected by the substrates. Scanning electron State University, Tacloban City, microscopy showed some changes in biofilm formation. Mixing lactate and Philippines acetate at 30 mM gave the best performance with a power density of 5Department of Biotechnology and 956.75 mW m−2 and chemical oxygen demand removal of 87.8%. Furthermore, Animal Science, National Ilan University, effective substrate degradation, having COD removal of 91.4%, was observed Yilan City, Taiwan with acetate. Highlights Correspondence • Substrate type and concentration affect MFCs performance significantly Chin-Tsan Wang, Department of • Mixed substrate promotes robust mix bacterial culture Mechanical and Electro-Mechanical • Substrate concentration of 30 mM gave the best power generation Engineering, National Ilan University, • Adverse effect of high substrate concentration on power generation was Yilan City, Taiwan. Email: [email protected] confirmed • Effective substrate degradation with COD removal of 91.4% was observed Van Man Tran, Department of Physical Chemistry, Faculty of Chemistry, with acetate VNUHCM–University of Science, Ho Chi Minh City, Vietnam. KEYWORDS Email: [email protected] acetate, biofilm, energy, lactate, microbial fuel cells, substrate, wastewater treatment 1 | INTRODUCTION world is aiming to use renewable energy sources as one of the ways to help solve the problem of increasing Fossil fuels are used at a much higher rate than they are energy demand. MFCs are promising in wastewater created, making them non-renewable sources. Fossil fuel treatment and energy recovery applications.1 The extra- consumption worsens environmental concerns. The cellular microbes in MFCs have an essential role in the Int J Energy Res. 2021;45:10655–10666. wileyonlinelibrary.com/journal/er © 2021 John Wiley & Sons Ltd 10655

10656 DINH ET AL. transfer of electrons outside the cell. Exoelectrogenic anode bacteria to generate electricity. Lactate and acetate bacteria in the anode compartment oxidize the substrate are known fermentation products and a simple form of and convert the chemical energy in the organic com- carbon easily used by anodic bacteria. Therefore, in this pounds energy sources to electrical energy.2 Unlike tradi- study, acetates and lactates were applied to an HCMFC tional fuel cells, MFCs can function using a wide range to examine the effect of substrate type and concentration of operations and a variety of substrates, making it a ver- on the biofilm formation on its anode electrode using a satile technology.3 The microorganisms in electrically mixture of bacteria available in domestic wastewater active biofilms are the main actors in any biochemical treatment plants. Recirculation was applied to the reactor, which involves complex electrochemical and cat- HCMFC and substrate utilization efficiency was studied. alytic reactions mediated by microorganisms. The The combination of native bacteria available in real growth and development of electrochemically active bac- wastewater with the application of continuous flow teria (EAB) on an anode electrode oxidize organic matter brings MFC technology closer to application based on the and transfers electrons to the anode by several existing conditions in the wastewater treatment plant. mechanisms.4 The HCMFC design is considered to be very benefi- The microbial community developed in the MFC cial in maintaining a uniform, symmetrical, and homoge- anode chamber showed diversity, not only limited to neous flow shape of the liquid inside the reactor. This common electrochemical active bacteria such as Geo- design affects the mass transfer of the substrate and ions bacter or Shewanella, but also other species.5 However, and ultimately improves the efficiency of MFCs. Until microorganisms are susceptible to changes in environ- now, the use of honeycomb MFCs as a flow rectifier is mental factors. Small fluctuations in the environment relatively new. The studies that have been published also cause biomes to change structure and diversity, include the study of MFC performance and its relation- which affects the output of the MFC.6 A new approach ship with flow properties and biofilm formation. The is to use a community of indigenous microorganisms. reactors achieved their maximum voltage and current density with reduced internal resistance when the opti- Many studies have analyzed the diversity of microbial mal flow rate was applied to the reactors having a value communities in wastewater. Zhang et al (2019) examined of 40 mL min−1.15-17 The influence of the hydrodynamic the microbiological community structure of activated boundary layer on the biofilm formation and the mass sludge in the urban wastewater treatment system transfer of the substrate in MFCs were observed. The (Zhuzhou City, China). Results showed that Prote- results show that a thin hydrodynamic boundary with a obacteria, Actinobacteria, Chloroflexi, Acidobacteria, Bac- boundary layer thickness of 1.6 cm creates a high voltage teroidetes, Actinobacteria, and Firmicutes were the most output.16 Furthermore, the effect of the distance of elec- dominant phyla of the five activated sludge samples.7 trodes on the performance of HCMFCs has also been Yang et al (2011) has reported Proteobacteria, Bacte- studied.18 roidetes, and Firmicutes as the most abundant phylum clusters at the phylum level in sludge in wastewater treat- 2 | MATERIALS AND METHODS ment plants.8 Many species of bacteria have been detected in wastewater such as Salmonella, Shigella, 2.1 | MFCs system design Escherichia coli, Aeromonas, Legionella, pneumonia, Lepto- spira, Achromobacter, Bacillus, Enterococcus, Erwinia, Dual-chambered reactors were assembled with Pseudomonas, Shigella, Staphylococcus, Streptococcus, Bur- polymethyl methacrylate (PMMA) sheets. The anode kholderia, Shewanella, Enterobacter cloacae, Enterococcus, compartment with a total working volume of 1 L con- Klebsiella Proteus vulgarism, and Shigellagenera.9,10 Most sisted of several chambers: A flow chamber (10 cm × of them are EAB and were applied in MFCs studies. The 10 cm × 12 cm) in the middle of the reactor, one reser- use of diverse existing indigenous microorganism culture voir with a honeycomb structure (5 cm × 5 cm × 8 cm) is a practical approach. on each side of the flow chamber and one surge tank (7 cm × 5 cm × 8 cm) next to each honeycomb reservoir Furthermore, the substrate is one of the most critical (Figure 1). The flow chamber contains the anode elec- factors affecting electricity generation because it serves as trode. The honeycomb structure was made up of equal a source of nutrients and energy for microorganisms' length plastic cylindrical tubes (5 cm) and radius growth. It affects the bacterial diversity in the anode bio- (0.025 cm) arranged to create a uniform and straightened film of MFC.11 Recent studies have mainly focused on substrate flow. Another chamber with a working volume available carbon sources: bamboo fermentation of 0.125 L (5 cm × 5 cm × 6 cm) attached to the anode effluent,12 pretreated latex wastewater,13 and seafood industrial wastewater.14 These complex molecular sub- strates require fermentation before being used by the

DINH ET AL. 10657 F I G U R E 1 Perspective view of recirculation dual chambers HCMFC [Colour figure can be viewed at wileyonlinelibrary.com] flow chamber served as the cathode compartment. It is saline (PBS) solution in a ratio of 1:1. The wastewater separated from the anode by a Nafion-117 PEM film had the following properties: COD: 273 mg L−1, bacterial (projected area of 5 cm × 5 cm; DuPont Co., USA). To count in LB agar: 1.86 × 108 CFU mL−1, pH: 7.42. PBS remove impurities, the PEM was soaked in 3% H2O2 was composed of the following: Na2HPO4, 9.16 g L−1; solution at 75C for 1 hour, washed with deionized NaH2PO4Á2H2O, 5.56 g L−1; NH4Cl, 0.638 g L−1; and KCl, water, immersed in 0.5 M H2SO4 at 75C for 1 hour, and 0.278 g L−1 with the pH value of 7.0.20 All MFCs were rinsed with deionized water. The membranes were operated and tested at 25C with the anolyte continu- submerged in deionized water to preserve membrane ously recirculated at 40 mL min−1 using a peristaltic conductivity by filling the anode and cathode compart- ments.19 Graphite felts with titanium wire head (pro- pump (Masterflex, model no. 7524-50, Cole-Parmer jected area of 5 cm × 5 cm, CeTech Co., Ltd., Taiwan) Instrument Company, USA).17 The catholyte was a 1:1 were used as anode and cathode electrodes, with elec- mixture of 100 mM K3[Fe(CN)6] (32.924 g L−1) and trode spacing at 0.0 cm.18 To enhance bacterial attach- 100 mM PBS solution. Six MFCs having different sub- ment, these were treated in 10% H2O2 solution at 90C to 100C for 3 hours and air-dried for 2 days. A titanium strates and concentrations were employed and labeled wire was connected to a 1000-Ω external resistance dur- ing cell operation.20 as follows: MFC1 - acetate, 10 mM; MFC2 - lactate, 2.2 | MFCs inoculation and operation 10 mM; MFC3 - acetate and lactate, 10 mM; MFC4 - The anolyte composed of a mixture of domestic wastewa- acetate and lactate, 20 mM; MFC5 - acetate, and lac- ter taken from Lou Dong wastewater treatment plant, Yilan City, Taiwan, and 100 mM phosphate-buffered tate, 30 mM; and, MFC6 - acetate and lactate, 40 mM. The anolyte and catholyte were replaced with new solutions at the end of each fed-batch cycle indicated by a decline of production voltage to 10% of the maxi- mum generated voltage. MFC operation was con- ducted for 30 days to ensure that steady voltage is achieved for at least three consecutive cycles before output measurements.

10658 DINH ET AL. 2.3 | Electrochemical analysis Agilent 1200 HPLC system with a refractive index detector. 1-mL aliquots of the anolyte solutions were collected at the The current (I), unit: ampere (A) was calculated using start and end of each fed-batch cycle. A Bio-Rad HPX-87 H Ohm's law, I = U/R, where U represents the voltage out- column was used with sulfuric acid as the mobile phase at put, unit: volts (V) and R is the external load resistance, a flow rate of 0.6 mL min−1.24 unit: Ohm (Ω). Power (P), unit: watts (W) was calculated by charging the voltage and amperage: P = U × I. The 3 | RESULTS AND DISCUSSION power density was calculated by the surface of the elec- trode; PD = P/Aanode, where Aanode is the total surface 3.1 | Effects of substrate type and area of the anode electrode in square meter (m2). concentration on MFCs performance Electrochemical Impedance Spectroscopy (EIS) was The voltage from the MFCs was allowed to stabilize for done by applying a 10-mV AC signal using HIOKY three consecutive cycles, known as the acclimation 3522-50 LCR Hi-TESTER (Japan) within a frequency phase, in which the biofilm in the anode matures. The range of 100 kHz to 0.1 Hz. The signal was chosen to be polarization curves showing current density as a function small enough to prevent the biofilm from detaching from of voltage are presented in Figure 2. The graph shows the electrode and minimize the disturbance on the sys- that the MFCs do not significantly differ in the maximum tem's stability.21,22 An equivalent circuit was used to sim- voltage generated, which is about 720 ± 20 mV. ulate EIS spectra and calculate internal resistance values in EC-Lab software. An in-depth analysis of reactor performance was done using the power density curves in Figure 3. The polariza- 2.4 | Chemical characterization tion and power curves determine the reactor's perfor- mance under external load and show losses in the reactor Chemical oxygen demand (COD) removal analysis was (activation, ohmic, and concentration).25 conducted using a TR-1100 COD thermoreactor (Suntex Instruments Co., Ltd., Taiwan). The anolyte liquid was The power density was calculated from the current collected at the start and end of each fed-batch cycle, density data. The influence of substrate type can be seen which usually takes 24 hours. The filtered 2-mL samples from the results in Figure 3A. The graph shows a maxi- were taken and then added to the COD vial. These were mum power density of 554.9 mW m−2 and 593.4 mW m−2 heated to 150C for 2 hours and cooled down for another produced from lactate and acetate, respectively. The max- 2 hours. Finally, the sample concentrations were mea- imum power density of 855.8 mW m−2 was achieved sured using a spectrophotometer (pHotoFlex, WTW).23 using the MFCs with a mixture of acetate and lactate at the same concentration (10 mM). These results show that The substrate concentrations in the anolyte liquid were using a mixture of the two has superior performance than measured using a high-performance liquid chromatography using each substrate individually. This difference in F I G U R E 2 Polarization curves A, different substrate types at 10 mM B, mixtures of acetate and lactate (1:1 ratio) at different concentrations [Colour figure can be viewed at wileyonlinelibrary.com]

DINH ET AL. 10659 F I G U R E 3 Power density curves A, different substrate types at 10 mM, B, mixtures of acetate and lactate (1:1 ratio) at different concentrations [Colour figure can be viewed at wileyonlinelibrary.com] performance is due to the microbial community's struc- in both acetate and lactate is more resilient in load con- ture and composition developed according to the sub- dition changes than those grown in a single substrate. strate, as suggested by a previous study.26 Studies have shown that a mix of cultures is better than a single culture.29 The result may indicate that the bacte- To investigate the effects of substrate concentration rial culture grown with the mixed substrate is not domi- on power generation performance, four MFCs con- nated by a single bacteria type but a rich culture of taining both acetate and lactate at a 1:1 ratio were oper- different kinds of bacteria. Mixed substrate leads to the ated batch-wise with recirculation and fed with growth of a mixed bacterial culture seen in previous substrates at concentrations of 10 mM, 20 mM, 30 mM, studies as more robust than pure culture.30 It was and 40 mM, respectively. It can be seen from Figure 3B observed that the higher the substrate concentration that as substrate concentration was increased from 10 to used was, the higher the power losses and the slower the 30 mM, power density increased because of the increased MFC power output recovery, and the less robust the availability of organic supply. Power density increased and MFC was. peaked at around 30 mM of 956.75 mW m−2. The decrease in power density at 40 mM suggests that a high substrate 3.2 | The overshoot phenomenon concentration harms microorganisms' growth, which leads to a reduction in MFC performance. At high concentra- Through specific experimental design and data analysis, tions of substrate, the substrate inhibits microbial growth, it was found that extracellular electron transfer mainly which decreases its rate. This substrate inhibition may be caused resistance in the anode, which reflects a power competitive or non-competitive similar to enzyme kinetics. overshoot in the polarization curve. In Figure 3 it was If the rate-limiting step during microbial growth is a observed that at high current densities on the polariza- single-substrate enzyme-catalyzed reaction, then the inhi- tion curves after the maximum power point, voltage and bition of the enzyme activity also prevents microbial current drop drastically (the curve retracts or “bends growth in a similar pattern.27 inwards”), resulting in lower power produced than previ- ously measured at lower current densities. It leads to High power output observed at laminar velocities was underestimating the best possible performance of reac- due to the sufficient distribution of substrate in the anode tors at higher current densities, and the reason for this leading to an effective transfer of organic supply to the spike remains a concern in many MFC studies. This phe- bacteria. This leads to more electrochemical activity and, nomenon, although not ideal, is widely seen in literature consequently, power production improvement. A laminar and is termed “power overshoot” or “Type D over- flow could eventually lead to mature and robust biology shoot.”31 This power overshoot is seen in a study using a with maximum performance.28 mixed bacterial culture.32 Type D overshoots could be In Figure 3, the MFCs with mixed substrate have the least power loss and recovers more quickly in the power density curve. It means that the bacterial culture grown

10660 DINH ET AL. avoided with the long-term use of external resistances. this in the polarization curve may indicate possible pol- This suggests the crucial changes that occur during accli- lutants or toxic elements.33 mation that affect the biofilm's ability to generate current. 3.3 | Effects of the substrate on anodic biofilm growth In Figure 3, after the power dips, the MFCs seem to recover their power generation even during the ongoing Voltage output was continuously monitored to investi- polarization sweep. At this period, the supply and gate the growth of microorganisms and biofilm formation demand of electrons and ions are balanced, and the cur- at the anode with Jiehan 5020 (Jiehan Technology Corpo- rent rises again. This recovery illustrates the bacterial ration, Taiwan), an automatic data logging system con- community's robustness and ability to adapt to changes nected with MFCs and 1000-Ω external resistors at a in conditions, even in hostile environments.31 The over- sampling rate of 5 minutes per point. Figure 4A presents shoot phenomenon was influenced by the internal resis- the voltage output and Figure 4B power density over time tance in MFCs. A healthy biofilm can reduce internal for MFCs at each substrate type with a 1000-Ω external resistance and eliminate any power overshoot. In con- resistance. The results showed that at a substrate concentra- trast, the destruction or inhibition of the microbial com- tion of 10 mM (MFC1, MFC2, and MFC3), the mixture of munity on the anode can cause increased internal acetate and lactate (MFC3) caused the best microorganism resistance, which decreases the performance of the MFCs and causes an overshoot. Furthermore, the presence of F I G U R E 4 Voltage output A, and power density B, of the MFCs during bacteria with the function of time acclimation at a 1000-Ω external resistance [Colour figure can be viewed at wileyonlinelibrary.com]

DINH ET AL. 10661 growth, as evident by higher output voltage values. Out- bacteria was observed for biofilm using acetate substrate put voltage started to become stable after 7 days for (Figure 5A). In contrast, a smooth biofilm was observed on 30 mM (MFC5) and 40 mM (MFC6) substrate concentra- the anode's surface using a lactate substrate (Figure 5B). tions. The other biofilms grown in lower substrate con- These only show that the type of substrate determines the centrations took more time to achieve stability around dominant bacteria types in the mixed culture. A combina- 10 days into the acclimation process. tion of smooth and clumpy growth was seen on the anode using a mixture of acetate and lactate substrates The current output depends only on the microbial (Figure 5C), indicating that both acetate- and lactate-loving community profile, the electrode materials, and the sub- bacteria are present. The dominant group of microbial strate type. The microbial communities identified in the communities was dependent on substrate type.40 various studies were very diverse.34 The same mixed morphology was seen in MFCs with Biofilm develops from the aggregation of a complex acetate and lactate at various substrate concentrations mass of microbial communities. The biofilm is formed in (Figure 6). A mixed morphology is expected to contribute three phases: attachment on the electrode surface, matu- to better MFC performance due to the presence of mixed ration, and dispersion.35 Using the microbe's adhesive bacterial culture, which has been observed from a previ- and protective matrix excretions, they attach to a solid ous study to enhance current by as much as six times substrate via self-immobilization growth. The biofilm is compared to pure culture.41 vital in the electrochemical process through its anodic reaction (oxidation). Furthermore, the electron transfer The electrical conductivity of biofilms plays an essen- mechanism is favored in cell-to-cell contact in high- tial role in the electricity generation in MFC. These density biofilms.36 The anode electrodes of the MFC must charge transfer losses in the anode indicate biofilm activ- contain stable and consistent biology to generate boosted ity, which may be caused by the difference in EAB pre- energy.37 The factors affecting bacterial biofilms' perfor- sent and the biofilm's anodophilic properties due to mance and stability are biofilm volume, biofilm rough- substrate fed, demonstrated by EIS analysis. The previous ness, cluster size, diffusion distance, and fractal studies reported that the generated electrons move from dimension.38 In this study, a scanning electron micro- bacteria/biofilm to an anode surface through the follow- scope (SEM) was used to analyze the anode electrode's ing mechanisms: indirect/mediated electron transfer biological morphology. (MET), direct electron transfer (DET), and inter-spacial electron transfer.42 After 1 month of stable MFC operations, the anode's electrodes were removed from all six MFCs and washed During a direct electron transfer, the electrons must using distilled water. The electrodes containing biofilm reach the cell's outer membrane, which should be physi- were cut using sterilized scissors and tweezers to the cally in contact with the anode. Biofilms from electrical 0.5 cm2 sample pieces. The electrode fragments were then substances or conductive nanowires (pili/flagella) are soaked with glutaraldehyde in 0.1 M phosphate buffer 3% formed on the anode's surface.43 Das et al reported bacte- at 4C for 2 hours. After fixing, the electrode samples rial pili on the anode electrode surface using the same were dehydrated by soaking in a series of graded ethanol: wastewater as the bacteria source.35 Microbial nanowires 30%, 50%, 70%, 80%, 90%, 100% for 3 × 20 minutes per provide the ability to enhance the interaction between concentration.39 Colonies were seen in patches among bacteria and electrodes significantly and play an essential the graphite felt fibers. “Clumpy” growth with rod-like role in the microorganisms' electronic interactions with F I G U R E 5 SEM images of anodes with electroactive bacteria biofilm grown with different substrates at 10 mM concentrations: A, acetate, B, lactate, and C, acetate and lactate

10662 DINH ET AL. inorganic electron acceptors in their environment and Developing a biofilm below the higher-than-optimal Rext the electron exchange between species.44 has a detrimental effect on its properties and perfor- During an indirect electron transfer, electronic media- mance. The highest energy efficiency was recorded from tors penetrate bacterial cells, separate electrons from the MFCs that matured using 1-kΩ Rext and produced low electrolytes' metabolic reactions, and supply them to the internal resistance (Rint).49 Hence in this study, a 1-kΩ MFC's anode.45 The biodegradable substrate serves as the Rext was used in the acclimation. The external resistance electron source in the MFC.46 Many bacteria species in value may also be related to the overshoot in concor- the domestic wastewater have been identified as capable of self-synthesizing intermediates such as E. coli47 and dance with the study of Hong et al. (2011) that shows Shewanella.48 MFCs acclimated to resistances from 500 to 5000 Ω pro- duced polarization curves with power overshoot.32 The mixed microorganism communities in wastewater are so complex that it is difficult to unravel how electrons High internal resistance is the main problem that transfer. However, it could be seen that both MET and DET mechanisms were present in the bacteria culture limits the MFC's power output. Therefore, efforts to used in this research, and they have enhanced the perfor- mance of the MFC vs using a single bacteria culture. understand should be made to improve MFC perfor- mance.50 An equivalent circuit shown in Figure 7 was used 3.4 | Internal resistance of MFCs to analyze the MFCs and calculate their corresponding Biofilm morphology is affected by external resistance internal resistance values,16 Rf (Ω) and its circuit compo- (Rext) and has an integral part in MFC efficiency. nents (Table 1). Nyquist plots (Figure 8) were generated, and the circuit components were calculated based on these. According to Figure 7, the ohmic loss in MFCs is represen- ted by R1. Its value is the linear resistance to the flow of electrons through different electrode materials and bonds and the resistance to the flow of ions through the proton F I G U R E 6 SEM images of anodes with electroactive bacteria biofilm was grown with different substrate concentrations: A, acetate and lactate at 20 mM, B, acetate and lactate at 30 mM, and C, acetate and lactate at 40 mM concentration F I G U R E 7 Equivalent circuit of MFC T A B L E 1 Internal resistance of MFCs and other circuit parameter values as a function of substrate type and concentration Rf (Ω) R1 (Ω) Ra,g (Ω) Ca,g (F) Ra,t (Ω) Ca,t (F) Rc,c (Ω) Cc,c (F) Wc ÀOhm=psffiÁ MFC1 100.24 2.41 2.34 5.11 × 10−2 76.34 3.343 × 10−2 19.15 3.74 × 10−5 385.30 MFC2 107.19 2.79 9.59 3.71 × 10−2 74.39 2.265 × 10−2 20.43 9.73 × 10−5 532.90 MFC3 95.04 1.79 3.63 8.02 × 10−4 75.84 2.552 × 10−2 13.79 1.25 × 10−5 135.70 MFC4 296.31 2.51 14.38 1.82 × 10−4 262.50 1.619 × 10−2 16.92 3.23 × 10−5 247.93 MFC5 299.79 2.53 3.51 5.66 × 10−5 265.80 1.623 × 10−2 27.95 7.32 × 10−5 243.20 MFC6 660.41 2.31 16.03 4.38 × 10−2 661.10 2.046 × 10−2 30.97 5.04 × 10−5 325.30

DINH ET AL. 10663 exchange membrane and concentration.17 The charge protons from the anode to the cathode. Therefore, the ohmic resistance of the MFCs in the study was mini- transfer losses of the anode are represented by Ra, which mal.54,55 Similar values in Ca,g, Ca,t, and Cc,c indicate an reflects biofilms' function.51 Two kinds of charge transfer insignificant difference in the electrical double layers at the interface with the anolyte/catholyte. The difference resistances were observed in the anode: from the graphite in internal resistance among the MFCs, therefore, could be explained by the difference in charge transfer losses in felt sheet (Ra,g) and from the titanium conductor (Ra,t). The the anode (Ra,t and Ra,g) and the cathode (Rc), and by the electrical double layer phenomenon of electrode materials concentration losses in the MFC (Wc). These charge and titanium conductor represented by capacitance Ca,g,52 transfer losses in the anode (Ra,t and Ra,g) indicate biofilm and Ca,t indicated that the electrode is exposed to the activity, which may be caused by the difference in EAB anode's fluid. The charge transfer losses of the anode are present and the anodophilic properties of the biofilm due represented by Rc and Cc, the electrical double layer.53 Wc to substrate fed. The EAB acclimate and colonize onto indicated concentration losses of the MFCs which repre- the anode electrode. They secrete matrix materials according to the type of EAB.56 This causes a difference sents the diffusion of the substrate, oxygen, or other reac- in the anolyte and catholyte content, which results in the tants in the MFCs.53 difference of Wc and Rc values. As shown in Table 1, the ohmic losses R1 are similar because the anolyte made of PBS solution and the elec- trode material (graphite felt) was the same in all MFCs. Generally, keeping a smaller distance between the anode and the cathode decreases the distance traveled by the 3.5 | Substrate degradation F I G U R E 8 Nyquist plots of MFCs with EC-Lab software Microorganisms, such as bacteria in MFCs, decompose [Colour figure can be viewed at wileyonlinelibrary.com] organic substances for cell maintenance and growth, leading to electrons and protons' production, which cau- ses bioelectricity generation.57 MFC's wastewater treatment efficiency was assessed by estimating the substrate's chemical oxygen demand (COD) removal efficiency during operation. The COD removal efficiency was assessed after each MFC opera- tion cycle. The results of Oh and Logan's research (2005) have shown that the concentration of organic pollutants available in the sample used is proportional to the cur- rent generated in the MFCs. They also suggested that the power produced is higher when the sample's COD value is also higher.58 The initial COD readings varied because of the presence of different substrate types (Figure 9A). The MFC fed with acetate was found to have more than 90% COD removal efficiency, which is the highest F I G U R E 9 COD removal efficiency for MFCs using A, different substrate types at 10 mM and B, mixtures of acetate and lactate (1:1 ratio) at different concentrations [Colour figure can be viewed at wileyonlinelibrary.com]

10664 DINH ET AL. obtained among the MFCs in this study. In comparison, that treat domestic wastewater requires a robust mixed mixed acetate and lactate (at ratio 1:1 and 10 mM con- substrate. centration) produced a lower result at 84 ± 4% (Figure 9A). This result implies that the dominant bacte- The performance of the MFCs was found to be rial species grown with acetate performed well in oxidiz- affected by the concentration of substrates. For systems ing acetate but not necessarily generating electricity; supplied with different concentrations of mixture acetate hence, it can be concluded that these bacteria are not and lactate, electricity production increased with COD electroactive. value. The MFCs with the mixture of acetate and lactate at 30 mM concentration had the best power generation pro- Comparing MFCs fed with both acetate and lactate ducing maximum power density up to 956.75 mW m−2, (1:1 ratio) at different concentrations (Figure 9B), it COD removal, and internal resistance among the four was observed that the highest COD removal was MFCs with different substrate concentrations. Eighty to obtained at 30 mM substrate concentration, which also ninety-one percentage COD removal efficiency was showed the best electricity generation performance attained for different concentrations. Furthermore, it among the MFCs. This result suggests that at 30 mM was found that internal resistance decreased with an mixed substrate, organic decomposition, and electricity increase in COD value. Additionally, among all the generation are enhanced, which implies that EAB MFCs tested, effective substrate degradation having growth was favored. However, COD removal decreased COD removal at 91.4% was observed with acetate. This at 40 mM substrate concentration. This result is consis- research also showed that high substrate concentrations tent with several studies where high substrate concen- could inhibit the development and formation of biofilm trations were found to inhibit power production in systems. The results suggest that there is an optimal MFCs.56,59 value for substrate concentration. These findings provide useful and progressive insights into future applications of The initial COD values were proportional to the sub- MFCs in wastewater treatment. strate concentration as expected (ie, higher initial COD at higher substrate concentration) (Figure 9B). However, ACKNOWLEDGEMENTS there is no trend found on the COD removal efficiency The authors gratefully acknowledge Taiwan MOST's determined by the final COD reading. The final COD financial support: 108-2221-E-197-015-MY3, 108-2622-E- value reflects the organic compounds that are present 197-002-CC3, 107-2221-E-197-022-MY3, and 106-2923-E- after the oxidation process in the anode. These are com- 197-001-MY3. pounds that may have remained un-decomposed together with byproducts of the bacterial culture present in the ORCID anode. The COD changes indicate the localized metabolism Chin-Tsan Wang https://orcid.org/0000-0003-0989- of the bacteria, but these do not differentiate consumption 076X by EAB against non-electrogenic microorganisms.60 The My Loan Phung Le https://orcid.org/0000-0003-1055- final COD values only show that the MFCs differed in the 3672 microbial mix culture that developed, which further implies that the byproducts also vary according to the bacterial spe- REFERENCES cies that dominate the mixed culture. Since identifying bac- terial species present was out of the scope of this study, 1. Logan BE, Hamelers B, Rozendal R, et al. 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