Charge transfer resistance (CTR) is a key operating parameter for regulating energy harvesting efficiency, electrochemically active biofilms (ECABs) development, and microbial diversity and metabolism in microbial fuel cell (MFC). However, the response behaviors in terms of electroactivity, spatio-temporal distribution, and catabolic pathway of mixed-culture ECABs when being exposed to different CTR have not been well understood. Herein, we comprehensively investigated the effects of CTR on the characteristics of ECABs by varying external resistance in MFC. Specifically, R3-500 exhibited short ECABs maturation (∼8 d), high energy output (∼0.37 W m−2, ∼0.4 mA cm−2), low internal resistance (∼355 Ω) when ECABs was mature, and highly efficient and stable operation. Exopolysaccharides and extracellular DNA play critical roles in cells attachment on electrode surface and development a steady biofilms skeleton. The electron transfer rate was controlled by biofilm resistance (Rfilm, > 90% of total internal resistance), and a typical two-layer ECABs structure (inner dead layer and outer live layer) colonized on carbon fiber, with the dead-cells bearing inner layer as the major resistance causing increased Rfilm. Metagenomics analysis revealed that R3-500 facilitated the proliferation and co-existence of electricigens (e.g., Geobacter), and putative electric-syntrophy bacteria (e.g., Trichococcus), building a syntrophic relationship for potential interspecies electron/message exchange. Furthermore, the key functional genes families associated with cell activity (such as ftsZ, and rps/rpl series), electron transfer (such as torC, rib series, and pilA), and energy metabolism (hdr, mcr, fwd/fmd, and ntp) were upregulated (1.33–6.67-fold) in R3-500. Together these insights into ECABs’ behaviors by regulating CTR are of potential significance to help to sustain the long-term, highly active operation in bioelectrochemical systems.
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