Microbial Dissimilatory Nitrate Reduction in Bioelectrochemical Systems with Electrode-respiring Biofilms and Wastewater Treatment Processes
Open Access
- Author:
- Kashima, Hiroyuki
- Graduate Program:
- Environmental Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 24, 2016
- Committee Members:
- John Michael Regan, Dissertation Advisor/Co-Advisor
John Michael Regan, Committee Chair/Co-Chair
Maryann Victoria Bruns, Committee Member
Ming Tien, Committee Member
Christopher Aaron Gorski, Committee Member - Keywords:
- Bioelectrochemical system
Nitrate reduction
Geobacter
nrfA
Wastewater treatment
Biofilm - Abstract:
- Some microorganisms dynamically alter their metabolic states in response to changes in the environmental conditions, such as availability of compatible electron donors and acceptors. A prominent example is nitrate, which is the most energetically favorable electron acceptor in the absence of oxygen and often dominantly controls microbial reactions taking place in anoxic systems. Solid electrodes present another substrate that can regulate microbial reactions, serving as an electron donor and/or electron acceptor for microbes capable of extracellular electron transfer (EET). Electrode-mediated microbial metabolisms are central in bioelectrochemical systems (BESs), an emerging technology with a variety of potential applications, including a novel experimental platform to study the dynamic change of microbial metabolisms with real-time monitoring under controlled conditions. A better characterization of microbial reactions associated with such key substrates has important implications to understand microbial reactions in anoxic environments and also developing biological processes that exploit relevant microbial metabolisms. This research investigated the capabilities and constraints of microbial metabolisms at the nexus of electrode- and nitrate-mediated respirations relevant to wastewater treatment processes. The facultative metabolic shift between anode electrode reduction and nitrate reduction was investigated in anode-reducing biofilms to study the effects of alternative metabolic options on exoelectrogenic biofilms in BESs. This has important implications not only to explain the fundamental ecology and performance of these systems, but also to develop reliable integrated nutrient removal strategies in BESs, which potentially involve nitrate that can support/induce alternative metabolisms. Using the exoelectrogenic nitrate reducer Geobacter metallireducens, the critical conditions controlling those alternative metabolisms were investigated in two-chamber, potentiostatically controlled BESs at various anode potentials, biofilm thicknesses, and nitrate concentrations. Results showed that anode-reducing biofilms preferentially reduced nitrate at all tested anode potentials (-150 to + 900 mV vs Standard Hydrogen Electrode) with a rapid metabolic shift, despite the fact that the biofilms had no prior nitrate exposure. The critical nitrate concentration that triggered a significant decrease in BES performance was a function of anode biofilm thickness but not anode potential. This indicates that these alternative metabolisms were controlled by the availability of nitrate, which is a function of nitrate concentration in the bulk solution and its diffusion into an anode-reducing biofilm. Coulombic recovery decreased as a function of nitrate dose due to electron-acceptor substrate competition, and nitrate-induced suspended biomass growth decreased the effluent quality. This nitrate-induced metabolic shift of anode-reducing biofilms was further investigated in the context of a shift between two different electrode-mediated metabolisms, electrode reduction and electrode oxidation. The characterization of metabolic shifts among different electrode-mediated reactions such as anode reduction and cathode oxidation is important to understand EETs in natural settings and also to develop stable BESs. This part of the research investigated the capability of anodically-grown G. metallireducens biofilms to shift from anode reduction to cathode oxidation. In tests with potentiostatically controlled graphite electrodes, G. metallireducens biofilms demonstrated a quick and alternative shift between anode reduction and cathode oxidation as a function of electrode potential and availability of the co-substrates nitrate and acetate. Cathodic electrode oxidation was coupled with nitrate reduction by metabolically active biofilms with a large cathodic current of ~ 3.68 A/m2. This metabolic shift from anode reduction to nitrate reduction took place quicker than the metabolic shift from ferric reduction to nitrate reduction. The presence of nitrate-reducing enzyme in the anode-reducing biofilms cells in the absence of nitrate, measured as specific in-vitro nitrate-reducing enzyme activity, was thought to enable such a quick metabolic shift to start nitrate reduction. Cyclic voltammetry and the analysis of its first derivative provided insights into the electron transfer mechanisms of these biofilms. Finally, the potential occurrence of dissimilatory nitrate reduction to ammonium (DNRA), a microbial nitrate-reducing metabolism that involves the sequential reduction of nitrate to nitrite and then nitrite to ammonium, was investigated in two full-scale wastewater treatment plants. DNRA in biological wastewater treatment systems and BESs is largely unstudied despite its potential impacts on system performance. This part of the research examined differential expression and diversity of nrfA, a key marker gene for DNRA, in activated sludge from full-scale domestic wastewater treatment plants with one designed for enhanced biological phosphorus removal (EBPR). Expression of nrfA, which encodes the penta-heme nitrite reductase NrfA catalyzing the nitrite ammonification step of DNRA, was observed in anaerobic and anoxic mixed liquor, but not in aerobic mixed liquor samples. The expression of nrfA under anaerobic and anoxic conditions suggests an overlooked potential for DNRA activity to occur in biological wastewater treatment systems. Some retrieved nrfA sequences were related to sequences associated with a microbial community with anammox activity, and the nrfA diversity in this wastewater treatment system differed from that observed in soil systems. Retrieved nrfA sequences both in genomic DNA and transcript samples were dominated by sequences associated with Actinobacteria, which are often abundant in EBPR processes. These results suggested potential occurrence of DNRA in wastewater activated sludge and encourage further studies in different types of wastewater treatment systems and with chemical tracer analyses to obtain comprehensive understanding of DNRA in this context. This research investigated dissimilatory nitrate reduction in electrode-respiring biofilms and full-scale wastewater treatment processes. Elucidating the dynamics of nitrate-dependent reactions of electrode-respiring G. metallireducens in the contexts of a competitive reaction to anode reduction and an alternative electrode-mediated reaction have implications for BES development. Moreover, the experimental frameworks that were developed to address those problems would be applicable to study other electrode-mediated microbial metabolisms. Findings of nrfA expression and its diversity in full-scale wastewater treatment processes indicated potential occurrences of DNRA in wastewater treatment processes, which would have implications for energy and chemical utilization in these systems, and broadened the representation of diversity in the rather limited nrfA database.