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Ecological and Transcriptional Responses of AnodeRespiring Communities to Nitrate in a Microbial Fuel Cell Varun N. Srinivasan, and Caitlyn S. Butler Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b06572 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017
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Ecological and Transcriptional Responses of AnodeRespiring Communities to Nitrate in a Microbial Fuel Cell Varun N. Srinivasan and Caitlyn S. Butler* Department of Civil and Environmental Engineering, University of Massachusetts-Amherst, Amherst MA 01003.
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ABSTRACT
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A poorly understood phenomenon with a potentially significant impact on electron recovery, is
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competition in microbial fuel cells (MFC) between anode-respiring bacteria and microorganisms
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that use other electron acceptors. Nitrate is a constituent of different wastewaters and can act as a
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competing electron acceptor in the anode. Studies investigating the impact of competition on
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population dynamics in mixed communities in the anode are lacking. Here, we investigated the
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impact of nitrate at different C/N ratios, 1.8, 3.7 and 7.4 mg-C/mg-N, on the electrochemical
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performance and the biofilm community in mixed-culture chemostat MFCs. The electrochemical
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performance of the MFC was not affected under electron donor non-limiting conditions, 7.4 mg-
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C/mg-N. At lower C/N, electron donor limiting, ratios electron recovery was significantly
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affected. The electrochemical performance recovered upon removal of nitrate at 3.7 mg-C/mg-N,
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but did not at 1.8 mg-C/mg-N.
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Deltaproteobacteria accompanied by increase in Betaproteobacteria in response to nitrate at low
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C/N ratios and no significant changes at 7.4 mg-C/mg-N. Transcriptional analysis showed
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increased transcription of nirK and nirS genes during nitrate flux suggesting that denitrification
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to N2, and not facultative nitrate reduction by Geobacter spp., might be the primary response to
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perturbation with nitrate.
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INTRODUCTION
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Bioelectrochemical systems (BESs) are a promising technology due to their ability to treat
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organic waste, decouple the electron donor and electron acceptor reactions and potential to
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produce an energetic product. BESs have great potential in applications as a mixed-culture
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bioprocess. However, before wide scale application, the robustness and resilience of anodic
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communities to environmental conditions have to be evaluated. In order to implement BESs in
Microbial community analysis showed a decrease of
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natural and engineered settings, there is a need for understanding the response of the anode-
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respiring communities to perturbations. Perturbations can have long-term or short-term effects
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which can adversely influence the performance of the biofilm and the technology.
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For example, oxygen crossover from the cathode of a microbial fuel cell (MFC) to the anode
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can cause a decrease in coulombic efficiencies (CEs) and lead to the growth of aerobic
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microorganisms in the anode which can outcompete anode-respiring bacteria (ARB).1,2
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Similarly, the presence of methanogenic communities can lead to decrease in CEs in MFCs or
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hydrogen yield in microbial electrolysis cells (MECs).3–6 ARB can also have alternate
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metabolisms such as nitrate, sulfate, and ferric respiration capabilities that can serve as
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competing metabolisms.7–9 In a mixed community, where both non-ARB and ARB exist, the
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primary response to induction of competition is unknown. An understanding of the response of
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the biofilm community to these perturbations can lead to a better design and optimization of this
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technology for scale-up and implementation.
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Nitrate is a regulated drinking water and wastewater contaminant. The common presence of
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nitrate in the environments in which MFCs can be potentially used such as for groundwater
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bioremediation10–12 and treatment of pre-processed secondary sludge13–17, makes it a co-
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contaminant of interest. The effect of nitrate on anode-respiring biofilms is of great interest
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since many bacteria harbor nitrate reduction capabilities including ARB such as Geobacter
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spp.9,18,19 and Shewenella spp..20,21 The effect of nitrate on mixed community anode respiring
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biofilms has been previously studied by Sukkasem et al (2008).22 CE was affected by the
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presence of nitrate in the anode while the maximum voltage output was not affected. Sukkasem
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et al. (2008) performed some preliminary investigations into the effect of nitrate on the microbial
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community using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis
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(DGGE). They postulated the existence of two distinct groups of bacteria: obligate ARB and
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facultative ARB capable of nitrate reduction. However, more quantitative investigations are
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required to understand the effect of nitrate on the biofilm community. The existence of
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facultative ARB capable of nitrate reduction has been documented by other studies.23 The effect
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of nitrate on electrode-respiring Geobacter metallireducens was studied by Kashima and Regan.9
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They observed that G.metallireducens biofilms reduced nitrate at a range of anode potentials.
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The critical nitrate concentration, at which a significant decrease in BES performance was
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observed, depended on the biofilm thickness. The use of nitrate as a competing electron acceptor
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by facultative ARBs was controlled by diffusional limitations in thicker biofilms.
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The above studies have investigated the effect of nitrate on anode-respiring biofilms using
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batch reactors. BESs designed for natural or engineered settings would require a chemostat based
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study which has more relevance to treatment outcomes. In a chemostat, there is continuous
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addition of the perturbing component (nitrate) to the MFC which could induce changes
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community structure over time not observed in batch-style experiments. Furthermore, previous
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studies have postulated the existence of both heterotrophic denitrifiers and facultative ARB
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capable of nitrate reduction, but a quantitative investigation into the community dynamics in the
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presence of nitrate has not been undertaken. An understanding of the effects of nitrate on the
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microbial community and the resiliency of the communities to recover from such perturbations
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could play an important role when MFCs are used for bioremediation or wastewater applications.
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Though researchers have often implicated competition in reduced MFC performance, few have
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explored the dynamics explicitly and those who have, have used batch configurations and/or pure
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cultures. This study describes the effect of an example competing electron acceptor, nitrate, on
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the structure of mixed microbial communities and resilience of putative anode-respiring species
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after the community’s response to a continuous input of nitrate in a chemostat over an extended
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period of time. The effect of three stoichiometrically relevant C/N ratios (1.8, 3.7 and 7.4 mg-
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C/mg-N) on the performance and microbial ecology of the anode in mixed culture chemostat-
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MFCs was examined using a combination of 16S rRNA gene sequencing and transcriptional
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profiling using reverse transcription-quantitative PCR. This approach could not only lead to a
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more quantitative understanding of the response of the communities to nitrate as an alternate
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electron acceptor but also point towards the predominant metabolic response to nitrate in the
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community which has previously not been identified. We hypothesize that at high C/N ratios
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(electron donor non-limiting), the presence of nitrate will not affect the electrochemical
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performance of the MFC while electron-donor limiting conditions will negatively affect the
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electrochemical performance of the MFC. We also hypothesize that the presence of nitrate in the
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bulk solution at low C/N ratios will cause significant changes in the biofilm community leading
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to decrease in the relative abundance of anode-respiring bacteria with time.
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MATERIALS AND METHODS
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MFC Design and Operation.
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Dual chamber H-type MFCs with a cation exchange membrane (CMI-7000, Membrane
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International Inc., Glen Rock) were used for all the experiments. Graphite cloth coupons (2.2 x
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11 x 0.32 cm) were used as electrodes with 5 electrode coupons in each chamber. Marine grade
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wire (Vertex Marine) was used to make all the electrical connections. An Ag/AgCl reference
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electrode (RE-6, BASi Inc. USA) was placed in each of the anode chambers to measure the
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anode potential.
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The anode chambers were inoculated with primary effluent from the Amherst Wastewater
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Treatment Plant, Amherst MA and acclimated in recycle batch with an external resistance of
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1500 Ω. The MFCs were inoculated with a 10:90 (by volume) mixture of inoculum and a
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phosphate-buffered minimal growth medium (see supplementary information for details) with
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1.66 g/L potassium acetate. The feed was sparged with filter-sterilized N2 gas for at least 45
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minutes prior to addition to the reactor. The feed solution was replaced when the voltage dropped
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below 0.05 V during the acclimation phase. The MFCs were operated in batch mode until
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reproducible maximum voltages were obtained for two successive batches. The MFCs were then
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switched to chemostat-mode at a flow rate of 0.21 mL/min (HRT=20 hours). The anode chamber
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was sparged continuously with filter-sterilized N2 gas to prevent oxygen diffusion into the anode
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and 2-bromoethanosulfonic acid (BES) was added at 3 mM to inhibit acetoclastic
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methanogenesis.4,24 All media was autoclaved. The anode chamber was continuously stirred
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throughout the experiment. The cathode contained 70 mM potassium ferricyanide in 80 mM
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phosphate buffer solution. All experiments were performed in duplicate.
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Experimental Design.
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The acetate concentration in the influent of the anode was decreased consecutively to
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determine Scritical of acetate required for maximum coulombic efficiency (analogous to minimum
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substrate concentration required for growth) in each reactor. The Scricitcal for the anode of an MFC
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is defined (in this study) as the minimum acetate concentration that would produce the maximum
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voltage obtained during the end of the acclimation phase. The Scritical has been shown to be
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important in other competition studies.25 The MFCs were run at each successively decreasing
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acetate concentrations for >3 HRTs. Once Scritical was determined and steady state conditions
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(steady performance for >3 HRTs) were achieved (Phase I), nitrate (as sodium nitrate) was
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introduced into the anode of the MFC at different C/N ratios with one of the reactors serving as a
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control (no nitrate). The C/N ratios used in this experiment were 1.8, 3.7 and 7.4 mg-C/mg-N
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(1.4, 2.8 and 5.7 meq e--donor/meq e- -NO3-). The different C/N ratios were tested in different
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MFC chemostats to avoid gradual adaptation of the community to nitrate. Nitrate was fed to the
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anode continuously for 43 days to study the long-term effect of nitrate on the anode-respiring
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biofilm (Phase II). Sodium chloride was added to normalize the conductivity of the media across
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different conditions. After 43 days, nitrate was removed from the influent to the anode and the
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MFC was operated without nitrate (Phase III) to test the resilience of the anode biofilms.
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Measurements and Analyses.
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Acetate was monitored in the influent and effluent of the anode using a Metrohm 850
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Professional Ion Chromatograph (Metrohm Inc., Switzerland) with a Metrosep A Supp 5-250
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Anion Column (Metrohm Inc., Switzerland) using a 3.2 mM Na2CO3 and 1.0 mM NaHCO3
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eluent. Samples were filtered using 0.45 µm syringe filters and stored at -20 °C before analysis.
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Nitrate, nitrite and ammonium were measured using a colorimeter (DR 890, Hach Company)
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according to the manufacturer’s instructions.
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The voltage across an external resistance of 1500 Ω was monitored every 15 minutes using a
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Keithley Model 2700 Multimeter with a 7700 Switching Module (Keithley Instruments Inc.,
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Cleveland, OH, USA). Polarization curves were performed using a Gamry Series G750
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Potentionstat/Galvanostat/ZRA (Gamry, USA). The voltage sweep was applied at a rate of 1
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mV/s.
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Coulombic efficiency (CE) was calculated using equation S1 (supplementary information).
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Biofilm Sampling and DNA/RNA Extraction.
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Anode electrode samples were collected at various stages of the experiment and preserved
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appropriately (see supplementary information for details). RNA and DNA were extracted from
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the electrode samples using the RNA Power Soil Cut (Mo Bio Laboratories Inc.) with a DNA
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Elution Accessory Kit (Mo Bio Laboratories Inc.). The extracted RNA was then treated with
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DNase Max Kit (Mo Bio Laboratories Inc.) to remove any DNA contamination. The extracted
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DNA and RNA were then quantified with a spectrophotometer (NanoDrop, ND-100, NanoDrop
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Technologies, Wilmington, DE). mRNA was then converted into cDNA using the High Capacity
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cDNA Reverse Transcription Kit (Life Technologies) and stored at -20 °C.
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Quantitative PCR.
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The activity of denitrifying genes was assessed using nitrite-reductase specific primers for nirK
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and nirS. The nirK activity was assessed using nirK876 and nirK1040.26 nirS activitiy was
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assessed using nirSCd3af and nirSR3cd.27,28 Bioelectrochemical activity was assessed using
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Geobacter spp. as model ARBs. The activity of Geobacter spp. was measured using Geobacter
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16S rRNA gene specific primers Geo564F and Geo840R.29 Even though this method can have
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potential limitations in terms of non-specific amplification from the 16S rRNA gene from other
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bacteria, this is the only method at present to quantify the activity of the Geobacter spp. due to
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lack of a specific functional gene for anode respiration. For performing relative quantification,
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16S rRNA gene was used as the reference transcript. The primer pair used for 16S rRNA gene
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quantification was 1114f and 1275r.30 Amplification of cDNA templates was carried out using a
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StepOne™ Real-Time PCR System (Applied Biosystems) using SYBR Green as a detection
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system. The reactions for each target were performed separately (more details are provided in
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supplementary information). Triplicate wells were run for each sample for each gene target.
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Standard curves, melting curves and negative controls were run for each qPCR run.
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Illumina MiSeq Sequencing and Analysis.
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The extracted DNA was sent to Research and Testing Facility (Lubbock, TX) for PCR
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amplification and sequencing targeting the V3-V4 region using the primers 338aF (5’-
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ACTCCTACGGGAGGCAGCAG-3’) and 785R (5’-GACTACHVGGGTATCTAATCC-3’) and
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the amplicons were sequenced on the Illumina MiSeq platform using V3 chemistry. The raw
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Fastq files were cleaned using Sickle 1.3331 with a minimum window quality score of 20. The
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quality-controlled sequences were analyzed using mothur32 using the protocol described in
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Kozich et al.33 The protocol is further described in supplementary information.
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Statistical Analyses.
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The anode potential, coulombic efficiency and acetate removal data were split into three
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phases: before nitrate flux, during nitrate flux (Day 0 to Day 43) and nitrate removed (days 44 to
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60). Statistical analysis comparing the three phases within each treatment was performed using
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1-way analysis of variance (ANOVA) followed by a Tukey’s Honestly Significant Difference
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(HSD). The HSD results are reported only if the 1-way ANOVA showed a significant effect.
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The normalized (using 16S rRNA transcripts as reference) relative quantities of gene
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transcripts (Rqtarget) were calculated for each target transcript and logarithmic (base 2) fold
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change values between sampling day (t) and the start of nitrate flux (Day 0) as follows: log Fold Change =
Rq Rq
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Statistical analysis of the fold change values within each C/N ratio treatment condition was
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performed using 1-way ANOVA using sampling day as the main effect. Significant interaction
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effects were further determined with Tukey’s HSD test. Non-metric multidimensional scaling
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(NMDS) analysis using the UniFrac weighted metric was performed in R34 using the phyloseq35
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package. Other R packages used in the analysis and plotting were ggplot236, vegan37, dplyr38 and
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ampvis.39
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RESULTS AND DISCUSSION
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The electrochemical performance of the MFCs is adversely affected by nitrate at low C/N
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ratios
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The MFCs were acclimated under optimal operating conditions with respect to power
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production and Scritical for each reactor (0.59 mM) were determined (Phase I). Acetate removal
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was not complete during this period. When nitrate was introduced into the anode, complete
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removal of nitrate was observed in the effluent of the anode across all the C/N ratios (data not
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shown). Neither nitrite nor ammonium were detected in the effluent during the period of nitrate
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flux. It is possible that ammonium produced through DNRA could have been assimilated by the
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cells and not detected in the effluent. Acetate removal increased significantly from 41.7 ± 9.4 %
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to 85.9 ± 10.6 % (p
