Harvesting energy from the marine sediment-water interface II. Kinetic activity of anode materials

Here, we report a comparative study on the kinetic activity of various anodes of a recently described microbial fuel cell consisting of an anode imbedded in marine sediment and a cathode in overlying seawater. Using plain graphite anodes, it was demonstrated that a significant portion of the anodic current results from oxidation of sediment organic matter catalyzed by microorganisms colonizing the anode and capable of directly reducing the anode without added exogenous electron-transfer mediators. Here, graphite anodes incorporating microbial oxidants are evaluated in the laboratory relative to plain graphite with the goal of increasing power density by increasing current density. Anodes evaluated include graphite modified by adsorption of anthraquinone-1,6-disulfonic acid (AQDS) or 1,4-naphthoquinone (NQ), a graphite-ceramic composite containing Mn2+ and Ni2+, and graphite modified with a graphite paste containing Fe3O4 or Fe3O4 and Ni2+. It was found that these anodes possess between 1.5- and 2.2-fold greater kinetic activity than plain graphite. Fuel cells were deployed in a coastal site near Tuckerton, NJ (USA) that utilized two of these anodes. These fuel cells generated ca. 5-fold greater current density than a previously characterized fuel cell equipped with a plain graphite anode, and operated at the same site.     

Relating MEC population dynamics to anode performance from DGGE and electrical data

The microbial electrolysis cell (MEC) is a promising system for H₂ production, but little is known about the active microbial population in MEC systems. Therefore, the microbial community of five different MEC graphite felt anodes was analyzed using denaturing gradient gel electrophoresis (DGGE) profiling. The results showed that the bacterial population was very diverse and there were substantial differences between microorganisms in anolyte and anode samples. The archaeal population in the anolyte and at the anodes, and between the different MEC anodes, was very similar. SEM and FISH imaging showed that Archaea were mainly present in the spaces between the electrode fibers and Bacteria were present at the fiber surface, which suggested that Bacteria were the main microorganisms involved in MEC electrochemical activity. Redundancy analysis (RDA) and QR factorization-based estimation (QRE) were used to link the composition of the bacterial community to electrochemical performance of the MEC. The operational mode of the MECs and their consequent effects on current density and anode resistance on the populations were significant. The results showed that the community composition was most strongly correlated with current density. The DGGE band mostly correlated with current represented a Clostridium sticklandii strain, suggesting that this species had a major role in current from acetate generation at the MEC anodes. The combination of RDA and QRE seemed especially promising for obtaining an insight into the part of the microbial population actively involved in electrode interaction in the MEC.     

Electrochemical treatment of graphite to enhance electron transfer from bacteria to electrodes

In this paper, graphite felts were continuously electrochemically oxidized to increase the current generation in microbial fuel cells (MFCs). The treated and untreated graphite felts were utilized as anodes in MFCs and current production was compared. The current production on electrochemically treated graphite felt anodes was about 1.13 mA, 39.5% higher compared with that of MFCs containing untreated anodes. The results demonstrated that the electronic coupling between graphite felt electrodes and electrogenic bacteria could be enhanced by electrochemical oxidization of the electrodes. Further study showed that the newly generated carboxyl containing functional groups from electrochemical oxidization were responsible for the enhanced electron transfer, due to their strong hydrogen bonding with peptide bonds in bacterial cytochromes.     

A method for high throughput bioelectrochemical research based on small scale microbial electrolysis cells

There is great interest in studying exoelectrogenic microorganisms, but existing methods can require expensive electrochemical equipment and specialized reactors. We developed a simple system for conducting high throughput bioelectrochemical research using multiple inexpensive microbial electrolysis cells (MECs) built with commercially available materials and operated using a single power source. MECs were small crimp top serum bottles (5 mL) with a graphite plate anode (92 m2/m(3)) and a cathode of stainless steel (SS) mesh (86 m2/m3), graphite plate, SS wire, or platinum wire. The highest volumetric current density (240 A/m3, applied potential of 0.7 V) was obtained using a SS mesh cathode and a wastewater inoculum (acetate electron donor). Parallel operated MECs (single power source) did not lead to differences in performance compared to non-parallel operated MECs, which can allow for high throughput reactor operation (>1000 reactors) using a single power supply. The utility of this method for cultivating exoelectrogenic microorganisms was demonstrated through comparison of buffer effects on pure (Geobacter sulfurreducens and Geobacter metallireducens) and mixed cultures. Mixed cultures produced current densities equal to or higher than pure cultures in the different media, and current densities for all cultures were higher using a 50 mM phosphate buffer than a 30 mM bicarbonate buffer. Only the mixed culture was capable of sustained current generation with a 200 mM phosphate buffer. These results demonstrate the usefulness of this inexpensive method for conducting in-depth examinations of pure and mixed exoelectrogenic cultures.     

Lack of electricity production by Pelobacter carbinolicus indicates that the capacity for Fe(III) oxide reduction does not necessarily confer electron transfer ability to fuel cell anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Hydrogen production profiles using furans in microbial electrolysis cells

Microbial electrochemical cells including microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) are novel biotechnological tools that can convert organic substances in wastewater or biomass into electricity or hydrogen. Electroactive microbial biofilms used in this technology have ability to transfer electrons from organic compounds to anodes. Evaluation of biofilm formation on anode is crucial for enhancing our understanding of hydrogen generation in terms of substrate utilization by microorganisms. In this study, furfural and hydroxymethylfurfural (HMF) were analyzed for hydrogen generation using single chamber membrane-free MECs (17 mL), and anode biofilms were also examined. MECs were inoculated with mixed bacterial culture enriched using chloroethane sulphonate. Hydrogen was succesfully produced in the presence of HMF, but not furfural. MECs generated similar current densities (5.9 and 6 mA/cm² furfural and HMF, respectively). Biofilm samples obtained on the 24th and 40th day of cultivation using aromatic compounds were evaluated by using epi-fluorescent microscope. Our results show a correlation between biofilm density and hydrogen generation in single chamber MECs.     

Microbial fuel cell energy from an ocean cold seep

Benthic microbial fuel cells are devices that generate modest levels of electrical power in seafloor environments by a mechanism analogous to the coupled biogeochemical reactions that transfer electrons from organic carbon through redox intermediates to oxygen. Two benthic microbial fuel cells were deployed at a deep-ocean cold seep within Monterey Canyon, California, and were monitored for 125 days. Their anodes consisted of single graphite rods that were placed within microbial mat patches of the seep, while the cathodes consisted of carbon-fibre/titanium wire brushes attached to graphite plates suspended ∼0.5 m above the sediment. Power records demonstrated a maximal sustained power density of 34 mW·m⁻² of anode surface area, equating to 1100 mW m⁻² of seafloor. Molecular phylogenetic analyses of microbial biofilms that formed on the electrode surfaces revealed changes in microbial community composition along the anode as a function of sediment depth and surrounding geochemistry. Near the sediment surface (20–29 cm depth), the anodic biofilm was dominated by micro-organisms closely related to Desulfuromonas acetoxidans. At horizons 46–55 and 70–76 cm below the sediment–water interface, clone libraries showed more diverse populations, with increasing representation of δ-proteobacteria such as Desulfocapsa and Syntrophus, as well as ɛ-proteobacteria. Genes from phylotypes related to Pseudomonas dominated the cathode clone library. These results confound ascribing a single electron transport role performed by only a few members of the microbial community to explain energy harvesting from marine sediments. In addition, the microbial fuel cells exhibited slowly decreasing current attributable to a combination of anode passivation and sulfide mass transport limitation. Electron micrographs of fuel cell anodes and laboratory experiments confirmed that sulfide oxidation products can build up on anode surfaces and impede electron transfer. Thus, while cold seeps have the potential to provide more power than neighbouring ocean sediments, the limits of mass transport as well as the proclivity for passivation must be considered when developing new benthic microbial fuel cell designs to meet specific power requirements.     

Genes for iron–sulphur cluster assembly are targets of abiotic stress in rice,Oryza sativa

Iron–sulphur (Fe–S) cluster assembly occurs in chloroplasts, mitochondria and cytosol, involving dozens of genes in higher plants. In this study, we have identified 41 putative Fe–S cluster assembly genes in rice (Oryza sativa) genome, and the expression of all genes was verified. To investigate the role of Fe–S cluster assembly as a metabolic pathway, we applied abiotic stresses to rice seedlings and analysed Fe–S cluster assembly gene expression by qRT‐PCR. Our data showed that genes for Fe–S cluster assembly in chloroplasts of leaves are particularly sensitive to heavy metal treatments, and that Fe–S cluster assembly genes in roots were up‐regulated in response to iron toxicity, oxidative stress and some heavy metal assault. The effect of each stress treatment on the Fe–S cluster assembly machinery demonstrated an unexpected tissue or organelle specificity, suggesting that the physiological relevance of the Fe–S cluster assembly is more complex than thought. Furthermore, our results may reveal potential candidate genes for molecular breeding of rice.     

Comparative Metagenomics of Anode-Associated Microbiomes Developed in Rice Paddy-Field Microbial Fuel Cells

In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.     

Enhancement of hydrogen production in a single chamber microbial electrolysis cell through anode arrangement optimization

Reducing the inner resistances is crucial for the enhancement of hydrogen generation in microbial electrolysis cells (MECs). This study demonstrates that the optimization of the anode arrangement is an effective strategy to reduce the system resistances. By changing the normal MEC configuration into a stacking mode, namely separately placing the contacted anodes from one side to both sides of cathode in parallel, the solution, biofilm and polarization resistances of MECs were greatly reduced, which was also confirmed with electrochemical impedance spectroscopy analysis. After the anode arrangement optimization, the current and hydrogen production rate (HPR) of MEC could be enhanced by 72% and 118%, reaching 621.3 ± 20.6 A/m³ and 5.56 m³/m³ d respectively, under 0.8 V applied voltage. A maximum current density of 1355 A/m³ with a HPR of 10.88 m³/m³ d can be achieved with 1.5 V applied voltage.     

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of ?483 ± 1 mV and ?187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.     

Photosynthetic microbial fuel cells with positive light response

The current study introduces an aerobic single‐chamber photosynthetic microbial fuel cell (PMFC). Evaluation of PMFC performance using naturally growing fresh‐water photosynthetic biofilm revealed a weak positive light response, that is, an increase in cell voltage upon illumination. When the PMFC anodes were coated with electrically conductive polymers, the rate of voltage increased and the amplitude of the light response improved significantly. The rapid immediate positive response to light was consistent with a mechanism postulating that the photosynthetic electron‐transfer chain is the source of the electrons harvested on the anode surface. This mechanism is fundamentally different from the one exploited in previously designed anaerobic microbial fuel cells (MFCs), sediment MFCs, or anaerobic PMFCs, where the electrons are derived from the respiratory electron‐transfer chain. The power densities produced in PMFCs were substantially lower than those that are currently reported for conventional MFC (0.95 mW/m² for polyaniline‐coated and 1.3 mW/m² for polypyrrole‐coated anodes). However, the PMFC did not depend on an organic substrate as an energy source and was powered only by light energy. Its operation was CO₂‐neutral and did not require buffers or exogenous electron transfer shuttles. Biotechnol. Bioeng. 2009; 104: 939–946. © 2009 Wiley Periodicals, Inc.