1. Electrochemical Performance and Microbial Characterization from a Thermophilic Microbial Fuel Cell K.C. Wrighton 1, P. Agbo 1, F. Warnecke 3, E.L. Brodie 2, Y.M. Piceno 2, K.A. Weber 1, C. Chow 1, T.Z. DeSantis 2, G.L Andersen 2, and J.D. Coates 1 1 Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 2 Lawrence Berkeley National Laboratory, Berkeley, CA 3 Joint Genome Institute, Walnut Creek, CA Abstract Significant research effort is currently focused on microbial fuel cells (MFC) as a source of renewable energy. To date, most of these efforts have concentrated on MFCs operating at mesophilic temperatures. However, many previous studies have reported on the superiority of thermophilic conditions in anaerobic digestion and demonstrated a net gain in energy yield, in terms of methane, relative to the increased energy requirements of operation. Because of this, our recent studies on MFCs focused on investigating the operation and microbiology associated with thermophilic MFCs operating at 55°C. Over 100-day operation, these MFCs were highly stable and achieved a maximum power density of 24mW/m 2 and a columbic efficiency of 89 percent with acetate as the sole electron donor. In order to characterize the microbial community involved in thermophilic electricity generation, DNA and RNA were isolated from the electrode. PhyloChip and clone library analyses were performed to describe anode bacterial community membership and dynamics. PhyloChip analysis using 16S rDNA and rRNA sequences revealed an increase in relative abundance of populations belonging to the Firmicutes and Chloroflexi by at least one order of magnitude in current producing reactors. Clone library analysis confirmed the dominance of Firmicutes and identified specific genera enriched only in current producing reactors. To better characterize the active microbial populations, we enriched and isolated novel Firmicutes, strain JR and strain S2E, from samples collected from an operating MFC. Based on 16S rRNA gene analysis strain JR was a member of the family Peptococcaceae clustering with Thermincola ferriacetica (>99 percent similarity) while strain S2E was a member of the family Bacillaceae clustering with Geobacillus (>98% similar to Geobacillus pallidus). Phenotypic characterization revealed that both strains were capable of thermophilic dissimilatory reduction of insoluble electron acceptors such as amorphous Fe(III); as well as reduction of the model quinone 2,6-anthraquinone disulfonate (AQDS). Thermincola strain JR was also capable of producing current by coupling acetate oxidation to anodic electron transfer. This represents the first organism isolated from a thermophilic microbial fuel cell and also the first Firmicute representative capable of independent anodic electron transfer. The results of this study indicate the potential advantages for thermophilic MFCs and the novel microbiology associated with their operation. Reactor Design and Model CEM Acetate + 2H 2 0 2CO 2 + 8H+ 8e- 4H 2 O 2O 2 + 8H+ 8e- e- A microbial fuel cell (MFC) is a device that uses bacteria as catalysts to oxidize organic matter to generate current. In this MFC, bacteria in the sludge completely oxidize acetate to carbon dioxide and protons. In metabolizing the acetate, the bacteria subsequently liberate electrons from the acetate onto the graphite anode. Electrons then flow through an external circuit to the cathode where they combine with oxygen and protons that have diffused through the cation exchange membrane (CEM) from the anode. Conclusions   Thermophilic current production is sustainable over a 100 days   PhyloChip analysis using 16S rRNA and rDNA response identified members of the Firmicutes and Chloroflexi that increased by at least one order of magnitude only in electricity producing MFC   Firmicutes sequences dominated (74%) the 16S rDNA clone library   specifically sequences belonging to genera Alicyclobacillus, Bacillus, Geobacillus, and Thermincola were enriched only in current producing reactors   Thermincola strain JR and Geobacillus Strain S2E, members of the Firmicutes, can reduce solid phase iron as well as transfer electrons to the electrode   Thermincola Strain JR produces 0.181 mA of current independent of an electron shuttle   Geobacillus Strain S2E produces 0.04 mA of current dependent on the addition of an exogenous electron shuttle   Together these results confirm the role of the Firmicutes in MFCs and expand the known role of Firmicutes to include both independent and dependent methods of current generation Clone Library Electrochemical Performance A total of six MFC reactors were constructed to evaluate thermophilic performance and microbiology. Three treatments (acetate amended, no electron donor control, and open circuit control) were applied with duplicate reactors. Shown below is representative current production from acetate and non acetate treatments.   Current was produced continually for 100 days in MFC amended with acetate, where MFCs lacking exogenous electron donor failed to produce current after 4 days   Duplicate reactors were functionally reproducible Current Production for 100 Days   471 ohms internal resistance (Ri= -m; y=mx+b)   24.2 mW/m 2 power density   89% of the electrons available in acetate were recovered as current Electrochemical Characterization A polarization is calculated by measuring potential and current changes across various external loads. It is used to calculate the internal resistance as well to describe the power as a function of current.. Firmicute Isolates Capable of Electricity Generation Geobacillus Strain S2E Characterization   Geobacillus strain S2E was isolated from a thermophilic MFC on solid phase iron and acetate   Gram positive, spore forming, obligately thermophilic rod belonging to class Bacillus, family Bacillaceae   Facultative anaerobe capable of fermentation as well as the reduction of solid phase iron and AQDS but cannot reduce soluble forms of iron (III), sulfate, chlorate, and perchlorate Thermincola Strain JR Current Production Thermincola Strain JR Characterization   Thermincola strain JR was isolated from a thermophilic MFC on AQDS and acetate   Gram positive and obligately thermophilic rod belonging to class Clostridia, family Peptococcaceae   Obligate anaerobe capable of reduction of solid phase iron and AQDS but cannot reduce soluble forms of iron (III), sulfate, chlorate, perchlorate, and nitrate Geobacillus Strain S2E Current Production   Strain JR generated a maximum of 0.181 mA (mean 0.1522 mA, n=3 ±0.025) without the presence of a mediator   Geobacter sulfurreducens 0.251 mA (n=3, ±0.04)   Shewanella putrefaceins IR-1 0.04 mA (n=3, ± not reported)   Strain S2E generated a small amount of current (0.04 mA) when provided with an electron shuttle PhyloChip Current Producing Community Structure   The high similarity between 16S rRNA and rDNA response from electricity producing anodes (distance between red and black labels in dark blue circle) signifies that the active community (rRNA) is similar to the persistent community (rDNA) Multidimensional Scaling (MDS) distances are based on relative similarity; samples that are most similar will appear closer on the MDS map. A stress value = 0.01 indicates the MDS accurately reflects the Bray-Curtis similarity matrix. Bacterial Dynamics   Members of the Firmicutes, Chloroflexi, and alpha Proteobacteria increased by at least one order of magnitude in relative abundance in rDNA response only in current producing reactors   Based on the enrichment of Firmicutes in rRNA and rDNA response, and the lack of enrichment of these OTUs in control reactor communities, strongly suggests Firmicutes play an active role in the current generating community A 1000 unit change in hybridization intensity represents a one log change in relative abundance between the initial starting inoculum and the electrode community after 100 days of current production. Change in intensity with time -5000 -3000 -1000 3000 5000 - - - - - - Cyanobacteria Verrucomicrobia Chloroflexi Chlorobia Bacteroidetes Actinobacteria Proteobacteria Firmicutes Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Cyanobacteria Verrucomicrobia Chloroflexi Chlorobia Bacteroidetes Actinobacteria Cyanobacteria Verrucomicrobia Chloroflexi Chlorobia Bacteroidetes Actinobacteria Proteobacteria Firmicutes Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Proteobacteria Firmicutes Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Change in intensity with time -5000 -3000 -1000 3000 5000 - - - - - - - -3000 -1000 3000 5000 - - - - - - - -3000 -1000 3000 5000 - - - - - - Cyanobacteria Verrucomicrobia Chloroflexi Chlorobia Bacteroidetes Actinobacteria Proteobacteria Firmicutes Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Cyanobacteria Verrucomicrobia Chloroflexi Chlorobia Bacteroidetes Actinobacteria Cyanobacteria Verrucomicrobia Chloroflexi Chlorobia Bacteroidetes Actinobacteria Proteobacteria Firmicutes Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Proteobacteria Firmicutes Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other Alpha Beta Delta Epsilon Gamma Bacillus Clostridia Other   Bacteria electrode communities cluster by electricity production and not temporally   Unique bacterial community associated with current production Hierarchical clustering to compare differences in overall community structure of anode communities and initial inoculum. Initial Inoculum Current Producing No Current Controls Linking Bacterial Dynamics with Activity Phylogenetic distribution of 42 OTUs identified by PhyloChip 16S rDNA analysis that increase by one log in relative abundance. Firmicutes (16) Proteobacteria (8) Chloroflexi (5) Cyanobacteria (3) Planctomycetes (2) AD3 (1) Chlorobi (1) Lentisphaearae (1) OP10 (1) OP9/JSI (1) Spirochaetes (1) Termite Group 1 (1) Chloroflexi Unclassified Thermomicrobia Chlroflexi-4 Chloroflexi-3 Proteobacteria 0 2 4 6 8 10 12 14 16 18 Gamma Delta Alpha Firmicutes Symbiobacteria Desulfotomaculum Unclassified Bacilli Clostridia Firmicutes (7) Chloroflexi (3) Proteobacteria (1) Planctomycetes (1) Chlorobi (1) Termite Group 1 (1) Firmicutes Desulfotomaculum Clostridia Bacilli Chloroflexi Unclassified Thermomicrobia Anaerolineae 0 1 2 3 4 5 6 7 8 Proteobacteria Delta Phylogenetic distribution of 14 OTUs identified by PhyloChip 16S rRNA analysis that increase by one log in relative abundance. Current Production Initial Inoculum No Current Controls Clusters are >87% similar (Bray-Curtis Similarity) Acetate No Acetate Acetate Open Acetate No Acetate Acetate Inoc. DNA RNA Inoc. Identification of Dominant Bacteria A maximum likelihood tree showing phylogenetic position of 16S rDNA clones generated from current producing electrodes. A total of 302 clones were sequenced representing 25 OTUs which are indicated in bold. Values in parentheses indicate number of clones from each OTU. Sequences from electrode biofilm isolates are also included on the tree and are denoted in red. The scale bar represents 0.1 inferred nucleotide changes per 100 substitutions.   Firmicutes sequences are enriched (77% of clones) only in clone libraries constructed from current generating reactors   Dominant Firmicute genera   Bacillus sp. (81 OTUs, 27%)   Alicyclobacillus sp. (73 OTUs, 24%)   Thermincola sp. (65 OTUs, 22%)   In contrast to the Firmicutes, Coprothermobacter sequences represent (16%) of the clone library sequences in current generating and (14%) of the initial inoculum   PhyloChip described 35 times more diversity than detected by clone library analysis B11C-0620