Thermophiles; or, the Modern Prometheus: The Importance of Extreme Microorganisms for Understanding and Applying Extracellular Electron Transfer.

Approximately four billion years ago, the first microorganisms to thrive on earth were anaerobic chemoautotrophic thermophiles, a specific group of extremophiles that survive and operate at temperatures ∼50 - 125°C and do not use molecular oxygen (O2) for respiration. Instead, these microorganisms performed respiration via dissimilatory metal reduction by transferring their electrons extracellularly to insoluble electron acceptors. Genetic evidence suggests that Gram-positive thermophilic bacteria capable of extracellular electron transfer (EET) are positioned close to the root of the Bacteria kingdom on the tree of life. On the contrary, EET in Gram-negative mesophilic bacteria is a relatively new phenomenon that is evolutionarily distinct from Gram-positive bacteria. This suggests that EET evolved separately in Gram-positive thermophiles and Gram-negative mesophiles, and that EET in these bacterial types is a result of a convergent evolutionary process leading to homoplasy. Thus, the study of dissimilatory metal reducing thermophiles provides a glimpse into some of Earth's earliest forms of respiration. This will provide new insights for understanding biogeochemistry and the development of early Earth in addition to providing unique avenues for exploration and discovery in astrobiology. Lastly, the physiological composition of Gram-positive thermophiles, coupled with the kinetic and thermodynamic consequences of surviving at elevated temperatures, makes them ideal candidates for developing new mathematical models and designing innovative next-generation biotechnologies. KEY CONCEPTS Anaerobe: organism that does not require oxygen for growth. Chemoautotroph: organism that obtains energy by oxidizing inorganic electron donors. Convergent Evolution: process in which organisms which are not closely related independently evolve similar traits due to adapting to similar ecological niches and/or environments. Dissimilatory Metal Reduction: reduction of a metal or metalloid that uses electrons from oxidized organic or inorganic electron donors. Exoelectrogen: microorganism that performs dissimilatory metal reduction via extracellular electron transfer. Extremophiles: organisms that thrive in physical or geochemical conditions that are considered detrimental to most life on Earth. Homoplasy: a character shared by a set of species that is not shared by a common ancestor Non-synonymous Substitutions (K a ): a substitution of a nucleotide that changes a codon sequence resulting in a change in the amino acid sequence of a protein. Synonymous Substitutions (K s ): a substitution of a nucleotide that may change a codon sequence, but results in no change in the amino acid sequence of a protein. Thermophiles: a specific group of extremophiles that survive and operate at temperatures ∼50-125°C.

pH Dependency in Anode Biofilms of Thermincola ferriacetica Suggests a Proton-Dependent Electrochemical Response.

Monitoring the electrochemical response of anode respiring bacteria (ARB) helps elucidate the fundamental processes of anode respiration and their rate limitations. Understanding these limitations provides insights on how ARB create the complex interfacing of biochemical metabolic processes with insoluble electron acceptors and electronics. In this study, anode biofilms of the thermophilic (60 °C) Gram-positive ARB Thermincola ferriacetica were studied to determine the presence of a proton-dependent electron transfer response. The effects of pH, the presence of an electron donor (acetate), and biofilm growth were varied to determine their influence on the electrochemical midpoint potential ( EKA) and formal redox potential ( E°') under nonturnover conditions. The EKA and E°' are associated with an enzymatic process within ARB's metabolism that controls the rate and energetic state of their respiration. Results for all conditions indicate that pH was the major contributor to altering the energetics of T. ferriacetica anode biofilms. Electrochemical responses measured in the absence of an electron donor and with a minimal proton gradient within the anode biofilms resulted in a 48 ± 7 mV/pH unit shift in the E°', suggesting a proton-dependent rate-limiting process. Given the limited energy available for anode respiration (<200 mV when using acetate as electron donor), our results provide a new perspective in understanding proton-transport limitations in ARB biofilms, one in which ARB are thermodynamically limited by pH gradients. Since the anode biofilms of all ARB that perform direct extracellular electron transfer (EET) investigated thus far exhibit an n = 1 Nernstian behavior, and because this behavior is affected by changes in pH, we hypothesize that the Nernstian response is associated with membrane proteins responsible for proton translocation. Finally, this study shows that the EKA and E°' are a function of pH within the physiological range of ARB, and thus, given the significant effect pH has on this parameter, we recommend reporting the EKA and E°' of ARB biofilms at a specific bulk pH.

Simultaneous fermentation of cellulose and current production with an enriched mixed culture of thermophilic bacteria in a microbial electrolysis cell.

An enriched mixed culture of thermophilic (60°C) bacteria was assembled for the purpose of using cellulose to produce current in thermophilic microbial electrolysis cells (MECs). Cellulose was fermented into sugars and acids before being consumed by anode-respiring bacteria (ARB) for current production. Current densities (j) were sustained at 6.5 ± 0.2 A m-2 in duplicate reactors with a coulombic efficiency (CE) of 84 ± 0.3%, a coulombic recovery (CR) of 54 ± 11% and without production of CH4 . Low-scan rate cyclic voltammetry (LSCV) revealed a mid-point potential (Eka ) of -0.17 V versus SHE. Pyrosequencing analysis of the V4 hypervariable region of 16S rDNA and scanning electron microscopy present an enriched thermophilic microbial community consisting mainly of the phylum Firmicutes with the Thermoanaerobacter (46 ± 13%) and Thermincola (28 ± 14%) genera occupying the biofilm anode in high relative abundance and Tepidmicrobium (38 ± 6%) and Moorella (11 ± 8%) genera present in high relative abundance in the bulk medium. The Thermoanaerobacter (15 ± 16%) and Brevibacillus (21 ± 30%) genera were also present in the bulk medium; however, their relative abundance varied by reactor. This study indicates that thermophilic consortia can obtain high CE and CR, while sustaining high current densities from cellulose in MECs.

The effect of pH and buffer concentration on anode biofilms of Thermincola ferriacetica.

We assessed the effects of pH and buffer concentration on current production and growth of biofilms of Thermincola ferriacetica - a thermophilic, Gram-positive, anode-respiring bacterium (ARB) - grown on anodes poised at a potential of -0.06V vs. SHE in microbial electrolysis cells (MECs) at 60°C. T. ferriacetica generated current in the pH range of 5.2 to 8.3 with acetate as the electron donor and 50mM bicarbonate buffer. Maximum current density was reduced by ~80% at pH5.2 and ~14% at 7.0 compared to pH8.3. Increasing bicarbonate buffer concentrations from 10mM to 100mM resulted in an increase in the current density by 40±6%, from 6.8±1.1 to 11.2±2.7Am(-2), supporting that more buffer alleviated pH depression within T. ferriacetica biofilms. Confocal laser scanning microscopy (CLSM) images indicated that higher bicarbonate buffer concentrations resulted in larger live biofilm thicknesses: from 68±20µm at 10mM bicarbonate to >150µm at 100mM, supporting that buffer availability was a strong influence on biofilm thickness. In comparison to mesophilic Geobacter sulfurreducens biofilms, the faster transport rates at higher temperature and the ability to grow at relatively lower pH allowed T. ferriacetica to produce higher current densities with lower buffer concentrations.

Characterization of Electrical Current-Generation Capabilities from Thermophilic Bacterium Thermoanaerobacter pseudethanolicus Using Xylose, Glucose, Cellobiose, or Acetate with Fixed Anode Potentials.

Thermoanaerobacter pseudethanolicus 39E (ATCC 33223), a thermophilic, Fe(III)-reducing, and fermentative bacterium, was evaluated for its ability to produce current from four electron donors-xylose, glucose, cellobiose, and acetate-with a fixed anode potential (+ 0.042 V vs SHE) in a microbial electrochemical cell (MXC). Under thermophilic conditions (60 °C), T. pseudethanolicus produced high current densities from xylose (5.8 ± 2.4 A m(-2)), glucose (4.3 ± 1.9 A m(-2)), and cellobiose (5.2 ± 1.6 A m(-2)). It produced insignificant current when grown with acetate, but consumed the acetate produced from sugar fermentation to produce electrical current. Low-scan cyclic voltammetry (LSCV) revealed a sigmoidal response with a midpoint potential of -0.17 V vs SHE. Coulombic efficiency (CE) varied by electron donor, with xylose at 34.8% ± 0.7%, glucose at 65.3% ± 1.0%, and cellobiose at 27.7% ± 1.5%. Anode respiration was sustained over a pH range of 5.4-8.3, with higher current densities observed at higher pH values. Scanning electron microscopy showed a well-developed biofilm of T. pseudethanolicus on the anode, and confocal laser scanning microscopy demonstrated a maximum biofilm thickness (Lf) greater than ~150 µm for the glucose-fed biofilm.

Draft Genome Sequence of the Gram-Positive Thermophilic Iron Reducer Thermincola ferriacetica Strain Z-0001T.

A 3.19-Mbp draft genome of the Gram-positive thermophilic iron-reducing Firmicutes isolate from the Peptococcaceae family, Thermincola ferriacetica Z-0001, was assembled at ~100× coverage from 100-bp paired-end Illumina reads. The draft genome contains 3,274 predicted genes (3,187 protein coding genes) and putative multiheme c-type cytochromes.

Kinetic, electrochemical, and microscopic characterization of the thermophilic, anode-respiring bacterium Thermincola ferriacetica.

Thermincola ferriacetica is a recently isolated thermophilic, dissimilatory Fe(III)-reducing, Gram-positive bacterium with capability to generate electrical current via anode respiration. Our goals were to determine the maximum rates of anode respiration by T. ferriacetica and to perform a detailed microscopic and electrochemical characterization of the biofilm anode. T. ferriacetica DSM 14005 was grown at 60 °C on graphite-rod anodes poised at -0.06 V (vs) SHE in duplicate microbial electrolysis cells (MECs). The cultures grew rapidly until they achieved a sustained current density of 7-8 A m(-2) with only 10 mM bicarbonate buffer and an average Coulombic Efficiency (CE) of 93%. Cyclic voltammetry performed at maximum current density revealed a Nernst-Monod response with a half saturation potential (EKA) of -0.127 V (vs) SHE. Confocal microscopy images revealed a thick layer of actively respiring cells of T. ferriacetica (~38 µm), which is the first documentation for a gram positive anode respiring bacterium (ARB). Scanning electron microscopy showed a well-developed biofilm with a very dense network of extracellular appendages similar to Geobacter biofilms. The high current densities, a thick biofilm (~38 µm) with multiple layers of active cells, and Nernst-Monod behavior support extracellular electron transfer (EET) through a solid conductive matrix - the first such observation for Gram-positive bacteria. Operating with a controlled anode potential enabled us to grow T. ferriacetica that can use a solid conductive matrix resulting in high current densities that are promising for MXC applications.