Emily R. Petersen

Probing single- to multi-cell level charge transport in Geobacter sulfurreducens DL-1

Microbial fuel cells, in which living microorganisms convert chemical energy into electricity, represent a potentially sustainable energy technology for the future. Here we report the single-bacterium level current measurements of Geobacter sulfurreducens DL-1 to elucidate the fundamental limits and factors determining maximum power output from a microbial fuel cell. Quantized stepwise current outputs of 92(±33) and 196(±20) fA are generated from microelectrode arrays confined in isolated wells. Simultaneous cell imaging/tracking and current recording reveals that the current steps are directly correlated with the contact of one or two cells with the electrodes. This work establishes the amount of current generated by an individual Geobacter cell in the absence of a biofilm and highlights the potential upper limit of microbial fuel cell performance for Geobacter in thin biofilms.

The utility of Shewanella japonica for microbial fuel cells

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.

Aggrandizing power output from Shewanella oneidensis MR-1 microbial fuel cells using calcium chloride

There are several interconnected metabolic pathways in bacteria essential for the conversion of carbon electron sources directly into electrical currents using microbial fuel cells (MFCs). This study establishes a direct exogenous method to increase power output from a Shewanella oneidensis MR-1 containing MFC by adding calcium chloride to the culture medium. The current output from each CaCl(2) concentration tested revealed that the addition of CaCl(2) to 1400 μM increased the current density by >80% (0.95-1.76 μA/cm(2)) using sodium lactate as the sole carbon source. Furthermore, polarization curves showed that the maximum power output could be increased from 157 to 330 μW with the addition of 2080 μM CaCl(2). Since the conductivity of the culture medium did not change after the addition of CaCl(2) (confirmed by EIS and bulk conductivity measurements), this increase in power was primarily biological and not based on ionic effects. Thus, controlling the concentration of CaCl(2) is a pathway to increase the efficiency and performance of S. oneidensis MR-1 MFCs.

Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output.

The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28 μW/cm(2)) compared to Luria-Bertani (LB) (0.07 μW/cm(2)) or a defined growth minimal medium (MM) (0.02 μW/cm(2)). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors.

Neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. These circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. N. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. We report a novel method for disrupting circadian gene transcription while maintaining light responsiveness in N. crassa when held in a steady metabolic state using bioreactors. The arrhythmic transcription of core circadian genes and downstream clock-controlled genes was observed in constant darkness (DD) as determined by reverse transcription-quantitative PCR (RT-qPCR). Nearly all core circadian clock genes were up-regulated upon exposure to light during 11hr light/dark cycle experiments under identical conditions. Our results demonstrate that the natural timing of the robust circadian clock in N. crassa can be disrupted in the dark when maintained in a consistent metabolic state. Thus, these data lead to a path for the production of industrial scale enzymes in the model system, N. crassa, by removing the endogenous negative feedback regulation by the circadian oscillator.

Optimal method for efficiently removing extracellular nanofilaments from Shewanella oneidensis MR-1

The identification, production, and potential electron conductivity of bacterial extracellular nanofilaments is an area of great study, specifically in Shewanella oneidensis MR-1. While some studies focus on nanofilaments attached to the cellular body, many studies require the removal of these nanofilaments for downstream applications. The removal of nanofilaments from S. oneidensis MR-1 for further study requires not only that the nanofilaments be detached, but also for the cell bodies to remain intact. This is a study to both qualitatively (AFM) and quantitatively (LC/MS-MS) assess several nanofilament shearing methods and determine the optimal procedure. The best method for nanofilament removal, as judged by maximizing extracellular filamentous proteins and minimizing membrane and intracellular proteins, is vortexing a washed cell culture for 10 min.