A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation o... more A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.
SUMMARYExtracellular electron transfer (EET), the process whereby cells access electron acceptors... more SUMMARYExtracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in select organisms, a widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. How these shuttles catalyze electron transfer within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) inPseudomonas aeruginosabiofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons i...
Electrically excitable cells harness voltage coupled calcium influx to transmit intracellular sig... more Electrically excitable cells harness voltage coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects of this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in E. coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports both voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and calcium transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux inhibited protein concentration differences, though the identity of the calcium effectors remains unknown. Thus, voltage and calcium relay a bacterial sense of touch, and alter cellular lifestyle. These data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other potential signals conveyed by voltage and calcium. These data also provide evidence that dynamic electrophysiological flux exists as a signaling modality in the oldest kingdom of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.
Physical chemistry chemical physics : PCCP, Jan 23, 2018
Protein molecular conductance has attracted attention from researchers for the possibility of con... more Protein molecular conductance has attracted attention from researchers for the possibility of constructing innovative flexible biocompatible nanoscale electronic devices and smart hybrid materials. Due to protein complexity, most evaluations of protein conductivity are based on the simple estimation of protein's molecular orbital energy levels and spatial distributions without analysing its protein interaction with electrodes and the calculation of the rates of electron transfer (ET). In the present work, we included in our density functional theory (DFT) analysis an approach based on the non-equilibrium Green's function (NEGF) allowing for calculation from the first principles the molecular interaction with electrodes and thus the role of electrode materials, Fermi level, the thermal distribution of electronic energy levels, and the coupling efficiency between the molecule and the electrodes. Compared to proteins studied so far, mainly artificial peptides, heme-containing c...
Atomic force microscopy and confocal resonance Raman microscopy (CRRM) of singlecells were used t... more Atomic force microscopy and confocal resonance Raman microscopy (CRRM) of singlecells were used to study the transition of anode-grown Geobacter sulfurreducens biofilms from lag phase (initial period of low current) to exponential phase (subsequent period of rapidly increasing current). Results reveal that lag phase biofilms consist of lone cells and tightly packed single-cell thick clusters crisscrossed with extracellular linear structures that appears to be comprised of nodules approximately 20 nm in diameter aligned end to end. By early exponential phase, cell clusters expand laterally and a second layer of closely packed cells begins to form on top of the first. Abundance of c-type cytochromes (c-Cyt) is threefold greater in two-cell thick regions than in one-cell thick regions. The results indicate that early biofilm growth involves two transformations. The first is from lone cells to two-dimensionally associated cells during lag phase when current remains low. This is accompanied by formation of extracellular linear structures. The second is from two-to three-dimensionally associated cells during early exponential phase when current begins to increase rapidly. This is accompanied by a dramatic increase in c-Cyt abundance.
The electron-transfer (ET) parameters for oriented and aligned monolayers of the bacterial photos... more The electron-transfer (ET) parameters for oriented and aligned monolayers of the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides formed on the top of self-assembled monolayers (SAMs) of alkanethiols of various lengths immobilized on gold ...
Proceedings of the National Academy of Sciences, 2012
Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extr... more Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extracellular electron transfer to an anode (an electrode poised at a metabolically oxidizing potential), forming a biofilm extending many cell lengths away from the anode surface. It has been proposed that long-range electron transport in such biofilms occurs through a network of bound redox cofactors, thought to involve extracellular matrix c -type cytochromes, as occurs for polymers containing discrete redox moieties. Here, we report measurements of electron transport in actively respiring Geobacter sulfurreducens wild type biofilms using interdigitated microelectrode arrays. Measurements when one electrode is used as an anode and the other electrode is used to monitor redox status of the biofilm 15 μm away indicate the presence of an intrabiofilm redox gradient, in which the concentration of electrons residing within the proposed redox cofactor network is higher farther from the anode su...
Results are presented for rate constants (k") and reorganizational energy barriers (1) for interf... more Results are presented for rate constants (k") and reorganizational energy barriers (1) for interfacial electron transfer at ultralow-temperatures (120-150 K) across mixed C~F~C~CO~(CH~),SWCH~(CH~),-ISH monolayers (n = 8, 12, 16). The monolayers are kinetically disperse, i.e., the ferrocene sites exhibit a range of rate constants. Average values of k" were measured by cyclic voltammetry with application of Marcus theory corrected for the density of electronic states in the gold electrode. The k" and pre-exponential @e) values exhibit exponential dependencies on alkane chain length characterized by exponential coefficients of 1.06 and 1.44KH2, respectively. The former value agrees with aqueous phase results by others for analogous but more highly ordered monolayers near ambient temperatures; the latter result corresponds to an electronic coupling coefficient of BEL of 1.1 The activation analysis-derived reorganizational barrier energies decrease somewhat with increasing chain length, contrary to theoretical expectations.
All regents were used as received unless otherwise indicated. Acetone, dimethylformamide (DMF), E... more All regents were used as received unless otherwise indicated. Acetone, dimethylformamide (DMF), EtOH, ether, hydrazine, 11-mercaptoundecanoic acid (MUA), sodium tetraphenylborate (NaBPh 4), 4-(aminomethyl)pyridine, CM-Sephadex C-25 and [Ru(NH 3) 5 Cl]Cl 2 were purchased from Aldrich. N-(3-dimethylaminopropyl)-N
pK, value of G" as determined by using Br,'-or SO;-as oxidants. The observations are described by... more pK, value of G" as determined by using Br,'-or SO;-as oxidants. The observations are described by, at pH 6, OH' + T1+-TIOH+ Note Added in Proof. Since TI(I1) has been suggested to one-electron oxidize 9-methylguanine,I9 this reagent was used (as a third oxidant) with guanosine, and the reaction was monitored with conductance in the pH range 3-6. At pH 3 (where the of H+, whereas at pH 6 (where the oxidizing species is T10H+),3y the net change of [H'] was zero.4o The inflection point of the conductance vs pH plot was 3.9, in perfect agreement with the oxidizing species is the reaction led to removal of 1 equiv TIOH' + G-.+ T1+ + H2O + G(-H)' at pH 3 3
he union between biologists, physical scientists, and engineers has yielded accelerated output in... more he union between biologists, physical scientists, and engineers has yielded accelerated output in all three of these areas for some time. New approaches to instrumentation [1], as well as new ideas in disease detection [2], [3], have been put forward and proven as a result of interdisciplinary teaming. The general public has been helped by new concepts in drug discovery-a field greatly benefitted by high-speed computation. Biological macromolecules (i.e., proteins (including toxins, hormones, antibodies, enzymes and those on surfaces and/or within cells, bacteria, and viruses), DNA, and RNA) are central to biological processes. Their presence and state of flux (i.e., concentration and/or structural change versus time) provide important signatures of disease and threat exposure. Detecting specific biological macromolecules in vivo or from samples derived from untreated body fluids or an environment is a challenging but worthwhile endeavor. Relevant concentrations of biological macromolecules are often low (often femtomolar), and they exist in complex media containing many other macromolecules, some of which may interfere with detection. In addition it is desirable to detect multiple macromolecules simultaneously to ensure a high confidence level in disease diagnosis or threat assessment and to determine their progression [2]. In this article, we review recent progress in the area of biological macromolecular sensors and we present several research activities aimed at achieving such a device. Here we distinguish sensors from assays-the former describing devices capable of
Benthic microbial fuel cells are devices that generate modest levels of electrical power in seafl... more 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 carbonfibre/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 microorganisms 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 ε-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.
A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation o... more A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.
A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation o... more A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.
SUMMARYExtracellular electron transfer (EET), the process whereby cells access electron acceptors... more SUMMARYExtracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in select organisms, a widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. How these shuttles catalyze electron transfer within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) inPseudomonas aeruginosabiofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons i...
Electrically excitable cells harness voltage coupled calcium influx to transmit intracellular sig... more Electrically excitable cells harness voltage coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects of this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in E. coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports both voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and calcium transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux inhibited protein concentration differences, though the identity of the calcium effectors remains unknown. Thus, voltage and calcium relay a bacterial sense of touch, and alter cellular lifestyle. These data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other potential signals conveyed by voltage and calcium. These data also provide evidence that dynamic electrophysiological flux exists as a signaling modality in the oldest kingdom of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.
Physical chemistry chemical physics : PCCP, Jan 23, 2018
Protein molecular conductance has attracted attention from researchers for the possibility of con... more Protein molecular conductance has attracted attention from researchers for the possibility of constructing innovative flexible biocompatible nanoscale electronic devices and smart hybrid materials. Due to protein complexity, most evaluations of protein conductivity are based on the simple estimation of protein's molecular orbital energy levels and spatial distributions without analysing its protein interaction with electrodes and the calculation of the rates of electron transfer (ET). In the present work, we included in our density functional theory (DFT) analysis an approach based on the non-equilibrium Green's function (NEGF) allowing for calculation from the first principles the molecular interaction with electrodes and thus the role of electrode materials, Fermi level, the thermal distribution of electronic energy levels, and the coupling efficiency between the molecule and the electrodes. Compared to proteins studied so far, mainly artificial peptides, heme-containing c...
Atomic force microscopy and confocal resonance Raman microscopy (CRRM) of singlecells were used t... more Atomic force microscopy and confocal resonance Raman microscopy (CRRM) of singlecells were used to study the transition of anode-grown Geobacter sulfurreducens biofilms from lag phase (initial period of low current) to exponential phase (subsequent period of rapidly increasing current). Results reveal that lag phase biofilms consist of lone cells and tightly packed single-cell thick clusters crisscrossed with extracellular linear structures that appears to be comprised of nodules approximately 20 nm in diameter aligned end to end. By early exponential phase, cell clusters expand laterally and a second layer of closely packed cells begins to form on top of the first. Abundance of c-type cytochromes (c-Cyt) is threefold greater in two-cell thick regions than in one-cell thick regions. The results indicate that early biofilm growth involves two transformations. The first is from lone cells to two-dimensionally associated cells during lag phase when current remains low. This is accompanied by formation of extracellular linear structures. The second is from two-to three-dimensionally associated cells during early exponential phase when current begins to increase rapidly. This is accompanied by a dramatic increase in c-Cyt abundance.
The electron-transfer (ET) parameters for oriented and aligned monolayers of the bacterial photos... more The electron-transfer (ET) parameters for oriented and aligned monolayers of the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides formed on the top of self-assembled monolayers (SAMs) of alkanethiols of various lengths immobilized on gold ...
Proceedings of the National Academy of Sciences, 2012
Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extr... more Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extracellular electron transfer to an anode (an electrode poised at a metabolically oxidizing potential), forming a biofilm extending many cell lengths away from the anode surface. It has been proposed that long-range electron transport in such biofilms occurs through a network of bound redox cofactors, thought to involve extracellular matrix c -type cytochromes, as occurs for polymers containing discrete redox moieties. Here, we report measurements of electron transport in actively respiring Geobacter sulfurreducens wild type biofilms using interdigitated microelectrode arrays. Measurements when one electrode is used as an anode and the other electrode is used to monitor redox status of the biofilm 15 μm away indicate the presence of an intrabiofilm redox gradient, in which the concentration of electrons residing within the proposed redox cofactor network is higher farther from the anode su...
Results are presented for rate constants (k") and reorganizational energy barriers (1) for interf... more Results are presented for rate constants (k") and reorganizational energy barriers (1) for interfacial electron transfer at ultralow-temperatures (120-150 K) across mixed C~F~C~CO~(CH~),SWCH~(CH~),-ISH monolayers (n = 8, 12, 16). The monolayers are kinetically disperse, i.e., the ferrocene sites exhibit a range of rate constants. Average values of k" were measured by cyclic voltammetry with application of Marcus theory corrected for the density of electronic states in the gold electrode. The k" and pre-exponential @e) values exhibit exponential dependencies on alkane chain length characterized by exponential coefficients of 1.06 and 1.44KH2, respectively. The former value agrees with aqueous phase results by others for analogous but more highly ordered monolayers near ambient temperatures; the latter result corresponds to an electronic coupling coefficient of BEL of 1.1 The activation analysis-derived reorganizational barrier energies decrease somewhat with increasing chain length, contrary to theoretical expectations.
All regents were used as received unless otherwise indicated. Acetone, dimethylformamide (DMF), E... more All regents were used as received unless otherwise indicated. Acetone, dimethylformamide (DMF), EtOH, ether, hydrazine, 11-mercaptoundecanoic acid (MUA), sodium tetraphenylborate (NaBPh 4), 4-(aminomethyl)pyridine, CM-Sephadex C-25 and [Ru(NH 3) 5 Cl]Cl 2 were purchased from Aldrich. N-(3-dimethylaminopropyl)-N
pK, value of G" as determined by using Br,'-or SO;-as oxidants. The observations are described by... more pK, value of G" as determined by using Br,'-or SO;-as oxidants. The observations are described by, at pH 6, OH' + T1+-TIOH+ Note Added in Proof. Since TI(I1) has been suggested to one-electron oxidize 9-methylguanine,I9 this reagent was used (as a third oxidant) with guanosine, and the reaction was monitored with conductance in the pH range 3-6. At pH 3 (where the of H+, whereas at pH 6 (where the oxidizing species is T10H+),3y the net change of [H'] was zero.4o The inflection point of the conductance vs pH plot was 3.9, in perfect agreement with the oxidizing species is the reaction led to removal of 1 equiv TIOH' + G-.+ T1+ + H2O + G(-H)' at pH 3 3
he union between biologists, physical scientists, and engineers has yielded accelerated output in... more he union between biologists, physical scientists, and engineers has yielded accelerated output in all three of these areas for some time. New approaches to instrumentation [1], as well as new ideas in disease detection [2], [3], have been put forward and proven as a result of interdisciplinary teaming. The general public has been helped by new concepts in drug discovery-a field greatly benefitted by high-speed computation. Biological macromolecules (i.e., proteins (including toxins, hormones, antibodies, enzymes and those on surfaces and/or within cells, bacteria, and viruses), DNA, and RNA) are central to biological processes. Their presence and state of flux (i.e., concentration and/or structural change versus time) provide important signatures of disease and threat exposure. Detecting specific biological macromolecules in vivo or from samples derived from untreated body fluids or an environment is a challenging but worthwhile endeavor. Relevant concentrations of biological macromolecules are often low (often femtomolar), and they exist in complex media containing many other macromolecules, some of which may interfere with detection. In addition it is desirable to detect multiple macromolecules simultaneously to ensure a high confidence level in disease diagnosis or threat assessment and to determine their progression [2]. In this article, we review recent progress in the area of biological macromolecular sensors and we present several research activities aimed at achieving such a device. Here we distinguish sensors from assays-the former describing devices capable of
Benthic microbial fuel cells are devices that generate modest levels of electrical power in seafl... more 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 carbonfibre/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 microorganisms 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 ε-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.
A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation o... more A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.
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Papers by Leonard Tender