Engineering topics

Fredrickson, Jim K

Publications and source records attributed to Fredrickson, Jim K.

At least 19 records

DOE OSTI2013

Production of bio-based materials using photobioreactors with binary cultures

A method, device and system for producing preselected products, (either finished products or preselected intermediary products) from biobased precursors or CO.sub.2 and/or bicarbonate. The principal features of the present invention include a method wherein a binary culture is incubated with a biobased precursor in a closed system to transform at least a portion of the biobased precursor to a preselected product. The present invention provides a method of cultivation that does not need sparging of a closed bioreactor to remove or add a gaseous byproduct or nutrient from a liquid medium. This improvement leads to significant savings in energy consumption and allows for the design of photobioreactors of any desired shape. The present invention also allows for the use of a variety of types of waste materials to be used as the organic starting material.

36 MATERIALS SCIENCE↗

DOE OSTI2013

Single-Cell Imaging and Spectroscopic Analyses of Cr(VI) Reduction on the Surface of Bacterial Cells

We investigate single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic and highly soluble Cr(VI) can be efficiently reduced to the less toxic and non-soluble Cr2O3 nanoparticles by MR-1. Cr2O3 is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr2O3 and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1 surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of the MR-1 is: wild type > single mutant @mtrC or mutant @omcA > double mutant (@omcA-@mtrC). Moreover, our results also suggest that the direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may co-exist in the reduction processes.

bacterial cells↗

DOE OSTI2013

Dynamic Modeling of Aerobic Growth of Shewanella oneidensis. Predicting Triauxic Growth, Flux Distributions and Energy Requirement for Growth

A model-based analysis is conducted to investigate metabolism of Shewanella oneidensis MR-1 strain in aerobic batch culture, which exhibits an intriguing growth pattern by sequentially consuming substrate (i.e., lactate) and by-products (i.e., pyruvate and acetate). A general protocol is presented for developing a detailed network-based dynamic model for S. oneidensis based on the Lumped Hybrid Cybernetic Model (LHCM) framework. The L-HCM, although developed from only limited data, is shown to accurately reproduce exacting dynamic metabolic shifts, and provide reasonable estimates of energy requirement for growth. Flux distributions in S. oneidensis predicted by the L-HCM compare very favorably with 13C-metabolic flux analysis results reported in the literature. Predictive accuracy is enhanced by incorporating measurements of only a few intracellular fluxes, in addition to extracellular metabolites. The L-HCM developed here for S. oneidensis is consequently a promising tool for the analysis of intracellular flux distribution and metabolic engineering.

ATP requirement for growth↗

DOE OSTI2012

Mtr Extracellular Electron Transfer Pathways in Fe(III)-reducing or Fe(II)-oxidizing Bacteria: A Genomic Perspective

Originally discovered in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), the Mtr (i.e., metal-reducing) pathway exists in all characterized strains of metal-reducing Shewanella. The protein components identified to date for the Mtr pathway of MR-1 include four multi-heme c-type cytochromes (c-Cyts), CymA, MtrA, MtrC and OmcA, and a porin-like, outer membrane protein MtrB. They are strategically positioned along the width of the MR-1 cell envelope to mediate electron transfer from the quinone/quinol pool in the inner-membrane to the Fe(III)-containing minerals external to the bacterial cells. A survey of microbial genomes revealed homologues of the Mtr pathway in other dissimilatory Fe(III)-reducing bacteria, including Aeromonas hydrophila, Ferrimonas balearica and Rhodoferax ferrireducens, and in the Fe(II)-oxidizing bacteria Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1. The widespread distribution of Mtr pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria emphasizes the importance of this type of extracellular electron transfer pathway in microbial redox transformation of Fe. Their distribution in these two different functional groups of bacteria also emphasizes the bi-directional nature of electron transfer reactions carried out by the Mtr pathways. The characteristics of the Mtr pathways may be shared by other pathways used by microorganisms for exchanging electrons with their extracellular environments.

c-type cytochromes↗

DOE OSTI2012

Analysis of Structural MtrC Models Based on Homology with the Crystal Structure of MtrF

The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to have functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the β-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.

Edwards, Marcus↗

DOE OSTI2012

Molecular Structure and Free Energy Landscape for Electron Transport in the Deca-Heme Cytochrome MtrF

The free energy profile for electron flow through the bacterial deca-heme cytochrome MtrF has been computed using thermodynamic integration and classical molecular dynamics. The extensive calculations on two versions of the structure help validate the method and results, because differences in the profiles can be related to differences in the charged amino acids local to specific heme groups. First estimates of reorganization free energies λ yield a range consistent with expectations for partially solvent exposed cofactors, and reveal an activation energy range surmountable for electron flow. Future work will aim at increasing the accuracy of λ with polarizable force field dynamics and quantum chemical energy gap calculations, as well as quantum chemical computation of electronic coupling matrix elements.

Environmental Molecular Sciences Laboratory↗

DOE OSTI2012

The roles of CymA in support of the respiratory flexibility of Shewanella oneidensis MR-1

Shewanella species are isolated from the oxic/anoxic regions of seawater and aquatic sediments where redox conditions fluctuate in time and space. Colonization of these environments is by virtue of flexible respiratory chains, many of which are notable for the ability to reduce extracellular substrates including the Fe(III) and Mn(IV) contained in oxide and phyllosilicate minerals. Shewanella oneidensis MR-1 serves as a model organism to consider the biochemical basis of this flexibility. In the present paper, we summarize the various systems that serve to branch the respiratory chain of S. oneidensis MR-1 in order that electrons from quinol oxidation can be delivered the various terminal electron acceptors able to support aerobic and anaerobic growth. This serves to highlight several unanswered questions relating to the regulation of respiratory electron transport in Shewanella and the central role(s) of the tetrahaem-containing quinol dehydrogenase CymA in that process.

cytochrome↗

DOE OSTI2012

Electron Transport at the Microbe–Mineral Interface: A Synthesis of Current Research Challenges

Many bacterial and archaeal species can couple growth to the respiratory reduction or oxidation of insoluble mineral oxides of transition metals. These solid substrates are abundant electron sinks and sources for life on Earth, but, since they are insoluble in water, they cannot enter the bacterial cells. So, to exploit these electron sinks and sources, specific respiratory electron-transfer mechanisms must overcome the physical limitations associated with electron transfer between a microbe and extracellular metal oxides. Recent microbiological, geochemical, biochemical, spectroscopic and structural work is beginning to shed light on the molecular mechanism and impacts of electron transfer at themicrobe–mineral interface from a nanometre to kilometre scale. The research field is attracting attention in applied quarters from those with interests in nanowires, microbial fuel cells, bioremediation and microbial cell factories.

Richardson, David↗

DOE OSTI2012

Fe(III) Reduction and U(VI) Immobilization by Paenibacillus sp. Strain 300A, Isolated from Hanford 300A Subsurface Sediments

A facultative iron-reducing (Fe(III)-reducing) Paenibacillus sp. strain was isolated from Hanford 300A subsurface sediment biofilms that was capable of reducing soluble Fe(III) complexes (Fe(III)-NTA and Fe(III)-citrate) but unable to reduce poorly crystalline ferrihydrite (Fh). However, Paenibacillus sp. 300A was capable of reducing Fh in the presence of low concentrations (2 µM) of either of electron transfer mediators (ETMs) flavin mononucleotide (FMN) or anthraquinone-2,6-disulfonate (AQDS). Maximum initial Fh reduction rates were observed at catalytic concentrations (<10 µM) of either FMN or AQDS. Higher FMN concentrations inhibited Fh reduction, while increased AQDS concentrations did not. We found that Paenibacillus sp. 300A also could reduce Fh in the presence of natural ETMs from Hanford 300A subsurface sediments. In the absence of ETMs, Paenibacillus sp. 300A was capable of immobilizing U(VI) through both reduction and adsorption. The relative contributions of adsorption and microbial reduction to U(VI) removal from the aqueous phase were ~7:3 in PIPES and ~1:4 in bicarbonate buffer. Our study demonstrated that Paenibacillus sp. 300A catalyzes Fe(III) reduction and U(VI) immobilization and that these reactions benefit from externally added or naturally existing ETMs in 300A subsurface sediments.

Environmental Molecular Sciences Laboratory↗

DOE OSTI2012

Biofilm Shows Spatially Stratified Metabolic Responses to Contaminant Exposure

The objective of this study was to elucidate the spatiotemporal responses of live S. oneidensis MR-1 biofilms to U(VI) (uranyl, UO22+) and Cr(VI) (chromate, CrO42-), important environmental contaminants at DOE contaminated sites. Toward this goal, we applied noninvasive nuclear magnetic resonance (NMR) imaging, diffusion, relaxation and spectroscopy techniques to monitor in situ spatiotemporal responses of S. oneidensis biofilms to U(VI) and Cr(VI) exposure in terms of changes in biofilm structures, diffusion properties, and cellular metabolism. Exposure to U(VI) or Cr(VI) did not appear to change the overall biomass distribution but caused changes in the physicochemical microenvironments inside the biofilm as indicated by diffusion measurements. Changes in the diffusion properties of the biofilms in response to U(VI) and Cr(VI) exposure imply a novel function of the extracellular polymeric substances (EPS) affecting the biotransformation and transport of contaminants in the environment. In the presence of U(VI) or Cr(VI), the anaerobic metabolism of lactate was inhibited significantly, although the biofilms were still capable of reducing U(VI) and Cr(VI). Local concentrations of Cr(III)aq in the biofilm suggested relatively high Cr(VI) reduction activities at the top of the biofilm, near the medium-biofilm interface. The depth-resolved metabolic activities of the biofilm suggested higher diversion effects of gluconeogenesis and C1 metabolism pathways at the bottom of the biofilm and in the presence of U(VI). This study provides a noninvasive means to investigate spatiotemporal responses of biofilms, including surface-associated microbial communities in engineering, natural and medical settings, to various environmental perturbations including exposure to environmental contaminants and antimicrobials.

biofilm↗

DOE OSTI2012

Pore-Scale Characterization of Biogeochemical Controls on Iron and Uranium Speciation under Flow Conditions

Etched silicon microfluidic pore network models (micromodels) with controlled chemical and redox gradients, mineralogy, and microbiology under continuous flow conditions are used for the incremental development of complex microenvironments that simulate subsurface conditions. We demonstrate the colonization of micromodel pore spaces by an anaerobic Fe(III)-reducing bacterial species (Geobacter sulfurreducens) and the enzymatic reduction of a bioavailable Fe(III) phase within this environment. Using both X-ray Microprobe and X-ray Absorption Spectroscopy, we investigate the combined effects of the precipitated Fe(III) phases and the microbial population on uranium biogeochemistry under flow conditions. Precipitated Fe(III) phases within the micromodel were most effectively reduced in the presence of an electron shuttle (AQDS), and Fe(II) ions adsorbed onto the precipitated mineral surface without inducing any structural change. In the absence of Fe(III), U(VI) was effectively reduced by the microbial population to insoluble U(IV), which was precipitated in discrete regions associated with biomass. In the presence of Fe(III) phases, however, both U(IV) and U(VI) could be detected associated with biomass, suggesting re-oxidation of U(IV) by localized Fe(III) phases. These results demonstrate the importance of the spatial localization of biomass and redox active metals, and illustrate the key effects of pore-scale processes on contaminant fate and reactive transport.

Environmental Molecular Sciences Laboratory↗

DOE OSTI2012

Sustained H2 Production Driven by Photosynthetic Water Splitting in a Unicellular Cyanobacterium

Continuously illuminated nitrogen-deprived Cyanothece sp. ATCC 51142 evolved H2 via dinitrogenase at rates up to 400 μmol•mg Chl-1•h-1 in parallel with photosynthetic O2 production. Notably, sustained co-production of H2 and O2 occurred over 100 h in the presence of CO2, with both gases displaying inverse oscillations which eventually dampened to stable rates. Oscillations were not observed when CO2 was omitted, while H2 and O2 evolution rates were positively correlated. In situ light saturation analyses of H2 production displayed dose-dependence and lack of O2 inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect the short-term H2 production indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. Collectively, our results demonstrate that uninterrupted H2 production in unicellular diazotrophic cyanobacteria can be fueled by water photolysis without the detrimental effects of O2 and have important implications for sustainable production of biofuels.

photosynthetic water↗

DOE OSTI2012

The Crystal Structure of the Extracellular 11-heme Cytochrome UndA Reveals a Conserved 10-heme Motif and Defined Binding Site for Soluble Iron Chelates.

Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure is the first outer membrane cytochrome to be crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

A Functional Description of CymA, an Electron Transfer Hub Supporting Anaerobic Respiratory Flexibility in Shewanella

CymA is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis MR-1 identifies three low-spin His/His coordinated c-hemes and a single high-spin c-heme with His/H{sub 2}O coordination lying adjacent to the quinol binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (ca. {approx}240 mV) and low-spin (ca. {approx}110, {approx}190 and {approx}265 mV) hemes that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E{sub m} = {approx} 80 mV) in the presence of NADH (E{sub m} = {approx} 320 mV) and an NADH:menadione oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H{sub 2} oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a nonheme cofactor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc{sub 3}. The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outermembrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

The ‘porin–cytochrome’ model for microbe-to-mineral electron transfer

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as 'nanowires', or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

Genome-Scale Modeling of Light-Driven Reductant Partitioning and Carbon Fluxes in Diazotrophic Unicellular Cyanobacterium Cyanothece sp. ATCC 51142

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When photosystem II flux is high, terminal oxidases of respiratory electron transport are predicted to be an important mechanism for removing excess electrons. When photosystem I flux is high cyclic electron transport becomes important. Model predictions of growth rates were in good quantitative agreement with measured growth rates, and predictions of reaction usage were ualitatively consistent with protein and mRNA expression data, when these latter datasets were used to constrain the model.

51142↗

DOE OSTI2012

Stochastic and Deterministic Assembly Processes in Subsurface Microbial Communities

A major goal of microbial community ecology is to understand the forces that structure community composition. Deterministic selection by specific environmental factors is sometimes important, but in other cases stochastic or ecologically neutral processes dominate. Lacking is a unified conceptual framework aiming to understand why deterministic processes dominate in some contexts but not others. Here we work towards such a framework. By testing predictions derived from general ecological theory we aim to uncover factors that govern the relative influences of deterministic and stochastic processes. We couple spatiotemporal data on subsurface microbial communities and environmental parameters with metrics and null models of within and between community phylogenetic composition. Testing for phylogenetic signal in organismal niches showed that more closely related taxa have more similar habitat associations. Community phylogenetic analyses further showed that ecologically similar taxa coexist to a greater degree than expected by chance. Environmental filtering thus deterministically governs subsurface microbial community composition. More importantly, the influence of deterministic environmental filtering relative to stochastic factors was maximized at both ends of an environmental variation gradient. A stronger role of stochastic factors was, however, supported through analyses of phylogenetic temporal turnover. While phylogenetic turnover was on average faster than expected, most pairwise comparisons were not themselves significantly non-random. The relative influence of deterministic environmental filtering over community dynamics was elevated, however, in the most temporally and spatially variable environments. Our results point to general rules governing the relative influences of stochastic and deterministic processes across micro- and macro-organisms.

community assembly↗