Paul D. Frymier

Self-organized photosynthetic nanoparticle for cell-free hydrogen production.

There is considerable interest in making use of solar energy through photosynthesis to create alternative forms of fuel. Here, we show that photosystem I from a thermophilic bacterium and cytochrome-c(6) can, in combination with a platinum catalyst, generate a stable supply of hydrogen in vitro upon illumination. The self-organized platinization of the photosystem I nanoparticles allows electron transport from sodium ascorbate to photosystem I via cytochrome-c(6) and finally to the platinum catalyst, where hydrogen gas is formed. Our system produces hydrogen at temperatures up to 55 degrees C and is temporally stable for >85 days with no decrease in hydrogen yield when tested intermittently. The maximum yield is approximately 5.5 micromol H(2) h(-1) mg(-1) chlorophyll and is estimated to be approximately 25-fold greater than current biomass-to-fuel strategies. Future work will further improve this yield by increasing the kinetics of electron transfer, extending the spectral response and replacing the platinum catalyst with a renewable hydrogenase.

Estimating the toxicities of organic chemicals to bioluminescent bacteria and activated sludge

Toxicity assays based on bioluminescent bacteria have several advantages including a quick response and an easily measured signal. The Shk1 assay is a procedure for wastewater toxicity testing based on the bioluminescent bacterium Shk1. Using the Shk1 assay, the toxicity of 98 organic chemicals were measured and EC50 values were obtained. Quantitative structure-activity relationship (QSAR) models based on the logarithm of the octanol-water partition coefficient (log(Kow)) were developed for individual groups of organic chemicals with different functional groups. The correlation coefficients for different groups of organic compounds varied between 0.69 and 0.99. An overall QSAR model without discriminating the functional groups, which can be used for a quick estimate of the toxicities of organic chemicals, was also developed and model predictions were compared to experimental data. The model accuracy was found to be one order of magnitude from the observed values.

Kinetics of the toxicity of metals to luminescent bacteria

Abstract Bioluminescent bacteria are widely used in the toxicity assessment of aqueous solutions of heavy metals. They have also been used to screen wastewater treatment plant influent for toxicity resulting from the presence of heavy metals in the influent. However, some studies have indicated that certain strains of bioluminescent bacteria are not appropriate for influent toxicity screening for wastewater treatment plants. Shk1 is a bioluminescent bacterial strain genetically engineered for the specific purpose of monitoring influent wastewater to wastewater treatment plants for toxicity. Heavy metals at sufficient concentrations are toxic to Shk1 cells as they are to activated sludge microorganisms, and the exposure of Shk1 cells to heavy metal ions results in bioluminescence repression. The kinetics of the toxic effects of the heavy metals to Shk1 can be mathematically described in a manner similar to the non-competitive inhibition of enzymes. We determined the inhibition coefficients K i of seven heavy metals. We arranged K i and EC 50 values (a frequently used indicator of toxicity) of the seven heavy metals in increasing order and found that the sequences were in good agreement. We also show that under appropriate conditions, predictions of toxicity can be made based on EC 50 values that contain kinetic information similar to that contained in predictions using K i .

Use of multidimensional scaling in the selection of wastewater toxicity test battery components

In aquatic toxicity testing, no single test species is sensitive to all toxicants. Therefore, test batteries consisting of several individual assays are becoming more common. The organisms in a test battery should be representative of the entire system of interest. The results of the assays should be complementary to other components in the test battery to avoid redundancy. With the aid of multidimensional scaling (MDS), a multivariate statistical method, we examined the toxicity data of five bioassays (the continuous Shk1, Polytox, activated sludge respiration inhibition, Nitrosomonas, and Tetrahymena assays) that could serve as test battery components for the assessment of wastewater toxicity to activated sludge. MDS mapped the five assays into a two-dimensional space and showed that the Nitrosomonas assay should be included in test batteries plus one of the remaining four assays for assessing wastewater toxicity to activated sludge.

CELLULAR DYNAMICS SIMULATIONS OF BACTERIAL CHEMOTAXIS

Abstract The temporal and spatial evolution of the density of populations of chemotactic bacteria have previously been modeled by phenomenological cell balance equations based on cell motion restricted to one dimension. These one-dimensional balance equations have been used to interpret the results of experiments involving three-dimensional motion of bacterial populations with symmetry in two of the three dimensions. We develop a computer simulation to rigorously model the movement of a large population of individual chemotactic bacteria in three dimensions. Results of the simulation are compared with results using a one-dimensional phenomenological model in order to verify the range of validity of this model under situations involving one-dimensional gradients of chemical attractants.

Using factorial experiments to study the toxicity of metal mixtures

Two-level factorial experiments were employed in this study for understanding and predicting the toxicity of binary and ternary metal mixtures. Toxicity of metal mixtures with concentrations between the respective EC10 and EC80 values was experimentally measured. Models were fit to the experimental data and the resultant models were of high quality as reflected by R2 (coefficient of determination). Interactions between mixture components were indicated by the existence of statistically significant interaction terms in the models. Toxicity predictions based on the models were compared with observed toxicity for binary and ternary metal mixtures. The models developed did not assume additivity between metals, were simple and interpretable, and gave satisfactory predictions of the toxicity of metal mixtures in aqueous solutions without requiring knowledge on synergism or antagonism.

An exploratory study of the use of multivariate techniques to determine mechanisms of toxic action

The most successful quantitative structure-activity relationships have been developed by separating compounds by their mechanisms of toxic action (MOAs). However, to correctly determine the MOA of a compound is often not easy. We investigated the usefulness of discriminant analysis and logistic regression in determining MOAs. The discriminating variables used were the logarithm of octanol-water partition coefficients (logKow) and the experimental toxicity data obtained from Pimephales promelas and Tetrahymena pyriformis assays. Small total error rates were obtained when separating nonpolar narcotic compounds from other compounds, however, relatively high total error rates were obtained when separating less reactive compounds (polar, ester, and amine narcotics) from more reactive compounds (electrophiles, proelectrophiles, and nucleophiles).

Comparison of Three Bacterial Toxicity Assays for Imidazolium-Derived Ionic Liquids

Ionic liquids have become leading candidates for replacing many common organic solvents used in the chemical process industry. There is, however, a general lack of toxicology data relevant to wastewater treatment facility microbes for these compounds. In this study, we performed three bacterial-based toxicity assays on several imidazolium-derived compounds as well as the precursor compound 1-methylimidazole. Two of the assays, the Shk1 and Microtox assays, are used as surrogate assays for toxicity to bacterial respiration in activated sludge wastewater treatment plants. The third assay was a direct measure of the effect of toxicity on mixed bacterial culture respiration, using a commercially available consortium of naturally occurring bacteria to obtain I C50 values for direct comparison to the E C50 values from the surrogate assays. The Shk1 assay is based on a genetically engineered bioluminescent Pseudomonas bacterium and is more highly correlated with the respiration inhibition than the Microtox assay...

Sortase-mediated ligation of PsaE-modified photosystem I from Synechocystis sp. PCC 6803 to a conductive surface for enhanced photocurrent production on a gold electrode.

Sortase-mediated ligation was used to attach the photosystem I (PSI) complex from Synechocystis sp. PCC 6803 in a preferential orientation to enhance photoinduced electron flow to a conductive gold surface. Ideally, this method can result in a uniform monolayer of protein, covalently bound unidirectionally to the electrode surface. The exposed C-termini of the psaE subunits of the PSI trimer were targeted to contain an LPETG-sortase recognition sequence to increase noncompeting electron transfer by uniformly orienting the PSI stromal side proximal to the surface. Surface characterization with atomic force microscopy suggested that monolayer formation and optimal surface coverage occurred when the gold surfaces were incubated with peptide at 100 to 500 μM concentrations. When photochronoamperometry with potassium ferrocyanide and ferricyanide as redox mediators was used, photocurrents in the range of 100 to 200 nA/cm(2) were produced, which is an improvement over other attachment techniques for photosystem monolayers that produce approximately 100 nA/cm(2) or less. This work demonstrated that sortase-mediated ligation aided in the control of PSI orientation on modified gold surfaces with a distribution of 94% stromal side proximal and 6% lumenal side proximal to the surface for current-producing PSI.

Analysis of the solution structure of Thermosynechococcus elongatus photosystem I in n-dodecyl-β-d-maltoside using small-angle neutron scattering and molecular dynamics simulation

Small-angle neutron scattering (SANS) and molecular dynamics (MD) simulation were used to investigate the structure of trimeric photosystem I (PSI) from Thermosynechococcus elongatus (T. elongatus) stabilized in n-dodecyl-β-d-maltoside (DDM) detergent solution. Scattering curves of detergent and protein-detergent complexes were measured at 18% D2O, the contrast match point for the detergent, and 100% D2O, allowing observation of the structures of protein/detergent complexes. It was determined that the maximum dimension of the PSI-DDM complex was consistent with the presence of a monolayer belt of detergent around the periphery of PSI. A dummy-atom reconstruction of the shape of the complex from the SANS data indicates that the detergent envelope has an irregular shape around the hydrophobic periphery of the PSI trimer rather than a uniform, toroidal belt around the complex. A 50 ns MD simulation model (a DDM ring surrounding the PSI complex with extra interstitial DDM) of the PSI-DDM complex was developed for comparison with the SANS data. The results suggest that DDM undergoes additional structuring around the membrane-spanning surface of the complex instead of a simple, relatively uniform belt, as is generally assumed for studies that use detergents to solubilize membrane proteins.