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Nanoscale sensing arrays utilizing the unique properties of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots are being developed for toxin detection applications. This paper describes an innovative method to activate bacteriorhodopsin-based electrodes with the optical output of quantum dots, producing an enhanced electrical response from the protein. Results show that the photonic emission of CdSe/ZnS quantum dots is absorbed by the bacteriorhodopsin retinal and initiates the proton pumping sequence, resulting in an electrical output from a bacteriorhodopsin-based electrode. It is also shown that activated quantum dots in sub-10 nm proximity to bacteriorhodopsin further amplify the photovoltaic response of the protein by approximately 23%, compared to without attached quantum dots, suggesting direct energy transfer mechanisms beyond photonic emission alone. The ability of quantum dots to activate nanoscale regions on bacteriorhodopsin-based electrodes could allow sub-micron sensing arrays to be created due to the ability to activate site-specific regions on the array.

Suppressive subtractive hybridization has been utilized to generate a cDNA library of genes differentially expressed in naphthalene grown cells of Pseudomonas fluorescens . The library was devoid of genes known to be associated with naphthalene catabolism, but was enriched in genes related to cellular uptake and efflux systems. The gene for OmpW, which was present in the cDNA library and has been proposed to encode a porin for the transport of hydrophobic molecules, was isolated, cloned, and sequenced. This gene was shown to be exclusively localized on a large catabolic plasmid possessed by the organism, and its specific mutation resulted in the loss of the organism’s ability to grow on naphthalene and several other polycyclic aromatic hydrocarbons. It is proposed that a primary response by P. fluorescens to the presence of naphthalene is the elevation of the cellular mechanism(s) required for its assimilation. The presence of genes related to the uptake and efflux mechanisms present following suppressive subtractive hybridization supports this proposal.

The utility of employing biogenerated ferric iron as an oxidant for the recycling of scrap metal has been demonstrated using continuously growing cells of the extremophilic organism Acidithiobacillus ferrooxidans. A ferric iron rich (70 mol%) lixiviant resulting from bioreactor based growth of A. ferrooxidans readily solubilized target scrap metal with the resultant generation of a leachate containing elevated ferrous iron levels and solubilized copper previously resident in the scrap metal. Recovery of the copper value was easily accomplished via a cementation reaction and the clarified leachate containing a replenished level of ferrous iron as growth substrate was shown to support the growth of A. ferrooxidans and be fully recyclable. The described process for scrap metal recycling and copper recovery was shown to be efficient and economically attractive. Additionally, the utility of employing the E(h) of the growth medium as a means for monitoring fluctuations in cell density in cultures of A. ferrooxidans is demonstrated.

Although lava-rock-based biofilters have demonstrated their efficiencies for hydrogen sulfide (H2S) removal found in odorous air emissions, the biogeochemical basis for this removal is unclear. In this study, samples of lava rock and rinse water from biofilters at Cedar Rapids Water Pollution Control Facilities (Iowa) were used to study the structure and chemical composition of lava rock and to identify the predominant microorganism(s) present in lava-rock-based biofilters. It was found that iron, in the form of Fe2+ and Fe3+, was present in lava rock. Although literature suggests that Acidithiobacillus thiooxidans are primarily responsible for gaseous H2S removal in biofilters, our study showed that Acidithiobacillus ferrooxidans was the dominant microorganism in the lava-rock-based biofilters. A novel mechanism for H2S removal in a lava-rock-based biofilter is proposed based on the biogeochemical analysis of lava rock.

A dynamic model that describes the biofiltration process for hydrogen sulfide removal from wastewater treatment plant air emissions was calibrated and validated using pilot- and full-scale biofilter data obtained from the Cedar Rapids (Iowa) Water Pollution Control Facilities. After calibration, the model was found to predict the dynamic effluent concentrations of the pilot- and full-scale biofilters well, with the measured data falling within 58 to 80% of the model output values. In addition, the model predicted the trend of the field data, even under field conditions of changing input concentration and at effluent concentrations below 1 ppm by volume. The model demonstrated that increasing gas residence time and temperature and decreasing influent concentration decreases effluent concentration. In addition, model simulations showed that a longer residence time is required to treat dynamic loading increases, indicating that biofilter design should account for the maximum influent concentration. These results can be used to help design and operate biofilters for controlling odorous and hazardous air emissions.

Contaminated sites often contain complex mixtures of aromatic compounds. This complex mixture and the system's redox condition may influence biodegradation patterns. In this study, the biodegradation of PAHs in varying mixture combinations by a pure culture of Pseudomonas putida strain KBM-1 under aerobic conditions showed that the presence of naphthalene (2-ringed PAH) stimulated phenanthrene (3-ringed PAH) degradation five-fold and pyrene (4-ringed PAH) degradation two-fold. However, the presence of phenanthrene inhibited pyrene degradation. Similar degradation patterns were observed under anaerobic, nitrate-reducing conditions; though the degradation rates were typically slower. For example, phenanthrene and pyrene degradation required 2–3 longer times to approach non-detectable levels under nitrate-reducing conditions. In contrast, the time to attain non-detectable levels using a different strain, Pseudomonas stutzeri SAG-R, was similar under both conditions. The results show the importance of improving our understanding of how to extrapolate single substrate biodegradation data obtained under aerobic conditions to multi-substrate situations in aerobic and anaerobic environments.

TThe uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing [1-14C]naphthalene. The uptake displayed an affinity constant (Kt) of 11 μM and a maximal velocity (Vmax) of 17 nmol·h-1·mg-1 cellular dry weight. Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth. Uptake was significantly inhibited (~90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene. The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol. The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 μM. It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.Key words: naphthalene, PAH, biodegradation, transport.

PAHs are thought to be particularly persistent in environments where anaerobic conditions exist. This study presents evidence for the biodegradation of three- and four-ringed PAHs (anthracene, phenanthrene, and pyrene) under strict anaerobic, denitrifying conditions. Three pseudomonad strains, isolated from contrasting environments, were used in this study. All three strains were known PAH degraders and denitrifiers. Degradation proceeded to nondetectable levels (

Biodegradation is a chemical transformation process that may result in the decontamination of sediments. A criterion for the potential success of biode gradation is the ability of indigenous microorganisms to catabolize contaminants, such as polycyclic aromatic hydrocarbons (PAHs). The number of microorganisms displaying this ability may be influenced by the extent of their exposure to PAHs. In this study, microorganisms from Keweenaw Bay (Lake Superior) and Trenton Channel (Detroit River) sediments were isolated and enumerated for their abilities to oxidize PAHs. The results revealed that total viable cell counts and PAH-degrading viable cell counts were similar in samples from both sites. The number of distinct biotypes differed, however. The total biotype count was higher for Keweenaw Bay than Trenton Channel; 68 versus 57, respectively. But, Trenton Channel sediment exhibited a greater diversity of PAH-degrading biotypes; 18 compared to only 3 for Keweenaw Bay sediment with the latter being confined to the upper 0 and 15 cm depths of the sediment core. Of the biotypes tested, 12 of 40 from Keweenaw Bay and 14 of 49 from Trenton Channel possessed the ability to denitrify. However, only 1 of 3 from Keweenaw Bay while 4 of 18 PAH-degrading biotypes from Trenton Channel could denitrify. Statistical analysis indicated that the number of PAH-degrading biotypes, the majority of which were identified as Gram negative rods, was site dependent. This suggested that contaminant exposure, an apparent major difference between the two sites, influences the relative percentage of PAH-degrading biotypes in sediments.

Le captage du naphtalene a ete etudie chez des cellules actives de Pseudomonas fluorescens en utilisant du [1- 14 C] naphtalene. Ce captage avait une constante d'affinite (K t ) de Il μM et une vitesse maximale (V max ) de 17 nmol.h -1 .mg -1 de poids sec bacterien. Le captage du naphtalene n'a pas ete observe chez la souche mutante TG-5 qui n'etait pas capable de pousser en utilisant le naphtalene comme seule source de carbone. L'entree du napthtalene dans la cellule etait significativement inhibee (∼90%) par l'azide ou le 2,4-dinitrophenol a des niveaux inhibiteurs de la croissance et elle etait sensible a la presence d'analogues structuraux du naphtalene. Les niveaux intracellulaires d'ATP ne baissaient pas significativement en presence d'azide ou de 2,4-dinitrophenol. L'-α-napthtol inhibait le captage du napthtalene de facon non-competitive avec une K i de 0,041 μM. Les resultats permettent de conclure que la premiere etape dans l'utilisation du napthtalene est son transport a l'interieur de la cellule par une systeme de transport specifique dependant de l'energie.

Hydrophobic organic chemicals (HOCs) discharged into soil and ground water will partition into gaseous, aqueous, oil, and sorbed phases. Knowledge of how bacteria assimilate HOCs is important to individuals involved in evaluating intrinsic, or engineered, bioremediation. The majority of bacteria isolated from the subsurface are gram-negative. The outer membrane of gram-negative organisms acts as a selective barrier to many solutes, including hydrophobic chemicals. Thus, diffusional transport of a hydrophobic solute through the outer membrane may be the rate-limiting step in biodegradation. Bacteria may also produce biosurfactants that can facilitate cell-oil contact or assist solubilization of oil and sorbed phases.

A model system was developed for evaluating naphthalene biosorption based on the use of a mutant (strain TG-5 Nah–) derived from a naphthalene-degrading Pseudomonas fluorescens isolate. Cells of strain TG-5 had a sorptive capacity for naphthalene (partition coefficient of 380 cm3/g) significantly higher than a soil with a 5.1% organic carbon content (partition coefficient of 41 cm3/g). However, experimental results and a mass balance model demonstrated that, in soil systems of high organic carbon content, the mass of naphthalene associated with biological solids is insignificant. In contrast, in a soil system of nonsorptive Ottawa sand, up to 10% of the initial naphthalene was demonstrated experimentally, and by modelin, to be associated with cells of strain TG-5.

The rate of adsorption and desorption of hydrophobic pollutants with lake sediments, or other particulate matter, may influence the ultimate fate of these chemicals. This study examined the kinetics of pyrene adsorption onto, and desorption from, a Great Lake sediment. The data were evaluated by two separate mathematical models. The batch pore surface diffusion model (BPSDM) predicted initial kinetics much better than a radial diffusion model (RDM). This is because the BPSDM incorporates film diffusion which the RDM neglects. In nonturbulent environments, or systems of low particle concentration, film diffusion has been shown to be the rate limiting step for the initial adsorption rate. Hysteresis was experimentally observed for pyrene desorption; therefore, both models overpredicted the rate of pyrene desorption. These results indicate that 1) hydrophobic chemicals may be less available for biological degradation and 2) particle concentration may influence the initial rates of adsorption and desorption.

The unique energy transfer interaction between the optical Utilizing the direct energy transfer mechanism existing between semiconductor quantum dots and the hydrogen ion protein pump bacteriorhodopsin, a multi-functional bioelectronics platform is demonstrated. Fluorescence resonance energy transfer coupled QD-bR systems have been proven in both aqueous and dried film states, allowing for the vast QD optical absorbance range to directly contribute energy to the bR proton pumping sequence. A nanoscale deposition technique was employed to construct hybrid QD-bR electrodes capable of harnessing the FRET phenomena and enhancing the bR electrical output by nearly 300%. A biosensing prototype system was created where the target molecule disrupts the QD-bR FRET relationship and is signaled by an altered bR electrical output. With an integrated TiO 2 electron generating substrate, the QD-bR hybrid functions as a sensitizer in a thin film bio solar cell design, which will be presented in more detail at the conference and in the full paper.

Harnessing the energy transfer interactions between the optical protein bacteriorhodopsin (bR) and CdSe/ZnS quantum dots (QDs) could provide a novel bio-nano electronics substrate with a variety of applications. In the present study, a polydimethyldiallyammonium chloride based I-SAM technique has been utilized to produce bilayers, trilayers and multilayers of alternating monolayers of bR, PDAC and QD's on a conductive ITO substrate. The construction of multilayer systems was directly monitored by measuring the unique A570 nm absorbance of bR, as well as QD fluorescence emission. Both of these parameters displayed a linear relationship to the number of monolayers present on the ITO substrate. The photovoltaic response of bilayers of bR/PDAC was observed over a range of 3 to 12 bilayers and the ability to efficiently create an electrically active multilayered substrate composed of bR and QDs has been demonstrated for the first time. Evaluation of QD fluorescence emission in the multilayer system strongly suggests that FRET coupling is occurring and, since the I-SAM technique provide a means to control the bR/QD separation distance on the nanometer scale, this technique may prove highly valuable for optimizing the distance dependent energy transfer effects for maximum sensitivity to target molecule binding by a biosensor. Finally, preliminary studies on the production of a sensor protein/bR hybrid gene construct are presented. It is proposed that the energy associated with target molecule binding to a hybrid sensor protein would provide a means to directly modulate the electrical output from a sensor protein/bR biosensor platform.

A novel technique for the patterning of bacteriorhodopsin (bR) films is presented. The photolithography based bacteriorhodopsin patterning technique (PBBPT) utilizes conventional photolithographic techniques to pattern purple membrane (PM) films containing bR. Several key process variables are investigated and characterized. The photoelectric response of PM films patterned using the PBBPT are presented, and the process is shown not to have a negative impact on the response of PM films to light. The possibility of integrating patterned PM films with single electron transistors is explored.

A novel bacteriorhodopsin (bR) patterning technique utilizing photolithographic processes is presented. This process allows the oriented deposition and patterning of dried bR thin films onto flat substrates. Standard lithographic techniques are used to pattern the substrate and electrodeposition is used to deposit and orient the purple membrane film, which contains bR. An acetone sonication bath is used to remove the resist and pattern the membrane. The physical and photoelectric properties of dried PM films are shown to be undamaged by the patterning process, and patterns with 60 mum feature sizes are shown. PM liquid suspension density prior to electrodeposition as well as acetone sonication time are reported on.

The unique energy transfer interaction between the optical protein bacteriorhodopsin (bR) and CdSe/ZnS quantum dots (QDs) provides a potential modulation mechanism for bio- nano electronic application. We have utilized ionic-self assembled monolayer (I-SAM) techniques to create a novel alternating monolayer system of QDs and bR on a conductive ITO substrate. Results demonstrate the ability to efficiently create bR/QD multilayer films along with the ability to control bR/QD spacing on the nanometer scale. I-SAM films of this nature demonstrate a sharp decrease in QD emission when deposited in close proximity to bR, suggesting possible fluorescence resonance energy transfer (FRET) effects in a bR/QD nanoscale system. The ability to modulate the QD photonic output based on proximity to bR in the I-SAM films could provide a direct method to modulate the electrical output for bio-nano sensing applications.

To effectively integrate bacteriorhodopsin (bR) with micro electromechanical systems (MEMS) and nano electromechanical systems (NEMS) devices, it is critical to know the electrical properties of the material and to understand how it will affect the functionality of the device. Inductance, capacitance and resistance (LCR) measurements can be used to determine material characteristics, such as permittivity, film thickness, and capacitance. In this paper, LCR measurements of dried films of oriented and unoriented bR with both light-on and light-off conditions are presented. Tests were performed on dried films of bR to determine if there is a relationship between LCR measurements and orientation, light-on/off, frequency, and time. The results indicated that the LCR measurements depended on the thickness and area of the film, but not on the orientation, as with other biological materials such as muscle. However, there was a transient LCR response for both oriented and unoriented bR which depends on light intensity. The possible effect of integrating this material with a single electron transistor (SET) is also briefly discussed.

Nanoscale sensing arrays utilizing the unique properties of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots are being developed to detect minute concentrations of airborne toxins. This paper describes an innovative method to activate bacteriorhodopsin-based sensors with the optical output of quantum dots, producing a measurable electrical response from the protein. The ability of quantum dots to activate nanoscale regions on bacteriorhodopsin-based electrodes allows sub-micron sensing arrays to be created due to the ability to activate site-specific regions on the array. A novel method to modulate the sensor's electrical output to obtain both "on" and "off" states is also achieved utilizing the fluorescence resonance energy transfer characteristics of a bacteriorhodopsin/quantum dot system. Apart from applying this technology to toxin detection arrays, the ability to readily manipulate the protein's electrical and optical characteristics could have implications in other areas of nanobiotronics.

A rapid and sensitive spectrophotometric procedure was developed for monitoring the growth of Thiobacillus ferrooxidans in liquid culture. Values determined for the optical densities at 500 nm of washed T. ferrooxidans cell suspensions were directly proportional to both total cell number and total cell protein concentration and provided an accurate measurement of culture growth rate. The utility of this procedure was demonstrated by conducting physiological studies on the influence of CO 2 and FeSO 4 availability on the growth of T. ferrooxidans . In addition, we describe a procedure for the long-term maintenance of cells T. ferrooxidans that ensures culture purity and genetic stability.

An experimental procedure for rapidly obtaining cell populations of phototrophically growing Rhodopseudomonas sphaeroides which display division and genomic synchrony has been developed. The basis of the procedure resides with the normal physiological response displayed by cells of R. sphaeroides that have been subjected to an immediate decrease in incident light intensity. After an abrupt high- to low-light transition of an asynchronously dividing cell population, an immediate cessation of increases in culture turbidity, total cell number, and net accumulations of culture deoxyribonucleic acid and phospholipid occurs. Total cell number remains constant for 2.5 h after the transition to low light, after which time, it undergoes a sharp increase. Reinitiation of high-light conditions of growth 1 h subsequent to this increase in total cell number results in a cell population possessing a high degree of division and genomic synchrony. A characterization of this procedure, together with a demonstration of its utility for studies on intracytoplasmic membrane assembly, is presented.

Acyl carrier protein (ACP) has been purified from the facultative phototrophic bacterium Rhodobacter sphaeroides. The ACP preparation was greater than 95% homogeneous as determined by native and disodium dodecyl sulfate (Na2DodSO4)-polyacrylamide gel electrophoreses and N-terminal amino acid analysis. Amino acid compositional analysis revealed that the protein contains approximately 75 amino acids, has a calculated minimum molecular weight of 8700, and lacks the amino acids tyrosine and tryptophan. The presence of the characteristic 4'-phosphopantetheine prosthetic group was indicated by the occurrence of equimolar quantities of beta-alanine and taurine in amino acid hydrolysates and was confirmed by independent chemical analysis. The protein displayed a pI of 3.8 and had a calculated partial specific volume of 0.732 mL/g. The primary structure of the protein has been determined for the first 46 amino acid residues from the N terminus of the molecule, and the region of the molecule encompassing the amino acids from residues 31 to 44 was found to have 100% homology with the identical residues in Escherichia coli ACP. In contrast to E. coli ACP, R. sphaeroides ACP migrated according to its molecular weight during Na2DodSO4 gel electrophoresis, was resistant to pH-induced denaturation, and comigrated with the cis-vaccenoyl-ACP derivative during native gel electrophoresis. It is proposed that the basis for these properties is the enhanced hydrophobic character of the protein.

A high molecular weight acyl coenzyme A (acyl-CoA) thioesterase, designated thioesterase II, has been purified 5300-fold from photoheterotrophically grown cells of Rhodopseudomonas sphaeroides. In contrast to R. sphaeroides acyl-CoA thioesterase I [Boyce, S.G., & Lueking, D.R. (1984) Biochemistry 23, 141-147], thioesterase II has a native molecular mass (Mr) of 120,000, is capable of hydrolyzing saturated and unsaturated acyl-CoA substrates with acyl chain lengths ranging from C4 to C18, and is completely insensitive to the serine esterase inhibitor diisopropyl fluorophosphate. Palmitoyl-CoA and stearoyl-CoA are the preferred (lowest Km) saturated acyl-CoA substrates and vaccenoyl-CoA is the preferred unsaturated substrate. However, comparable Vmax values were obtained with a variety of acyl-CoA substrates. Unlike a similar thioesterase present in cells of Escherichia coli [Bonner, W.M., & Bloch, K. (1972) J. Biol. Chem. 247, 3123-3133], R. sphaeroides thioesterase II displays a high ratio of decanoyl-CoA to palmitoyl-CoA activities and exhibits little ability to hydrolyze 3-hydroxyacyl-CoA substrates. Only 3-hydroxydodecanoyl-CoA supported a measurable rate of enzyme activity. With the purification of thioesterase II, the enzymes responsible for greater than 90% of the acyl-CoA thioesterase activity present in cell-free extracts of R. sphaeroides have now been identified.

The membrane localization and properties of the Rhodopseudomonas sphaeroides sn-glycerol-3-phosphate acyltransferase have been examined utilizing enzymatically prepared acyl-acyl carrier protein (acyl-ACP) substrates as acyl donors for sn-glycerol-3-phosphate acylation. Studies conducted with membranes prepared from chemotrophically and phototrophically grown cells show that sn-glycerol-3-phosphate acyltransferase activity is predominantly (greater than 80%) associated with the cell's cytoplasmic membrane. Enzyme activity associated with the intracytoplasmic membranes present in phototrophically grown R. sphaeroides was within the range attributable to cytoplasmic membrane contamination of this membrane fraction. Enzyme activity was optimal at 40 degrees C and pH 7.0 to 7.5, and required the presence of magnesium. No enzyme activity was observed with any of the long-chain acyl-CoA substrates examined. Vaccenoyl-ACP was the preferred acyl-ACP substrate and vaccenoyl-ACP and palmitoyl-ACP were independently utilized to produce lysophosphatidic and phosphatidic acids. With either vaccenoyl-ACP or palmitoyl-ACP as sole acyl donor substrate, the lysophosphatidic acid formed was primarily 1-acylglycerol-3-phosphate and the Km(app) for sn-glycerol-3-phosphate utilization was 96 microM. The implications of these results to the mode and regulation of phospholipid synthesis in R. sphaeroides are discussed.