Donald R. Lueking

Biodegradation of mixtures of polycyclic aromatic hydrocarbons under aerobic and nitrate-reducing conditions

Contaminated sites often contain complex mixtures of aromatic compounds. This complex mixture and the systems 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.

Naphthalene uptake by a Pseudomonas fluorescens isolate

The 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 microM 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 (approximately 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 microM. 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.

Quantum dot enhancement of bacteriorhodopsin-based electrodes

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-10nm 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.

Naphthalene biosorption in soil/water systems of low or high sorptive capacity

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.

Polycyclic Aromatic Hydrocarbon Degrading Microorganisms in Great Lakes Sediments

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.

Adsorption and Desorption Kinetics of Pyrene onto a Great Lakes Sediment

Abstract 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.

Förster Resonance Energy Transfer between Core/Shell Quantum Dots and Bacteriorhodopsin

An energy transfer relationship between core-shell CdSe/ZnS quantum dots (QDs) and the optical protein bacteriorhodopsin (bR) is shown, demonstrating a distance-dependent energy transfer with 88.2% and 51.1% of the QD energy being transferred to the bR monomer at separation distances of 3.5 nm and 8.5 nm, respectively. Fluorescence lifetime measurements isolate nonradiative energy transfer, other than optical absorptive mechanisms, with the effective QD excited state lifetime reducing from 18.0 ns to 13.3 ns with bR integration, demonstrating the Förster resonance energy transfer contributes to 26.1% of the transferred QD energy at the 3.5 nm separation distance. The established direct energy transfer mechanism holds the potential to enhance the bR spectral range and sensitivity of energies that the protein can utilize, increasing its subsequent photocurrent generation, a significant potential expansion of the applicability of bR in solar cell, biosensing, biocomputing, optoelectronic, and imaging technologies.

Multi-functional protein-QD hybrid substrates for photovoltaics and real-time biosensing

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 TiO2 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.

Photolithographic Patterning of Bacteriorhodopsin Films

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.

Integration of Optical Protein and Quantum Dot Films for Biosensing

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.