The impact of culture medium on the development and physiology of biofilms of Pseudomonas fluorescens formed on polyurethane paint.

Microbial biofilms cause the deterioration of polymeric coatings such as polyurethanes (PUs). In many cases, microbes have been shown to use the PU as a nutrient source. The interaction between biofilms and nutritive substrata is complex, since both the medium and the substratum can provide nutrients that affect biofilm formation and biodeterioration. Historically, studies of PU biodeterioration have monitored the planktonic cells in the medium surrounding the material, not the biofilm. This study monitored planktonic and biofilm cell counts, and biofilm morphology, in long-term growth experiments conducted with Pseudomonas fluorescens under different nutrient conditions. Nutrients affected planktonic and biofilm cell numbers differently, and neither was representative of the system as a whole. Microscopic examination of the biofilm revealed the presence of intracellular storage granules in biofilms grown in M9 but not yeast extract salts medium. These granules are indicative of nutrient limitation and/or entry into stationary phase, which may impact the biodegradative capability of the biofilm.

Biotechnology to reduce logistics burden and promote environmental stewardship for Air Force civil engineering requirements.

This review provides discussion of advances in biotechnology with specific application to civil engineering requirements for airfield and airbase operations. The broad objectives are soil stabilization, waste management, and environmental protection. The biotechnology focal areas address (1) treatment of soil and sand by biomineralization and biopolymer addition, (2) reduction of solid organic waste by anaerobic digestion, (3) application of microbes and higher plants for biological processing of contaminated wastewater, and (4) use of indigenous materials for airbase construction and repair. The consideration of these methods in military operating scenarios, including austere environments, involves comparison with conventional techniques. All four focal areas potentially reduce logistics burden, increase environmental sustainability, and may provide energy source, or energy-neutral practices that benefit military operations.

Bacterial sunscreen: layer-by-layer deposition of UV-absorbing polymers on whole-cell biosensors.

UV-protective coatings on live bacterial cells were created from the assembly of cationic and UV-absorbing anionic polyelectrolytes using layer-by-layer (LbL) methodology. A cationic polymer (polyallylamine) and three different anionic polymers with varying absorbance in the UV range (poly(vinyl sulfate), poly(4-styrenesulfonic acid), and humic acid) were used to encapsulate Escherichia coli cells with two different green fluorescent protein (GFP) expression systems: constitutive expression of a UV-excitable GFP (GFPuv) and regulated expression of the intensely fluorescent GFP from amphioxus (GFPa1) through a theophylline-inducible riboswitch. Riboswitches activate protein expression after specific ligand-RNA binding events. Hence, they operate as a cellular biosensor that will activate reporter protein synthesis after exposure to a ligand target. E. coli cells coated with UV-absorbing polymers demonstrated enhanced protection of GFP stability, metabolic activity, and viability after prolonged exposure to radiation from a germicidal lamp. The results show the effectiveness of LbL coatings to provide UV protection to living cells for biotechnological applications.

Design parameters for tuning the type 1 Cu multicopper oxidase redox potential: insight from a combination of first principles and empirical molecular dynamics simulations.

The redox potentials and reorganization energies of the type 1 (T1) Cu site in four multicopper oxidases were calculated by combining first principles density functional theory (QM) and QM/MM molecular dynamics (MD) simulations. The model enzymes selected included the laccase from Trametes versicolor, the laccase-like enzyme isolated from Bacillus subtilis, CueO required for copper homeostasis in Escherichia coli, and the small laccase (SLAC) from Streptomyces coelicolor. The results demonstrated good agreement with experimental data and provided insight into the parameters that influence the T1 redox potential. Effects of the immediate T1 Cu site environment, including the His(N(δ))-Cys(S)-His(N(δ)) and the axial coordinating amino acid, as well as the proximate H(N)(backbone)-S(Cys) hydrogen bond, were discerned. Furthermore, effects of the protein backbone and side-chains, as well as of the aqueous solvent, were studied by QM/MM molecular dynamics (MD) simulations, providing an understanding of influences beyond the T1 Cu coordination sphere. Suggestions were made regarding an increase of the T1 redox potential in SLAC, i.e., of Met198 and Thr232 in addition to the axial amino acid Met298. Finally, the results of this work presented a framework for understanding parameters that influence the Type 1 Cu MCO redox potential, useful for an ever-growing range of laccase-based applications.

Supramolecular assembly of a biomineralizing antimicrobial peptide in coarse-grained Monte Carlo simulations.

Monte Carlo simulations are used to model the self-organizing behavior of the biomineralizing peptide KSL (KKVVFKVKFK) in the presence of phosphate. Originally identified as an antimicrobial peptide, KSL also directs the formation of biosilica through a hypothetical supramolecular template that requires phosphate for assembly. Specificity of each residue and the interactions between the peptide and phosphate are considered in a coarse-grained model. Both local and global physical quantities are calculated as the constituents execute their stochastic motion in the presence and absence of phosphate. Ordered peptide aggregates develop after simulations reach thermodynamic equilibrium, wherein phosphates form bridging ligands with lysines and are found interdigitated between peptide molecules. Results demonstrate that interactions between the lysines and phosphate drive self-organization into lower energy conformations of interconnected peptide scaffolds that resemble the supramolecular structures of polypeptide- and polyamine-mediated silica condensation systems. Furthermore, the specific phosphate-peptide organization appears to mimic the zwitterionic structure of native silaffins (scaffold proteins of diatom shells), suggesting a similar template organization for silica deposition between the in vitro KSL and silaffin systems.

Impedance spectroscopy as a tool for non-intrusive detection of extracellular mediators in microbial fuel cells.

Endogenously produced, diffusible redox mediators can act as electron shuttles for bacterial respiration. Accordingly, the mediators also serve a critical role in microbial fuel cells (MFCs), as they assist extracellular electron transfer from the bacteria to the anode serving as the intermediate electron sink. Electrochemical impedance spectroscopy (EIS) may be a valuable tool for evaluating the role of mediators in an operating MFC. EIS offers distinct advantages over some conventional analytical methods for the investigation of MFC systems because EIS can elucidate the electrochemical properties of various charge transfer processes in the bio-energetic pathway. Preliminary investigations of Shewanella oneidensis DSP10-based MFCs revealved that even low quantities of extracellular mediators significantly influence the impedance behavior of MFCs. EIS results also suggested that for the model MFC studied, electron transfer from the mediator to the anode may be up to 15 times faster than the electron transfer from bacteria to the mediator. When a simple carbonate membrane separated the anode and cathode chambers, the extracellular mediators were also detected at the cathode, indicating diffusion from the anode under open circuit conditions. The findings demonstrated that EIS can be used as a tool to indicate presence of extracellular redox mediators produced by microorganisms and their participation in extracellular electron shuttling.

Encapsulation of bacterial spores in nanoorganized polyelectrolyte shells.

Layer-by-layer assembly uses alternating charged layers of polyionic polymers to coat materials sequentially in a sheath of functionalized nanofilms. Bacterial spores were encapsulated in organized ultrathin shells using layer-by-layer assembly in order to assess the biomaterial as a suitable core and determine the physiological effects of the coating. The shells were constructed on Bacillus subtilis spores using biocompatible polymers polyglutamic acid, polylysine, albumin, lysozyme, gelatin A, protamine sulfate, and chondroitin sulfate. The assembly process was monitored by measuring the electrical surface potential (zeta-potential) of the particles at each stage of assembly. Fluorescent laser confocal microscopy and scanning electron microscopy confirmed the formation of uniform coatings on the spores. The coating surface charge and thickness (20-100 nm) could be selectively tuned by using appropriate polymers and the number of bilayers assembled. The effect of each coating type on germination was assessed and compared to native spores. The coated spores were viable, but the kinetics and extent of germination were changed from control spores in all instances. The results and insight gained from the experiments may be used to design various bioinspired systems. The spores can be made dormant for a desired amount of time using the LbL encapsulation technique and can be made active when appropriate.

Entrapment of enzymes and carbon nanotubes in biologically synthesized silica: glucose oxidase-catalyzed direct electron transfer.

This work demonstrates a new approach for building bioinorganic interfaces by integrating biologically derived silica with single-walled carbon nanotubes to create a conductive matrix for immobilization of enzymes. Such a strategy not only allows simple integration into biodevices but presents an opportunity to intimately interface an enzyme and manifest direct electron transfer features. Biologically synthesized silica/carbon nanotube/enzyme composites are evaluated electrochemically and characterized by means of X-ray photoelectron spectroscopy. Voltammetry of the composites displayed stable oxidation and reduction peaks at an optimal potential close to that of the FAD/FADH(2) cofactor of immobilized glucose oxidase. The immobilized enzyme is stable for a period of one month and retains catalytic activity for the oxidation of glucose. It is demonstrated that the resulting composite can be successfully integrated into functional bioelectrodes for biosensor and biofuel cell applications.

Synthesis of bioinorganic antimicrobial peptide nanoparticles with potential therapeutic properties.

Amphiphilicity and cationicity are properties shared between antimicrobial peptides and proteins that catalyze biomineralization reactions. Merging these two functionalities, we demonstrate a reaction where a cationic antimicrobial peptide catalyzes self-biomineralization within inorganic matrices. The resultant antimicrobial peptide nanoparticles retain biocidal activity, protect the peptide from proteolytic degradation, and facilitate a continuous release of the antibiotic over time. Taken together, these properties demonstrate the therapeutic potential of self-synthesizing biomaterials that retain the biocidal properties of antimicrobial peptides.

Producing methane, methanol and electricity from organic waste of fermentation reaction using novel microbes.

Residual solid and liquid streams from the one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process were treated with two separate biochemical routes for renewable energy transformation. The solid residual stream was subjected to thermophilic anaerobic digestion (TAD), which produced 95 ±â€¯7 L methane kg-1 volatile solid with an overall energy efficiency of 12.9 ±â€¯1.7%. A methanotroph, Methyloferula sp., was deployed for oxidation of mixed TAD biogas into methanol. The residual liquid stream from CRUDE process was used in a Microbial Fuel Cell (MFC) to produce electricity. Material balance calculations confirmed the integration of biochemical routes (i.e. CRUDE, TAD, and MFC) for developing a sustainable approach of energy regeneration. The current work demonstrates the utilization of different residual streams originated after food waste processing to release minimal organic load to the environment.

Biosensor system for continuous monitoring of organophosphate aerosols.

An enzyme-based monitoring system provides the basis for continuous sampling of organophosphate contamination in air. The enzymes butyrylcholinesterase (BuChE) and organophosphate hydrolase (OPH) are stabilized by encapsulation in biomimetic silica nanoparticles, entrained within a packed bed column. The resulting immobilized enzyme reactors (IMERs) were integrated with an impinger-based aerosol sampling system for collection of chemical contaminants in air. The sampling system was operated continuously and organophosphate detection was performed in real-time by single wavelength analysis of enzyme hydrolysis products. The resulting sensor system detects organophosphates based on either enzyme inhibition (of BuChE) or substrate hydrolysis (by OPH). The detection limits of the IMERs for specific organophosphates are presented and discussed. The system proved suitable for detection of a range of organophosphates including paraoxon, demeton-S and malathion.

Enzyme-encapsulated silica monolayers for rapid functionalization of a gold surface.

We report a simple and rapid method for the deposition of amorphous silica onto a gold surface. The method is based on the ability of lysozyme to mediate the formation of silica nanoparticles. A monolayer of lysozyme is deposited via non-specific binding to gold. The lysozyme then mediates the self-assembled formation of a silica monolayer. The silica formation described herein occurs on a surface plasmon resonance (SPR) gold surface and is characterized by SPR spectroscopy. The silica layer significantly increases the surface area compared to the gold substrate and is directly compatible with a detection system. The maximum surface concentration of lysozyme was found to be a monolayer of 2.6 ng/mm(2) which allowed the deposition of a silica layer of a further 2 ng/mm(2). For additional surface functionalization, the silica was also demonstrated to be a suitable matrix for immobilization of biomolecules. The encapsulation of organophosphate hydrolase (OPH) was demonstrated as a model system. The silica forms at ambient conditions in a reaction that allows the encapsulation of enzymes directly during silica formation. OPH was successfully encapsulated within the silica particles and a detection limit for the substrate, paraoxon, using the surface-encapsulated enzyme was found to be 20 microM.

Enzyme Stabilization via Bio-Templated Silicification Reactions.

Effective entrapment of enzymes in solid phase materials is critical to their practical application. The entrapment generally stabilizes biological activity compared to soluble molecules and the material simplifies catalyst integration compared to other methods. A silica sol-gel process based upon biological mechanisms of inorganic material formation (biomineralization) supports protein immobilization reactions within minutes. The material has high protein binding capacity and the catalytic activity of the enzyme is retained. We have demonstrated that both oligopeptides and selected proteins will mediate the biomineralization of silica and allow effective co-encapsulation of other proteins present in the reaction mixture. The detailed methods described here provide a simple and effective approach for molecular biologists, biochemists and bioengineers to create stable, solid phase biocatalysts that may be integrated within sensors, synthetic processes, reactive barriers, energy conversion, and other biotechnology concepts.

Simultaneous hydrolysis and fermentation of unprocessed food waste into ethanol using thermophilic anaerobic bacteria.

The one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process was developed for simultaneous hydrolysis and fermentation of unprocessed food waste into ethanol using thermophilic (growing at 65°C) anaerobic bacteria. Unlike existing waste to energy technologies, the CRUDE process obviates the need for any pre-treatment or enzyme addition. A High-Temperature-High-Pressure (HTHP) distillation technique was also applied that facilitated efficient use of fermentation medium, inoculum recycling, and in-situ ethanol collection. For material balancing of the process, each characterized component was represented in terms of C-mol. Recovery of 94% carbon at the end confirmed the operational efficiency of CRUDE process. The overall energy retaining efficiency calculated from sugars to ethanol was 1262.7kJdryweightkg-1 of volatile solids using HTHP. These results suggest that the CRUDE process can be a starting point for the development of a commercial ethanol production process.

Silica-immobilized enzyme reactors; application to cholinesterase-inhibition studies.

A rapid and economical method is reported for the preparation of an immobilized enzyme reactor (IMER) using silica-encapsulated equine butyrylcholinesterase (BuChE) as a model system. Peptide-mediated silica formation was used to encapsulate BuChE, directly immobilizing the enzyme within a commercial pre-packed column. The silica/enzyme nanocomposites form and attach simultaneously to the metal affinity column via a histidine-tag on the silica-precipitating peptide. BuChE-IMER columns were integrated to a liquid chromatography system and used as a rapid and reproducible screening method for determining the potency of cholinesterase inhibitors. The IMER preparation method reported herein produces an inert silica-encapsulation matrix with advantages over alternative systems, including ease of preparation, high immobilization efficiency (70-100%) and complete retention of activity during continuous use.

Biosynthesis of hydroxydiphenylacetylene by regiospecific monooxygenation.

Bacterial monooxygenase enzymes catalyze a regiospecific single-step hydroxylation of diphenylacetylene to yield meta- and para-hydroxydiphenylacetylene.

Modification of carbon nanotube electrodes with 1-pyrenebutanoic acid, succinimidyl ester for enhanced bioelectrocatalysis.

Conductive materials functionalized with redox enzymes provide bioelectronic architectures with application to biological fuel cells and biosensors. Effective electron transfer between the enzyme (biocatalyst) and the conductive materials is imperative for function. Various nanostructured carbon materials are common electrode choices for these applications as both the materials' inherent conductivity and physical integrity aids optimal performance. The following chapter presents a method for the use of carbon nanotube buckypaper as a conductive architecture suitable for biocatalyst functionalization. In order to securely attach the biocatalyst to the carbon nanotube surface, the conductive buckypaper is modified with the heterobifunctional cross-linker, 1-pyrenebutanoic acid, succinimidyl ester. The technique effectively tethers the enzyme to the carbon nanotube which enhances bioelectrocatalysis, preserves the conductive nature of the carbon surface, and facilities direct electron transfer between the catalyst and material interface. The approach is demonstrated using phenol oxidase (laccase) and pyrroloquinoline quinone-dependent glucose dehydrogenase PQQ-GDH, as representative biocatalysts.

Immobilization of whole cells by chemical vapor deposition of silica.

Effective entrapment of whole bacterial cells onto solid-phase materials can significantly improve bioprocessing and other biotechnology applications. Cell immobilization allows integration of biocatalysts in a manner that maintains long-term cell viability and typically enhances process output. A wide variety of functionalized materials have been explored for microbial cell immobilization, and specific advantages and limitations were identified. The method described here is a simple, versatile, and scalable one-step process for the chemical vapor deposition of silica to encapsulate and stabilize viable, whole bacterial cells. The immobilized bacterial population is prepared and captured at a predefined physiological state so as to affix bacteria with a selected metabolic or catalytic capability to compatible materials and surfaces. Immobilization of Shewanella oneidensis to carbon electrodes and immobilization of Acinetobacter venetianus to adsorbent mats are described as model systems.

Microbial-enzymatic-hybrid biological fuel cell with optimized growth conditions for Shewanella oneidensis DSP-10.

In this work we present a biological fuel cell fabricated by combining a Shewanella oneidensis microbial anode and a laccase-modified air-breathing cathode. This concept is devised as an extension to traditional biochemical methods by incorporating diverse biological catalysts with the aim of powering small devices. In preparing the biological fuel cell anode, novel hierarchical-structured architectures and biofilm configurations were investigated. A method for creating an artificial biofilm based on encapsulating microorganisms in a porous, thin film of silica was compared with S. oneidensis biofilms that were allowed to colonize naturally. Results indicate comparable current and power densities for artificial and natural biofilm formations, based on growth characteristics. As a result, this work describes methods for creating controllable and reproducible bio-anodes and demonstrates the versatility of hybrid biological fuel cells.

Power generation from a hybrid biological fuel cell in seawater.

A hybrid biological fuel cell (HBFC) comprised of a microbial anode for lactate oxidation and an enzymatic cathode for oxygen reduction was constructed and then tested in a marine environment. Shewanella oneidensis DSP-10 was cultivated in laboratory medium and then fixed on a carbon felt electrode via a silica sol-gel process in order to catalyze anodic fuel cell processes. The cathode electrocatalyst was composed of bilirubin oxidase, fixed to a carbon nanotube electrode using a heterobifunctional cross linker, and then stabilized with a silica sol-gel coating. The anode and cathode half-cells provided operating potentials of -0.44 and 0.48 V, respectively (vs. Ag/AgCl). The HBFC maintained a reproducible open circuit voltage >0.7 V for 9 d in laboratory settings and sustained electrocatalytic activity for >24h in open environment tests.