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.

Effect of anaerobic digester inoculum preservation via lyophilization on methane recovery.

A robust anaerobic digestion (AD) inoculum is key to a successful digestion process by providing the abundant bacteria needed for converting substrate to useable methane (CH). While transporting digester contents from one AD to another for digester startup has been the norm, transportation costs are high, and it is not feasible to transport wet inoculum to remote locations. In this study, the impact of preservation of AD inoculum via lyophilization was investigated for the purposes of digester startup and restabilization.

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.

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.

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.

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.

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.

Facile fabrication of scalable, hierarchically structured polymer/carbon architectures for bioelectrodes.

This research introduces a method for fabrication of conductive electrode materials with hierarchical structure from porous polymer/carbon composite materials. We describe the fabrication of (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds doped with carbon materials that provide a conductive three-dimensional architecture that was demonstrated for application in microbial fuel cell (MFC) anodes. Composite electrodes from PHBV were fabricated to defined dimensions by solvent casting and particulate leaching of a size-specific porogen (in this case, sucrose).

Biodegradation of medium chain hydrocarbons by Acinetobacter venetianus 2AW immobilized to hair-based adsorbent mats.

The natural attenuation of hydrocarbons can be hindered by their rapid dispersion in the environment and limited contact with bacteria capable of oxidizing hydrocarbons. A functionalized composite material is described herein, that combines in situ immobilized alkane-degrading bacteria with an adsorbent material that collects hydrocarbon substrates, and facilitates biodegradation by the immobilized bacterial population. Acinetobacter venetianus 2AW was isolated for its ability to utilize hydrophobic n-alkanes (C10-C18) as the sole carbon and energy source.

Enzymatic fuel cells: integrating flow-through anode and air-breathing cathode into a membrane-less biofuel cell design.

One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD(+)-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication.

Bioelectrocatalytic generation of directly readable code: harnessing cathodic current for long-term information relay.

Here we present an exceptionally stable bioelectrocatalytic architecture for electrocatalytic oxygen reduction using a carbon nanotube electrode as the electron donor and a fungal enzyme as electrocatalyst. Controlling oxygen content in the electrolyte enables generation of a directly readable barcode from monitoring the enzyme response.

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 significantly. A silica sol-gel process based upon biological mechanisms of inorganic material formation (biomineralization) supports protein immobilization reactions within minutes.

Standardized microbial fuel cell anodes of silica-immobilized Shewanella oneidensis.

Populations of metabolically active bacteria were associated at an electrode surface via vapor-deposition of silica to facilitate in situ characterization of bacterial physiology and bio-electrocatalytic activity in microbial fuel cells.

High electrocatalytic activity of tethered multicopper oxidase-carbon nanotube conjugates.

Multicopper oxidases linked to multiwall carbon nanotubes via the molecular tethering reagent, 1-pyrenebutanoic acid, succinimidyl ester, displayed high bioelectrocatalytic activity for oxygen reduction.

Hybrid antimicrobial enzyme and silver nanoparticle coatings for medical instruments.

We report a method for the synthesis of antimicrobial coatings on medical instruments that combines the bacteriolytic activity of lysozyme and the biocidal properties of silver nanoparticles. Colloidal suspensions of lysozyme and silver nanoparticles were electrophoretically deposited onto the surface of stainless steel surgical blades and needles. Electrodeposited films firmly adhered to stainless steel surfaces even after extensive washing and retained the hydrolytic properties of lysozyme.

Lysozyme-mediated formation of protein-silica nano-composites for biosensing applications.

We demonstrate a rapid method for enzyme immobilization directly on a waveguide surface by encapsulation in a silica matrix. Organophosphate hydrolase (OPH), an enzyme that catalytically hydrolyzes organophosphates, was used as a model enzyme to demonstrate the utility of lysozyme-mediated silica formation for enzyme stabilization. Silica morphology and the efficiency of OPH encapsulation were directly influenced by the precursor choice used in silica formation.

Bioinspired enzyme encapsulation for biocatalysis.

Biocatalysis exploits the versatility of enzymes to catalyse a variety of processes for the production of novel compounds and natural products. Enzyme immobilization enhances the stability and hence applicability of biomolecules as reusable and robust biocatalysts. Biomimetic mineralization reactions have emerged as a versatile tool for generating excellent supports for enzyme stabilization.

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.

Three-dimensional immobilization of beta-galactosidase on a silicon surface.

Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica-based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support.

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.

Silica-immobilized enzymes for multi-step synthesis in microfluidic devices.

The combinatorial synthesis of 2-aminophenoxazin-3-one (APO) in a microfluidic device is reported. Individual microfluidic chips containing metallic zinc, silica-immobilized hydroxylaminobenzene mutase and silica-immobilized soybean peroxidase are connected in series to create a chemo-enzymatic system for synthesis. Zinc catalyzes the initial reduction of nitrobenzene to hydroxylaminobenzene which undergoes a biocatalytic conversion to 2-aminophenol, followed by enzymatic polymerization to APO.

Coimmobilization of a redox enzyme and a cofactor regeneration system.

The coimmobilization of nitrobenzene nitroreductase and glucose-6-phosphate dehydrogenase in silica particles enables the continuous conversion of nitrobenzene to hydroxylaminobenzene with NADPH recycling.

Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase.

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone.