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.

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 (CH4). 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. The effect of lyophilizing inoculum on CH4 production using food waste as the substrate was tested using biochemical methane potential (BMP) tests under the following conditions: (1) three inoculum sources, (2) two inoculum to substrate ratios (ISR), (3) two cryoprotectants, and (4) two inoculum growth phases. After lyophilization with skim milk, the three inocula produced 144-146 mL CH4/g volatile solids (VS) and 194-225 mL CH4/g VS at a 2:1 and 4:1 ISR, respectively, with 33-57% more CH4 at the 4:1 ISR. Preservation with 10% skim milk exhibited complete recovery of CH4 production, while 10% glycerol and 10% glycerol/skim milk mixture yielded 76% and 4% CH4 recovery, respectively. Inoculum growth phase before preservation (mid-exponential or stationary growth phase) did not significantly affect CH4 recovery. The study indicates that inoculum can be preserved via lyophilization using 10% skim milk as a cryoprotectant and reactivated for food waste digestion. The results provide a systematic quantification of the conditions needed to successfully preserve a mixed AD inoculum.

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. The methodology utilizes biological templates and synthetic analogues to catalyse the formation of inorganic oxides. Such materials provide biocompatible environments for enzyme immobilization. The utility of the method is further enhanced by entraining and attaching encapsulated catalysts to a variety of supports. This review discusses biomimetic and bioinspired mineral formation as a technique for the immobilization of enzymes with potential application to a wealth of biocatalytic processes.

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.

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. beta-galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three-dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5-fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 x 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized beta-galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24 degrees C. The ability to generate three-dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity.

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 immobilization in a biomimetic silica support.

Robust immobilization techniques that preserve the activity of biomolecules have many potential applications. Silicates, primarily in the form of sol-gel composites or functionalized mesoporous silica, have been used to encapsulate a wide variety of biomolecules but the harsh conditions required for chemical synthesis limit their applicability. Silaffin polypeptides from diatoms catalyze the formation of silica in vitro at neutral pH and ambient temperature and pressure. Here we show that butyrylcholinesterase entrapped during the precipitation of silica nanospheres retained all of its activity. Ninety percent of the soluble enzyme was immobilized, and the immobilized enzyme was substantially more stable than the free enzyme. The mechanical properties of silica nanospheres facilitated application in a flow-through reactor. The use of biosilica for enzyme immobilization combines the excellent support properties of a silica matrix with a benign immobilization method that retains enzyme activity.

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.

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.

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.

Continuous synthesis of aminophenols from nitroaromatic compounds by combination of metal and biocatalyst.

The combined action of immobilized hydroxylaminobenzene mutase and zinc in a flow-through system catalyzes the conversion of nitroaromatic compounds to the corresponding ortho-aminophenols, including a novel analog of chloramphenicol.

Biosynthesis of hydroxydiphenylacetylene by regiospecific monooxygenation.

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

Entrapment of enzymes and nanoparticles using biomimetically synthesized silica.

Entrapment of enzymes and nanoparticles using biosilicification reactions.

Enzyme-catalysed oxygenation and deoxygenation routes to chiral thiosulfinates.

Enantioenriched thiosulfinates have been obtained by dioxygenase- and chloroperoxidase-catalysed oxidation of 1,2-disulfides and dimethyl sulfoxide reductase-catalysed deoxygenation.

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.

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). The cellular biocompatibility of the resulting composite material facilitated effective immobilization of a defined preparation of Shewanella oneidensis DSP-10 as a model microbial catalyst. Bacterial cells were immobilized via chemical vapor deposition (CVD) of silica to create an engineered biofilm that exhibits efficient bioelectrocatalysis of a simple-carbon fuel in a MFC. The functionalized PHBV electrodes demonstrate stable and reproducible anodic open circuit potentials of -320 ± 20 mV (vs Ag/AgCl) with lactate as the electron donor. Maximum power densities achieved by the hierarchically structured electrodes (~5 mW cm(3)) were significantly higher than previously observed for graphite-felt electrodes. The methodology for fabrication of scalable electrode materials may be amenable to other bioelectrochemical applications, such as enzyme fuel cells and biosensors, and could easily be adapted to various design concepts.