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.

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.

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.

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.

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. Growth of strain 2AW also resulted in the production of a biosurfactant that aided in the dispersion of complex mixtures of hydrophobic compounds. Effective immobilization of strain 2AW to the surface of Ottimat™ adsorbent hair mats via vapor phase deposition of silica provided a stable and reproducible biocatalyst population that facilitates in situ biodegradation of n-alkanes. Silica-immobilized strain 2AW demonstrated ca. 85% removal of 1% (v/v) tetradecane and hexadecane within 24 h, under continuous flow conditions. The methodology for immobilizing whole bacterial cells at the surface of an adsorbent, for in situ degradation of hydrocarbons, has practical application in the bioremediation of oil in water emulsions. Published 2011 American Institute of Chemical Engineers Biotechnol Prog., 2011.

Lysozyme catalyzes the formation of antimicrobial silver nanoparticles.

Hen egg white lysozyme acted as the sole reducing agent and catalyzed the formation of silver nanoparticles in the presence of light. Stable silver colloids formed after mixing lysozyme and silver acetate in methanol and the resulting nanoparticles were concentrated and transferred to aqueous solution without any significant changes in physical properties. Activity and antimicrobial assays demonstrated lysozyme-silver nanoparticles retained the hydrolase function of the enzyme and were effective in inhibiting growth of Escherichia coli, Staphylococcus aureus, Bacillus anthracis, and Candida albicans. Remarkably, lysozyme-silver nanoparticles demonstrated a strong antimicrobial effect against silver-resistant Proteus mirabilis strains and a recombinant E. coli strain containing the multiple antibiotic- and silver-resistant plasmid, pMG101. Results of toxicological studies using human epidermal keratinocytes revealed that lysozyme-silver nanoparticles are nontoxic at concentrations sufficient to inhibit microbial growth. Overall, the ability of lysozyme to assemble silver nanoparticles in a one-step reaction offers a simple and environmentally friendly approach to form stable colloids of nontoxic silver nanoparticles that combine the antimicrobial properties of lysozyme and silver. The results expand the functionality of nanomaterials for biological systems and represent a novel antimicrobial composite for potential aseptics and therapeutic use in the future.

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.