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.

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.

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.

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.

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.

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.

Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output.

The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28 µW/cm(2)) compared to Luria-Bertani (LB) (0.07 µW/cm(2)) or a defined growth minimal medium (MM) (0.02 µW/cm(2)). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

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.

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. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 µW cm(-3) are reported, and characterization criteria that will aid in future optimization are discussed.

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.

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.

The utility of Shewanella japonica for microbial fuel cells.

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.

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. 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 materials, and other biotechnology concepts.

Biophysical properties of membrane-active peptides based on micelle modeling: a case study of cell-penetrating and antimicrobial peptides.

We investigated the molecular mechanisms of short peptides interacting with membrane-mimetic systems. Three short peptides were selected for this study: penetratin as a cell-penetrating peptide (CPP), and temporin A and KSL as antimicrobial peptides (AMP). We investigated the detailed interactions of the peptides with dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles, and the subsequent peptide insertion based on free energy calculations by using all-atomistic molecular dynamics simulations with the united atom force field and explicit solvent models. First, we found that the free energy barrier to insertion for the three peptides is dependent on the chemical composition of the micelles. Because of the favorable electrostatic interactions between the peptides and the headgroups of lipids, the insertion barrier into an SDS micelle is less than a DPC micelle. Second, the peptides' secondary structures may play a key role in their binding and insertion ability, particularly for amphiphilic peptides such as penetratin and KSL. The secondary structures with a stronger ability to bind with and insert into micelles are the ones that account for a smaller surface area of hydrophobic core, thus offering a possible criterion for peptide design with specific functionalities.