A model system for increasing the intensity of whole-cell biocatalysis: investigation of the rate of oxidation of D-sorbitol to L-sorbose by thin bi-layer latex coatings of non-growing Gluconobacter oxydans

We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations.     

A versatile method for preparation of hydrated microbial–latex biocatalytic coatings for gas absorption and gas evolution

We describe a latex wet coalescence method for gas-phase immobilization of microorganisms on paper which does not require drying for adhesion. This method reduces drying stresses to the microbes. It is applicable for microorganisms that do not tolerate desiccation stress during latex drying even in the presence of carbohydrates. Small surface area, 10-65?μm thick coatings were generated on chromatography paper strips and placed in the head-space of vertical sealed tubes containing liquid to hydrate the paper. These gas-phase microbial coatings hydrated by liquid in the paper pore space demonstrated absorption or evolution of H(2), CO, CO(2) or O(2). The microbial products produced, ethanol and acetate, diffuse into the hydrated paper pores and accumulate in the liquid at the bottom of the tube. The paper provides hydration to the back side of the coating and also separates the biocatalyst from the products. Coating reactivity was demonstrated for Chlamydomonas reinhardtii CC124, which consumed CO(2) and produced 10.2?±?0.2?mmol?O(2)?m(-2)?h(-1), Rhodopseudomonas palustris CGA009, which consumed acetate and produced 0.47?±?0.04?mmol?H(2)?m(-2)?h(-1), Clostridium ljungdahlii OTA1, which consumed 6?mmol CO?m(-2)?h(-1), and Synechococcus sp. PCC7002, which consumed CO(2) and produced 5.00?±?0.25?mmol O(2)?m(-2)?h(-1). Coating thickness and microstructure were related to microbe size as determined by digital micrometry, profilometry, and confocal microscopy. The immobilization of different microorganisms in thin adhesive films in the gas phase demonstrates the utility of this method for evaluating genetically optimized microorganisms for gas absorption and gas evolution.     

Engineering the microstructure and permeability of thin multilayer latex biocatalytic coatings containing E. coli.

The microstructure and permeability of rehydrated 20-100 microm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 microm thick model trilayer biocatalytic coating consisting of two sealant SF091 layers containing a middle layer of viable E. coli HB101 + latex were studied as delaminated films in a diffusion apparatus with KNO(3) as the diffussant. The permeability of the hydrated coatings is due to diffusive transport through the pore space between the partially coalesced SF091 latex particles. Coating microstructure was visualized by fast freeze cryogenic scanning electron microscopy (cryo-SEM). The effective diffusion coefficient of SF091 latex coatings (diffusive permeability/film thickness) was determined as the ratio of the effective diffusivity of KNO(3) to its diffusivity in water (D(eff)/D). Polymer particle coalescence was arrested by two methods to increase coating permeability. The first used glycerol with coating drying at 4 degrees C, near the glass transition temperature (T(g)). The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away. D(eff)/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time. D(eff)/D varied from 3 x 10(-4) for unmodified SF091 coatings to 6.8 x 10(-2) for coatings containing sucrose. D(eff)/D was reduced by the flattening of latex particles against the surface of the solid substrate, as well as by the presence of the colloid stabilizer hydroxyethylcellulose (HEC). When corrected for the flattened particle layer, D(eff)/D of HEC-free coatings was as high as 0.20, which agreed with the value predicted from analysis of cryo-SEM images of the coat surface. D(eff)/D decreased by one-half in approximately 5 days in rehydrated SF091 coatings, indicating that significant wet coalescence occurs after glycerol, sucrose, or trehalose are leached from the films. D(eff)/D of SF091 latex trilayer coatings containing viable E. coli HB101 cells decreased as cell loading was increased from 2.2 x 10(-2) for 64 g dry cell weight per liter of coat volume to 5 x 10(-3) for 151 g DCW/L of coat volume. The reduction in coating permeability with increasing cell loading is predicted by Maxwell's equation for D(eff)/D in periodic composites.     

Mars' surface is not universally biocidal

The Mars surface/near-surface is often considered to be biocidal. Here, diverse lines of evidence are presented indicating that some terrestrial microbes can survive the in-situ conditions albeit in an inactive state. For the purposes of planetary protection, it is important to consider what we mean by a planetary ‘surface’; this term has qualitatively distinct definitions fordifferent scientific disciplines, and can also have different meanings from a humanviewpoint versus that of a microbial cell. Most microbial cells spores or other cells deposited on Mars, even those that initially fall on the outward-facing part of the absolute surface, will fall within pores of the regolith or become covered by its dust. They are, therefore, protected from ultra-violet radiation. Desiccating conditions and low temperatures (−40 to −70°C) can act to preserve rather than kill all microbes, potentially maintaining cellular viability – especially for certain extremophiles – over geological timescales. Whereas salts are ubiquitous on Mars, many terrestrial microbes are highly tolerant to NaCl and other salts, and these substances (including potentially inhibitory chaotropes such as MgCl₂ and perchlorates) cannot access cells in the absence of a liquid milieu. Whereas the Mars regolith is nutrient-deplete and conditions may be acidic in places, oligotrophic conditions per se are not biocidal and many terrestrial microbes can thrive in acidic conditions (some acidophiles can proliferate at or below pH 0). The low temperatures of Mars' surface are not conducive to metabolic activity, but the biophysical sophistication and robust stress biology of many terrestrial microbes, and the protection afforded by Martian conditions, are likely to ensure the long-term viability of some extremophilic microbes if transported to Mars.     

Permeability and reactivity of Thermotoga maritima in latex bimodal blend coatings at 80°C: a model high temperature biocatalytic coating

Thermostable polymers cast as thin, porous coatings or membranes may be useful for concentrating and stabilizing hyperthermophilic microorganisms as biocatalysts. Hydrogel matricies can be unstable above 65°C. Therefore a 55-m thick, two layer (cell coat + polymer top coat) bimodal, adhesive latex coating of partially coalesced polystyrene particles was investigated at 80°C using Thermotoga maritima as a model hyperthermophile. Coating permeability (pore structure) was critical for maintaining T. maritima viability. The permeability of bimodal coatings generated from 0.8 v/v of a suspension of non-film-forming 800 nm polystyrene particles with high glass transition temperature (T_g= 94°C, 26.9% total solids) blended with 0.2 v/v of a suspension of film-forming 158 nm polyacrylate/styrene particles (T_g –5°C, 40.9% total solids) with 0.3 g sucrose/g latex was measured in a KNO₃ diffusion cell. Diffusivity ratio remained above 0.04 (D_eff/D) when incubated at 80°C in artificial seawater (ASW) for 5 days. KNO₃ permeability was corroborated by cryogenic-SEM images of the pore structure. In contrast, the permeability of a mono-dispersed acrylate/vinyl acetate latex Rovace SF091 (T_g~10°C) rapidly decreased and became impermeable after 2 days incubation in ASW at 80°C. Thermotoga maritima were entrapped in these coatings at a cell density of 49 g cell wet weight/liter of coating volume, 25-fold higher than the density in liquid culture. Viable T. maritima were released from single-layer coatings at 80°C but accurate measurement of the percentage of viable entrapped cells by plate counting was not successful. Metabolic activity could be measured in bilayer coatings by utilization of glucose and maltose, which was identical for latex-entrapped and suspended cells. Starch was hydrolyzed for 200 h by latex-entrapped cells due to the slow diffusion of starch through the polymer top coat compared to only 24 h by suspended T. maritima. The observed reactivity and stability of these coatings was surprising since cryo-SEM images suggested that the smaller low T_g polyacrylate/styrene particles preferentially bound to the T. maritima toga-sheath during coat formation. This model system may be useful for concentrating, entrapment and stabilization of metabolically active hyperthermophiles at 80°C.     

Preservation of H2 production activity in nanoporous latex coatings of Rhodopseudomonas palustris CGA009 during dry storage at ambient temperatures

To assess the applicability of latex cell coatings as an ‘off‐the‐shelf’ biocatalyst, the effect of osmoprotectants, temperature, humidity and O₂ on preservation of H₂ production in Rhodopseudomonas palustris coatings was evaluated. Immediately following latex coating coalescence (24 h) and for up to 2 weeks of dry storage, rehydrated coatings containing different osmoprotectants displayed similar rates of H₂ production. Beyond 2 weeks of storage, sorbitol‐treated coatings lost all H₂ production activity, whereas considerable H₂ production was still detected in sucrose‐ and trehalose‐stabilized coatings. The relative humidity level at which the coatings were stored had a significant impact on the recovery and subsequent rates of H₂ production. After 4 weeks storage under air at 60% humidity, coatings produced only trace amounts of H₂ (0–0.1% headspace accumulation), whereas those stored at < 5% humidity retained 27–53% of their H₂ production activity after 8 weeks of storage. When stored in argon at < 5% humidity and room temperature, R. palustris coatings retained full H₂ production activity for 3 months, implicating oxidative damage as a key factor limiting coating storage. Overall, the results demonstrate that biocatalytic latex coatings are an attractive cell immobilization platform for preservation of bioactivity in the dry state.     

Hydrogen production by photoreactive nanoporous latex coatings of nongrowing Rhodopseudomonas palustris CGA009

Nonuniform light distribution is a fundamental limitation to biological hydrogen production by phototrophic bacteria. Numerous light distribution designs and culture conditions have been developed to reduce self-shading and nonuniform reactivity within bioreactors. In this study, highly concentrated (2.0 x 108 CFU/muL formulation) nongrowing Rhodopseudomonas palustris CGA009 were immobilized in thin, nanoporous, latex coatings. The coatings were used to study hydrogen production in an argon atmosphere as a function of coating composition, thickness, and light intensity. These coatings can be generated aerobically or anaerobically and are more reactive than an equivalent number of suspended or settled cells. Rhodopseudomonas palustris latex coatings remained active after hydrated storage for greater than 3 months in the dark and over 1 year when stored at -80 degrees C. The initial hydrogen production rate of the microphotobioreactors containing 6.25 cm2, 58.4 mum thick Rps. palustris latex coatings illuminated by 34.1 PAR mumol photons m-2 s-1 was 6.3 mmol H2 m-2 h-1 and had a final yield of 0.55 mol H2 m-2 in 120 h. A dispersible latex blend has been developed for direct comparison of the specific activity of settled, suspended, and immobilized Rps. palustris.     

Progress toward a biomimetic leaf: 4,000 h of hydrogen production by coating‐stabilized nongrowing photosynthetic Rhodopseudomonas palustris

Intact cells are the most stable form of nature's photosynthetic machinery. Coating‐immobilized microbes have the potential to revolutionize the design of photoabsorbers for conversion of sunlight into fuels. Multi‐layer adhesive polymer coatings could spatially combine photoreactive bacteria and algae (complementary biological irradiance spectra) creating high surface area, thin, flexible structures optimized for light trapping, and production of hydrogen (H₂) from water, lignin, pollutants, or waste organics. We report a model coating system which produced 2.08 ± 0.01 mmol H₂ m^?2 h^?1 for 4,000 h with nongrowing Rhodopseudomonas palustris, a purple nonsulfur photosynthetic bacterium. This adhesive, flexible, nanoporous Rps. palustris latex coating produced 8.24 ± 0.03 mol H₂ m^?2 in an argon atmosphere when supplied with acetate and light. A simple low‐pressure hydrogen production and trapping system was tested using a 100 cm² coating. Rps. palustris CGA009 was combined in a bilayer coating with a carotenoid‐less mutant of Rps. palustris (CrtI^?) deficient in peripheral light harvesting (LH2) function. Cryogenic field emission gun scanning electron microscopy (cryo‐FEG‐SEM) and high‐pressure freezing were used to visualize the microstructure of hydrated coatings. A light interaction and reactivity model was evaluated to predict optimal coating thickness for light absorption using the Kubelka‐Munk theory (KMT) of reflectance and absorptance. A two‐flux model predicted light saturation thickness with good agreement to observed H₂ evolution rate. A combined materials and modeling approach could be used for guiding cellular engineering of light trapping and reactivity to enhance overall photosynthetic efficiency per meter square of sunlight incident on photocatalysts. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Partial incubation during egg laying reduces eggshell microbial loads in a temperate‐breeding passerine

Incubation prior to clutch completion may be adaptive if it maintains egg viability by inhibiting eggshell microbial growth, thus reducing the likelihood that the embryo becomes infected. To test this hypothesis, we examined the effect of partial incubation during egg laying on eggshell microbial loads in eastern bluebirds Sialia sialis breeding at a temperate‐zone site. We sampled eggshell microbes prior to and following four days of exposure to either partial incubation during the laying period or ambient environmental conditions without incubation (experimental eggs). Microbial colony counts declined significantly for eggs left in the nest during the laying period but did not vary significantly for eggs exposed to ambient conditions. Initial microbial loads were more similar to those previously reported from tropical than temperate environments, and microbes from potentially pathogenic groups were detected on 88% of first‐laid eggs on the day of laying. Egg viability was maintained when eggs were held indoors for four days without incubation but declined sharply thereafter. Our results suggest that partial incubation during egg laying may enhance egg viability in eastern bluebirds by reducing eggshell microbial loads; these effects appear stronger than those usually reported from the temperate zone.     

Enhancing bioplastic-substrate interaction via pore induction and directed migration of enzyme location

We demonstrate two novel approaches to enhance interactions of polymer-immobilized biomolecules with their substrates. In the first approach, diisopropylfluorophosphatase (DFPase) containing poly(urethane) (PU) coatings were made microporous by incorporating, then extracting, poly(ethylene glycol)-based diesters as porogens. Incorporation of 2% w/w porogen increased the effective diffusion coefficient of diisopropylfluorophosphate (DFP) through the coatings by 30% and increased the apparent turnover number of immobilized DFPase 3-fold. In the second approach, prior to immobilization, hydrophobic modification of DFPase was achieved through its conjugation with a dimer/trimer mixture of a uretdione based on 1,6-diisocyanatohexane. When the hydrophobically modified DFPase was immobilized in coatings, catalytic activity was 4-fold higher than that of the equivalent, immobilized, native DFPase. This activity enhancement was independent of the presence or absence of pores. Confocal microscopy images of coatings containing fluorescently labeled lysozyme show that the native enzyme is distributed uniformly over the entire thickness of the coatings. Hydrophobically modified and fluorescently labeled lysozyme is accumulated only in the upper 10 microm cross-sectional layer of a 100 microm-thick coating. Interactions of bioplastics with their substrates are tunable either by pore induction in a polymer or by directed migration of the hydrophobically modified biomolecule to the desired location. The latter approach has broad implications, including overcoming mass transfer limitations experienced by immobilized biocatalysts.     

Rapid assay for the assessment of a potential of chemical biocides to microbial destructors of industrial materials

A colorimetric rapid assay for estimating the biocide potential of various chemicals towards metal biocorrosive and petroleum product degrading microbes was developed based on the reducing potential of live microbial cell. A water-soluble organic redox indicator, blue in the oxidized form and pink in the reduced form, was used as an indicator of the reducing potential of microbial cells. Once added to a suspension of vital microbial cells, it was reduced and changed in color. A good correlation between the results of this assay and viability control was obtained by employing surfactants and heavy metal ions.     

Reagentless detection of microorganisms by intrinsic fluorescence

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading-edge technologies described in this and the accompanying paper. In this contribution, a hand-held prototype instrument is described which is capable of statistically sampling the environment for microbial contamination and determining cell viability. The technology is sensitive enough to detect very low levels ( approximately 20 cells/cm(2) or cm(3)) of microbes in seconds.     

Best practices for cryopreserving,thawing, recovering,and assessing cells

Long-term storage of cell stocks insures that cells are available for use whenever needed. Cryopreservation of cells is the method of choice for preservation of important or rare cell stocks. There are several factors to consider when establishing a protocol for freezing, thawing, and recovery of cells after storage. These parameters may include cell concentration, cryoprotectant choice and concentration, and thawing rate among others. Further, the assessment of cell viability and/or function prior to and following cryopreservation is imperative in order to accurately determine downstream utility as well for optimizing the cryopreservation process. This chapter is designed to provide guidance and insight into developing robust and successful protocols for preserving cells that will preserve cell stocks and provide optimal cell yield and viability.     

Effects and applications of sub-lethal ultrasound, electroporation and UV radiations in bioprocessing

Advances in bioprocess technology involving microbial cells have led to increased and improved production of beneficial new products and bioactive compounds. However, the semipermeable barrier of the cell membrane often retards the efficient productivity or reaction rate of the cells. Physical treatments such as ultrasound, electroporation and UV radiation provide an efficient approach to increase membrane permeability, leading to enhanced productivity of microbial cells. It is important to note that extensive membrane permeabilization by these physical treatments could be detrimental to cell viability leading to lower yield. An appropriate selection of sublethal dosage and intensity of these physical treatments are critical to preserve the viability of cells and at the same time maintain their bioprocess applications. Despite the promising applications of these physical treatments, safety issues related to possible genotoxicity or mutation of cells upon treatments have been raised. This genotoxic effect of physical treatments could be prevented if appropriate measures are taken, without compromising their bioprocess potentials. The current review highlights the effect of sublethal physical treatments such as ultrasound, electroporation and UV radiation on the viability of cells, their potential bioprocess applications, and the possibility of mutations.     

Effectiveness of sodium benzoate as a freshwater low toxicity antifoulant when dispersed in solution and entrapped in silicone coatings

The traditional solution for preventing organisms from attaching to submerged surfaces is to apply antifouling coatings or biocides. Based on the varied defence mechanisms exhibited by biofilms, the antifoulant needs to prevent bacterial attachment during the early stages of biofilm formation. The potential of benzoic acid and sodium benzoate (NaB) as antifoulants for deterring freshwater bacterial attachment was evaluated with the antifoulants dispersed in solution or entrapped in silicone coatings. Effectiveness was based on the decrease in microbial attachment, limited toxicity, and minimum alteration of the properties of the coatings. The optimal NaB concentration when dispersed in solution, 700 mg l-1, resulted in a biofilm surface coverage of only 3.34% after four weeks. The model silicone, Sylgard 184, demonstrated a better overall performance than the commercial coating, RTV11. Sylgard 184 containing sodium benzoate had 41-52% less biofilm in comparison to the control Sylgard 184, whereas both the control and NaB-entrapped RTV11 coatings had significant biofilm coverage.     

Testing in vitro viability of a thermophilic probiotic bacterial strain in simulated gastrointestinal conditions

The preservation of the viability of Streptococcus salivarius ssp. thermophilus strain IL 5.1 under gastrointestinal conditions is one of the more important microbial features for its use in probiotic products. The aim of this study was to test the viability of the bacterium under various gastrointestinal stressor conditions. The influence of pepsin at various pH levels and of pancreatin in the presence of bile salts was also determined in order to demonstrate the influence of these substances on the viability of the microorganism. Under identical conditions, the effect of both casein and mucin was also determined. Mucin was found to preserve microbial viability better than casein based on calculated mathematical parameters relating to viability and mortality.     

Biological Characteristics of the MG-63 Human Osteosarcoma Cells on Composite Tantalum Carbide/Amorphous Carbon Films

Tantalum (Ta) is a promising metal for biomedical implants or implant coating for orthopedic and dental applications because of its excellent corrosion resistance, fracture toughness, and biocompatibility. This study synthesizes biocompatible tantalum carbide (TaC) and TaC/amorphous carbon (a-C) coatings with different carbon contents by using a twin-gun magnetron sputtering system to improve their biological properties and explore potential surgical implant or device applications. The carbon content in the deposited coatings was regulated by controlling the magnetron power ratio of the pure graphite and Ta cathodes. The deposited TaC and TaC/a-C coatings exhibited better cell viability of human osteosarcoma cell line MG-63 than the uncoated Ti and Ta-coated samples. Inverted optical and confocal imaging was used to demonstrate the cell adhesion, distribution, and proliferation of each sample at different time points during the whole culture period. The results show that the TaC/a-C coating, which contained two metastable phases (TaC and a-C), was more biocompatible with MG-63 cells compared to the pure Ta coating. This suggests that the TaC/a-C coatings exhibit a better biocompatible performance for MG-63 cells, and they may improve implant osseointegration in clinics.     

Single cell electrophoresis in determining cell death: potential for use in organ transplant research

Viability of donor tissues is essential for the success of organ transplantation. Although much work has been done in the field of organ preservation, currently there are few objective methods for evaluating transplant organ viability, and thus preservation efficiency. In the field of cancer biology, single-cell gel electrophoresis (SCGE) is a technique commonly used to measure the efficacy of anti-tumor treatments by measuring the breakdown of tumor cell deoxyribonucleic acid (DNA). This assay has recently been applied to various organs from a postmortem porcine animal model, and cells were found to undergo postmortem breakdown in a way similar to apoptosis-induced DNA fragmentation. Collections of cells from each organ reached levels indicative of non-viability as the postmortem interval (PMI) progressed. The rates of cellular DNA degradation were found to be specific to each organ type at a given ambient temperature. We believe that following the application of various preservation techniques, SCGE assay has the potential to provide a clear indication of cell viability in an organ destined for transplant. As a readily available viability assay, this technique could provide transplant researchers with a useful tool to quantify the efficacy of their experimental organ preservation techniques.