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-μm 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 (Tg= 94°C, 26.9% total solids) blended with 0.2 v/v of a suspension of film-forming 158 nm polyacrylate/styrene particles (Tg≈ −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 (Deff/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 (Tg~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 Tg 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.

The microstructure and permeability of rehydrated 20−100 μm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 μm 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 KNO3 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 KNO3 to its diffusivity in water (Deff/D). Polymer particle coalescence was arrested by two methods to increase coating permeability. The first used glycerol with coating drying at 4 °C, near the glass transition temperature (Tg). The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away. Deff/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time. Deff/D varied from 3 × 10−4 for unmodified SF091 coatings to 6.8 × 10−2 for coatings containing sucrose. Deff/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, Deff/D of HEC-free coatings was as high as 0.20, which agreed with the value prediced from analysis of cryo-SEM images of the coat surface. Deff/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. Deff/D of SF091 latex trilayer coatings containing viable E. coliHB101 cells decreased as cell loading was increased from 2.2 × 10−2 for 64 g dry cell weight per liter of coat volume to 5 × 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 Deff/D in periodic composites.

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.