A model system for increasing the intensity of whole-cell biocatalysis: Investigation of the rate of oxidation ofD-sorbitol toL-sorbose by thin bi-layer latex coatings of non-growingGluconobacter oxydans

Abstract

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.

Ink-Jet Printing of Gluconobacter oxydans: Micropatterned Coatings As High Surface-to-Volume Ratio Bio-Reactive Coatings

“…The ink formulation for immobilization of G. oxydans was prepared according to the following ratio [29] and glycerol were added directly to the wet cell paste according to the abovementioned ratio and mixed gently with a sterile spatula for about 5 min. The latex binder emulsion was then added and mixed gently for an additional 5 min.…”

“…Thus, while for the preparation of coatings by the Mayer rod drawdown coating method [29] the viscosity of the emulsion is appropriate, for ink-jet printing with the GeSIM plotter, it was too high. In order to reduce ink viscosity, latex inks were prepared by diluting the original G. oxydans latex emulsion with deionized water, in agreement with Flickinger et al [6][7][8], who diluted several E. coli latex emulsions with water in order to obtain ink viscosity in the range of 1.5 to 3 mPa· s.…”

“…When a G. oxydans ink-jet printed dot is placed in contact with an oxygen-saturated solution containing D-sorbitol, the sugar diffuses through the nanoporous microstructure and is converted quantitatively by the membrane-bound sorbitol dehydrogenase located on the periplasmic side of the cytoplasmic membrane of the G. oxydans cells [29] into D-sorbitol without cell growth, according to the following reaction: The oxidation of D-sorbitol to L-sorbose by washed, 30 × 30 dot arrays of G. oxydans one-to-two diluted latex ink ink-jet printed on polyester was investigated in non-growth SP buffer for 83.5 h. After 64 h of incubation, the patches were removed from the wells of the plate in order to quantify the contribution to the bioconversion of any G. oxydans cells leaked from the dots into the liquid medium. Table 1 reports the catalytic properties of the dot array.…”

“…Previous coating studies using the same latex formulation have shown that without a porous polymer top coat, a small amount of G. oxydans cells can be released from the surface of the ink dots. It is not likely that cells are released from the inner part of the structure, where the nanoporous interstices between the cells and the partially coalesced polymer particles have dimensions less than G. oxydans cells [29].…”

“…This highly reactive cell coat was sealed by a second thin nanoporous adhesive polymer layer topcoat (~13-17 µm thick), and following rehydration, the coating selectively and quantitatively oxidized D-sorbitol to L-sorbose [29]. The kinetics of D-sorbitol oxidation with suspended cells and the catalytic parameters of the matrix (effective diffusion coefficients and active cell concentration) were estimated from independent experiments and used with a diffusion-reaction model to predict the effectiveness factor and the effective reaction rate as a function of cell coat thickness, topcoat thickness and dissolved oxygen concentration in the liquid medium.…”