An improved procedure for characterization of spatial and temporal evolution of immobilized cells in gel membranes

Abstract.

An improved procedure that allows the simple and reproducible characterization of spatial and temporal distribution of immobilized biomass in gel membranes was developed. This procedure involves three main steps in the preparation of membrane samples, the use of a standard microtome to obtain membrane slices, and the measurement of cell concentration by spectrophotometry. The key improvement in this procedure is to prepare the membrane samples by clamping them between two glass plates and storing them in a –80°C freezer for a specified period of time depending on the membrane thickness. With this simple pre-treatment, the membrane samples were frozen in an ideal physical state to be cut into flat, consistent, slices using a commercial freezing sledge microtome, thus providing accurate and reproducible results. As a validation case study, a gel membrane bioreactor was constructed in which an alginate gel membrane with immobilized Lactobacillus rhamnosus cells was flanked by two well-mixed chambers with identical fermentation media. The improved procedure was employed to experimentally determine the intra-membrane cell distribution in the alginate membranes during fermentation. The experimental results showed a heterogeneous "U-shape" biomass distribution across the membrane, with the highest cell concentration at the membrane-solution interface. High reproducibility and accuracy were verified by a low average standard deviation (<5%) and a high biomass recovery ratio (>90%), respectively.