Immobilization of cells and enzymes to LentiKats®.

Biocatalyst immobilization is one of the techniques, which can improve whole cells or enzyme applications. This method, based on the fixation of the biocatalyst into or onto various materials, may increase robustness of the biocatalyst, allows its reuse, or improves the product yield. In recent decades, a number of immobilization techniques have been developed. They can be divided according to the used natural or synthetic material and principle of biocatalyst fixation in the particle. One option, based on the entrapment of cells or enzymes into a synthetic polyvinyl alcohol lens with original shape, is LentiKats® immobilization. This review describes the preparation principle of these particles and summarizes existing successful LentiKats® immobilizations. In addition, examples are compared with other immobilization techniques or free biocatalysts, pointing to the advantages and disadvantages of LentiKats®.

Immobilised whole-cell recombinant monoamine oxidase biocatalysis.

This work demonstrates the first example of the immobilisation of MAO-N whole cells to produce a biocatalyst that remained suitable for repetitive use after 11 months of storage and stable up to 15 months after immobilisation. The production of Escherichia coli expressing recombinant MAO-N was scaled up to bioreactors under regulated, previously optimised conditions (10% DO, pH 7), and the amount of biomass was almost doubled compared to flask cultivation. Subsequently, pilot immobilisation of the whole-cell biocatalyst using LentiKats technology was performed. The amount of the immobilised biomass was optimised and the process was scaled up to a production level by immobilising 15 g of dry cell weight per litre of polyvinyl alcohol to produce 3 kg of whole-cell ready-to-use biocatalyst. The immobilised biocatalyst retained its initial activity over six consecutive biotransformations of the secondary amine model compound 3-azabicylo [3,3,0]octane, a building block of the hepatitis C drug telaprevir. Consecutive cultivation cycles in growth conditions not only increased the initial specific activity of biocatalyst produced on the industrial plant by more than 30%, but also significantly increased the rate of the biotransformation compared to the non-propagated biocatalyst.

Butanol production by immobilised Clostridium acetobutylicum in repeated batch, fed-batch, and continuous modes of fermentation.

Clostridium acetobutylicum immobilised in polyvinylalcohol, lens-shaped hydrogel capsules (LentiKats(®)) was studied for production of butanol and other products of acetone-butanol-ethanol fermentation. After optimising the immobilisation protocol for anaerobic bacteria, continuous, repeated batch, and fed-batch fermentations in repeated batch mode were performed. Using glucose as a substrate, butanol productivity of 0.41 g/L/h and solvent productivity of 0.63 g/L/h were observed at a dilution rate of 0.05 h(-1) during continuous fermentation with a concentrated substrate (60 g/L). Through the process of repeated batch fermentation, the duration of fermentation was reduced from 27.8h (free-cell fermentation) to 3.3h (immobilised cells) with a solvent productivity of 0.77 g/L/h (butanol 0.57 g/L/h). The highest butanol and solvent productivities of 1.21 and 1.91 g/L/h were observed during fed-batch fermentation operated in repeated batch mode with yields of butanol (0.15 g/g) and solvents (0.24 g/g), respectively, produced per gram of glucose.

Determination of low concentration of Paracoccus denitrificans encapsulated in polyvinyl alcohol LentiKat's pellets.

The aim of this work was to compare three methods to determinate low concentrations of Paracoccus denitrificans encapsulated in polyvinyl alcohol pellets, which is important for evaluation and optimization of pellet production as well as for monitoring of biomass growth. Pellets with different and well-defined biomass concentrations were used for experiments. The following fast and simple methods were tested: (1) dissolution of polyvinyl alcohol in hot water followed by dry weight estimation, (2) dissolution of polyvinyl alcohol in hot water followed by optical density measurement, (3) and extraction and quantification of proteins. Dry weight estimation proved to be problematic as it was difficult to separate biomass from polymeric carrier. Optical density measurement showed good linearity of dependence of optical density on biomass content, but determined limits of detection and limits of quantification were not within the range necessary for intended application. The only tested method meeting the requirements for sensitivity was determination of protein concentration after protein extraction.

Comparison of denitrification at low temperature using encapsulated Paracoccus denitrificans, Pseudomonas fluorescens and mixed culture.

The aim of this work was to compare denitrification activity of three types of encapsulated biomass containing pure culture of Paracoccus denitrificans or Pseudomonas fluorescens or mixed culture of psychrophilic denitrifiers cultivated at 5 °C from activated sludge. The experiments were held with synthetic wastewater containing 50 mg L(-1) N-NO(3)(-) under the temperature 15, 10, 8 and 5 °C. Specific denitrification rates related to the weight of pellets and to the protein content were calculated and the temperature coefficients describing the dependence of denitrification rate on the temperature were determined. Although the mixed culture showed the highest denitrification rate at the temperatures below 10 °C, using of pellets containing pure culture is recommended as the mixed culture has slow growth rate and its activity at temperatures above 10 °C is very low.