Cryogels Based on Crosslinked Cells as Green Biocatalysts for Biotechnology

Sammanfattning: Abstract Due to the finite nature of fossil resources and their environmental impact, such as greenhouse gases and climate change, the bio-based production of chemicals, materials and fuels is the subject of extensive research in industrial biotechnology. Biotransformation and fermentation are considered to be mild, “green” processes that make use of effective and reliable biocatalysts to provide bio-based chemicals. Immobilization is one approach used to improve the productivity and process performance of biocatalysts. Thanks to modern genetic techniques, it is now possible to produce mutant strains of microorganisms with superior activity allowing renewable bio-based feedstocks to be converted into valuable chemicals. This thesis describes studies on the development of green catalysts made from whole cells using cryostructuration. Various cells, including Escherichia coli, Clostridium acetobutylicum and Lactobacillus reuteri, were used in the formation of high-cell-density macroporous cryogel monoliths. The main focus was on obtaining highly efficient biocatalysts. For this purpose, aldehyde-containing macromolecular crosslinkers based on dextran, polyvinyl alcohol and polyethyleneimine, which allow retained cell viability after immobilization, were synthesized and evaluated. Cell viability was evaluated by monitoring glucose consumption and cell growth during the incubation of prepared cryogels. High metabolic activity was seen in the crosslinked cells; e.g. 80% of the initial specific ?-glucosidase activity was retained in immobilized E. coli cells. Viability allows protein expression in immobilized cells, providing opportunities to use sensitive and unstable enzymes, which do not usually survive immobiliz¬ation. It was possible to induce in crosslinked cells almost 40% enzymatic activity of ?-glucosidase found in free cells. The potential of cryogels for the production of biobutanol and some interesting bio-based chemicals was established in this work. Cryogels containing growing crosslinked C. acetobutylicum cells were used as a biocatalyst in repeated batch fermentation resulting in high butanol yields up to 15.4 g/l. Glycerol was converted into the chemicals 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propandiol in flow-through bioreactor mode using cryogels made from crosslinked L. reuteri cells, showing product yields of 281, 32 and 27 mg/gCDW'h, respectively. The biocatalysts demonstrated stable high activity for 40 hours in the continuous biotransformation of glycerol into these biochemicals. Improved tolerance of L. reuteri cells to propanol was seen after cryostructuration, indicating promising potential for this method of immobilization in biotechnological applications where cells are exposed to solvents and other toxic compounds.