Cryogels Based on Crosslinked Cells as Green Biocatalysts for Biotechnology

Abstract

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 β-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 β-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.

Abstract (Swedish)

Popular Abstract in English

Popular science

Microbial cells are small and difficult to handle in industrial processes, especially if they are to be reused subsequent batches. In order to make them easier to handle, these cells can be attached to a solid support, by so-called immobilization. This provides particles of sizes that are easy to filter and reuse. However, immobilization is associated with two main problems. The first is aggregation of cells with help of polymers, which leads to large clusters, reducing the rate of diffusion. That means that the substrate will not be very accessible to the cells, and the products formed will be enriched around the cells before leaking out into the reaction medium. The second problem is negative effects on the metabolic machinery of the cells, which may lead to cell death during the immobilization procedure.

Several mild processes can be used for immobilization, e.g. adsorption of the cells onto a solid surface. However, some cells will leak into the reaction medium, creating the need for an additional separation step. Furthermore, in order to obtain an immobilized preparation with viable cells that have good access to the substrate, and where the products formed are efficiently removed from the neighbourhood of the cells, good mass transfer must be ensured under conditions that are not hazardous to the cells.

Cryostructuration offers one such possibility. When an aqueous suspension of small particles is frozen, ice crystals made of pure water are formed and the suspended material collects between the ice crystals. It is possible to cross-link the suspended particles while the ice crystals are still present, and when the ice is melted, a network of interconnected pores with thin filaments of crosslinked cells will be formed. This material has interesting properties for biocatalysis.

The work described in this thesis is focused on the preparation of such cryostructured gels with retained metabolic activity, and the evaluation of some of the materials in lab-scale processes. If and when this technology is adapted for large-scale processing, it would provide an important contribution towards a greener society, by facilitating the sustainable conversion of biomass into value-added products.

• author: Zaushitsyna, Oksana
• supervisor: Rajni Hatti-Kaul
• opponent: Dr D'Souza, Stanislaus Francis, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India
• organization: Biotechnology
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Industrial Biotechnology
• keywords: Cells, Crosslinkers, PVA/PEI, Immobilization, Biocatalysts, Biobased chemicals, Biotransformation, stress tolerance
• categories: Higher Education
• defense location: Lecture Hall A at Center of Chemistry and Chemical Engineering, Lund University, Faculty of Engineering LTH, Getingevägen 60, Lund
• defense date: 2015-03-31 10:15:00
• ISBN: 978-91-7422-393-4
• language: English
• LU publication?: yes
• id: fdd42412-afc5-4762-afd1-9944b39a4bfb (old id 5149200)
• date added to LUP: 2016-04-04 13:37:04
• date last changed: 2018-11-21 21:15:09