Optimization of microbial encapsulation techniques for isolation of keratin degrading microbial species. – Section of Microbiology - University of Copenhagen

05. januar 2017

Optimization of microbial encapsulation techniques for isolation of keratin degrading microbial species.

Project type: Master project

Europe –especially Denmark- has a serious protein deficit as animal production which is reliant on soy protein imported from South America. Whereas, significant protein food chain loss is worsened by waste and inefficient by-product use. Waste management is a key industrial process, as it can significantly contribute to reducing global environmental footprint of an activity, while optimizing the process by valorization of non-noble materials into high quality products with significant added value. Therefore, it is necessary to develop novel technology to convert biological residual resources into new alternative high value products. Microbial cooperation, resulting from billions of years of evolution in multispecies consortia, is central to all natural processes and optimizes natural energy flow and decomposition. Investigation and manipulation of synergistic microbial interactions require development of high throughput techniques.

 Keratin packs into a semi-crystalline state in nature (hair and feather), where keratin monomers are highly cross-linked with disulphide bonds (R-S-S-R). Conversely, biological and biochemically-mediated processes have already been attempted with, for instance, proteases produced by pure cultures of Bacillus sp.

Fig.1. Agarose embedded GFP expressing bacteria after ~24h of growth

And several proteases (keratinases) able to degrade keratin and belonging to the subtilizing family of serine proteases have been already characterized. However, these technologies are hampered by the compact nature of keratin in the feather fibers and the extensive disulfide bonding.

Therefore, the one gene, one protein, one product dogma underpins most modern commercial industrial biotechnology. Application of state of the art techniques will take this further by elucidation and application of the roles of multiple microorganisms in consortia gained through millennia of evolution. Bead encapsulation of cells relies on the incorporation of cells into beads of 25-35 micron in diameter, small enough to be sorted in a fluorescence activated cell sorter (Figure 1). Although the beads are small, they are large enough to support growth of few cells up to a micro-colony of 500-5000 cells per bead. In effect, this is equivalent to a well in a microtiter dish allowing for assay of enzymes activities that are not sensitive enough to be scored on single cell level. This gives us the possibility to monitor the relative decrease in fluorescence signal, as the labelled substrate of interest gets degraded by active extracellular enzymes secreted by the encapsulated cells in comparison to control beads.

The aim of this project is to design and apply a high throughput screening approach coupling encapsulation of cells in micro-beads of agarose or alginate coupled to fluorescence-based activity assays to generate and screen a wide panel of synthetic consortia with flow cytometric identification of promising candidates coupled to development of innovative keratin degrading assays.

Søren J. Sørensen (sjs@bio.ku.dk)