Thesis
Evaluating models of cellulose degradation by Fibrobacter succinogenes S85
Washington State University
Master of Science (MS), Washington State University
12/2015
Handle:
https://hdl.handle.net/2376/103292
Abstract
Lignocellulose-based biofuel production is a promising interest for energy as fossil fuel energy becomes rapidly depleted and demands for energy increases. Conversion of lignocellulose biomass into biofuels has many significant benefits. However, postponement of utilizing lignocellulose for energy is due to the recalcitrant nature of cellulose. Hydrolysis of the cellulose is needed to break the cellulose into glucose monomers for fermentation to bioethanol. Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing bacterium with high potential for biofuel production. The mechanism utilized for cellulose degradation by this major cellulolytic bacterial species is unknown. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime and pilin proteins, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space via ABC transporters for further degradation. Here, to further elucidate the cellulolytic mechanism of F. succinogenes a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) was performed. RNA-sequence analysis revealed that genes encoding type II and III secretion systems, fibro-slime and pilin proteins are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose suggesting they are a sign of cell aging. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pilin proteins, produces cellulases such as endoglucanases that are secreted extracellularly by type II and III secretion systems and degrades the cellulose into cellodextrins that are imported back into the periplasm for further digestion by [Beta]-glucanases and other cellulases.
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Details
- Title
- Evaluating models of cellulose degradation by Fibrobacter succinogenes S85
- Creators
- Meagan Christine Burnet
- Contributors
- Allan S. Felsot (Chair)Stephen Jan Callister (Committee Member) - Washington State University, Biological Sciences, School ofMary Lipton (Committee Member) - Washington State University, Biological Chemistry, Institute ofKathleen McAteer (Committee Member) - Washington State University, Biological Sciences, School of
- Awarding Institution
- Washington State University
- Academic Unit
- Environment, School of the (CAHNRS)
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Number of pages
- 117
- Identifiers
- 99900525148301842
- Language
- English
- Resource Type
- Thesis