Progress 09/01/02 to 08/31/11
Outputs
Expression of 17 genes involved in cellulose degradation and primary catabolism by the thermophilic, anaerobic cellulose-degrading bacterium Clostridium thermocellum ATCC 27045 were determined, using real-time PCR of mRNA recovered from cells grown in continuous culture on cellulose or on a soluble cellulose building block, cellobiose. Relative to growth on cellulose, expression of genes encoding the cellulosome scaffoldin (CipA) and main exoglucanase (CelS), as well as a mannanase (ManA) and one alcohol dehydrogenase was decreased when cells were grown on cellobiose at high growth rates, but not at low growth rate, suggesting that expression of these genes was regulated by growth rate, not (as previously believed) by substrate type. Expression of genes encoding known catabolite regulatory proteins was not quantitatively affected by growth rate or by substrate type. Growth on cellulose, but not on cellobiose, was accompanied by production of a polysaccharide-rich
sticky substance (called a glycocalyx) that allowed cells to adhere tightly to cellulose (a requirement for cellulose degradation by this species). The glycocalyx from C. thermocellum and from a related bacterium, Ruminococcus albus, had similar carbohydrate compositions, suggesting a similar function and a similar evolutionary origin. Variable pressure scanning electron microscopy (VP-SEM), a technique not previously applied to the study of biofilms, allowed accurate visualization of the R. albus cells without physical disruption of the biofilm. The biofilm consisted of a single layer of bacterial cells attached by the glycocalyx to one another and to the cellulose substrate. Compositional and linkage analysis of the polysaccharide component of the glycocalyx revealed chains of six-carbon sugars of up to 26 units in length, with substantial branching of the main polysaccharide chain, indicating that the glycocalyx is structurally different from other bacterial polysaccharides studied
thus far. Analysis based on known biochemical pathways for microbial polymer assembly suggest that R. albus must invest only a small fraction (<4%) of the energy available from cellulose fermentation in order to assemble the polysaccharide component of the glycocalyx that it needs to attach to the cellulose.
Impacts
This research indicates that important cellulolytic functions in C. thermocellum, a potentially useful organism for biomass conversion to ethanol, are not exclusively controlled by substrate type. This, in turn, suggests novel strategies for regulating biomass conversion by this organism. This research also identifies the unique structure of the polysaccharide component of this glycocalyx, provides both a means of estimating glycocalyx yield and energy demands, provides guidance for future studies to link composition and structure of the glycocalyx-containing fermentation residues to the functional behavior of the glycocalyx as a potentially adhesive for manufacture of plywood and other bonded wood products.
Publications
- Weimer, P.J., N.P.J. Price, O. Kroukamp, L.M. Joubert, G.M. Wolfaardt, and W.H. Van Zyl. 2006. Studies of the extracellular glycocalyx of the anaerobic cellulolytic bacterium Ruminococcus albus. Appl. Environ. Microbiol. 72:7559-7566.
- Lynd, L.R., P.J. Weimer, G. Wolfaardt, and Y.H.P. Zhang. 2006. Cellulose hydrolysis by Clostridium thermocellum: A microbial perspective. In: I.A. Kataeva (ed.). Cellulosome: Molecular Anatomy and Physiology of Proteinaceous Machines (Uversky V.N., Series Editor). Nova Science Publishers, Hauppauge, New York (In press).
|
Progress 01/01/05 to 12/31/05
Outputs
We have determined an expanded range of biomass substrates that can be utilized by this bacterium for the production of ethanol and the bio-based adhesive. We have also conducted additional continuous cultures to examine the impact of microbial growth rate and substrate type on the the yield of microbial cells and the bio-based adhesive material (analyses are currently underway). We have scaled up the fermentation to 300 liter scale and produced sufficient quantity of residual fermentation solids for additional testing of its adhesive capabilities.
Impacts
This work established the effect of growth conditions on fermentation product yields and on the expression of genes involved in the production of ethanol and other products, including a novel adhesive compound. Control of gene expression may be one potential route to improving substrate utilization and product formation by this organism.
Publications
- Stevenson, D.M., and P.J. Weimer. 2005. Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. Appl. Environ. Microbiol. 71:4672-4678
|
Progress 01/01/04 to 12/31/04
Outputs
The anaerobic cellulolytic bacteria Ruminococcus albus and Clostridium thermocellum produce ethanol and acetic acid as major fermentation end products. Because they produce their own plant cell wall-degrading enzymes and ferment the oligosaccharide products to ethanol, microbial cell mass, and novel bio-based adhesive, these organisms may be useful in converting agricultural residues and other cellulosic biomass materials to value-added products. We found that both species produce an extracellular glycocalyx that contributes to their ability to adhere to the cellulose substrate. These materials contain polysaccharides of similar sugar compositions, with an unexpected abundance of xylose and mannose, but a relatively low content of galactose, the major sugar of most bacterial exopolysaccharides; this suggests that these glycocalyces may be atypical of those found in bacteria that do not adhere to cellulose. Calculations suggest that the organisms spend 2 to 4 per cent
of their total biosynthetic energy expenditure in producing the carbohydrate component of the glycocalyx. In order to better understand the regulation of end product formation, we examined the effect of growth conditions on gene expression in C. thermocellum strain ATCC 27405 using cells grown in continuous culture under cellobiose- or cellulose-limitation over a ten-fold range of dilution rates (0.013 to 0.16 per hour). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of seventeen genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes to determine relative expression. Thirteen genes displayed modest (five-fold or less) differences in
expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cAMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed over ten-fold reduced levels of expression when grown on cellobiose at dilution rates of over 0.05 per hour: cipA (cellulosomal scaffolding protein), celS(exoglucanase), manA (mannanase) and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated, but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution. Overall, the data provide suggestions of how these bacteria may be modified to improve their
ability to produce the biofuel ethanol, or a more environmentally-friendly type of wood adhesive.
Impacts
This research identifies the "glue" produced by these bacteria as important components of the cellulose-degradation process, and will serve as a basis for studies of the practical application of this material as a wood adhesive. In addition, this research is the most complete study to date, in any organism, of the effect of growth rate on the expression of genes involved in energy metabolism.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
The glycocalyx of cellulose-grown cultures of the thermophilic bacterium Clostridium thermocellum was shown to be remarkably similar in composition to that of the mesophilic rumen bacterium Ruminococcus albus. The exopolysaccharide (EPS) component of the glycocalyx contains large amounts of xylose and mannose (not found to date in glycocalyces from any other organisms), and only small amounts of galactose (the dominant sugar in EPS of most bacteria). Electron microscopic characterization has revealed aspects of glycocalyx structure that are similar in the two species, and that explain some of the functional properties of the glycocalyces. Calculations based on the amount of EPS synthesized and the presumed ATP requirements for synthesis of the EPS components indicate that both C. thermocellum and R. albus devote approximately 2 to 4 per cent of their biosynthetic energy demand to EPS synthesis. Based on the unexpected abundance of xylosyl and mannosyl residues in the
EPS, several primers have been designed for RT-PCR monitoring of expression of potential genes for EPS synthesis, and are currently being used for gene expression studies using cells grown on cellulose versus those grown on soluble sugars, during which substantial EPS synthesis is not observed.
Impacts
The data will allow the refinement of bioenergetic models of growth and metabolism for C. thermocellum. The PCR primers will permit studies of gene expression involved in EPS synthesis by this organism.
Publications
- No publications reported this period
|
|