Clostridium cellulolyticum strain H10 (ATCC 35319) is a non-ruminal mesophilic cellulolytic bacterium originally isolated from decayed grass compost (Petitdemange et al., 1984). An attractive feature of C. cellulolyticum is its capability of anaerobic fermentation of cellulosic plant materials, yielding acetate, ethanol, lactate, and H₂, which could be used as alternative energy and commodity chemicals (Giallo et al., 1983; Guedon et al., 1999; Mohand- Oussaid et al., 1999). Since cellulose and hemicellulose comprise about 40 to 50% of plant materials and are considered to be the largest components of the earth's biomass, efficient conversion of this material to alternative energy and chemicals would be highly desirable in order to achieve less dependence on imported petroleum as a fuel and chemical source (Doi et al., 2003). Fermentative cellulolysis, which effectively conserves the reducing equivalents present in plant biomass, has been envisioned as a promising strategy to this challenge (Lynd et al., 1999). However, efficient cellulose fermentation processes remain to be developed, despite progress made in extensive enzymatic and genetic studies using reductionist approaches. Sequencing the genome of C. cellulolyticum would provide us the best opportunity to the fundamental understanding of anaerobic microbial cellulose utilization and the development of cost-effective processes for the conversion of plant biomass into renewable energy and commodity chemicals.


Relevance to DOE missions.

Cellulose degradation.
Plant biomass is the only foreseeable sustainable source of fuels and materials available to humanity (Lynd et al., 1999). The central technological impediment to more widespread utilization of this important renewable resource is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. Microbial cellulose utilization exists as one of the biotechnological approaches to developing practical processes for the conversion of cellulose to fuels and commodity chemicals that are both promising and relatively unexplored. Fermentative cellulolytic microorganisms such as C. cellulolyticum occupy a place of choice because these bacteria digest cellulose very efficiently via an extracellular enzymatic complex called the cellulosome (Schwarz, 2001), and can convert cellulose into a variety of valuable metabolites including ethanol and hydrogen (Mitchell, 1998). However, the recursive interest in cellulose utilization for bioenergetic prospects, notably for the production of H₂ or bio-ethanol, requires a better understanding of the physiology and metabolism of these bacteria (Nandi and Sengupta, 1998; Zaldivar et al., 2001). The sequencing of C. cellulolyticum, a model organism of mesophilic cellulolytic Clostridia (Doi et al., 2003), should greatly facilitate a mechanistic understanding of how these bacteria convert plant polymers into alternative energy and commodity chemicals.

Energy production.
Cellulose fermentation by C. cellulolyticum yields ethanol and H₂ (Giallo et al., 1983; Guedon et al., 1999), both can be used as alternative energy sources. Since cellulosic plant biomass is an important renewable resource, it is particularly attractive to produce energy from this material, given the challenges of national energy security and global climate change facing this country. However, the bio-energy yields of cellulose fermentation by C. cellulolyticum and other cellulolytic microorganisms vary considerably depending on growth phase, substrate limitation, cultivation mode, nutrient condition, and pH (Desvaux et al., 2001; Guedon et al., 2000; Payot et al., 1998). Therefore, strategies to optimize metabolic conditions for hydrogen production from cellulose fermentation by C. cellulolyticum are of great interest, because H₂ and ethanol are attracting substantial attention as a replacement for fossil fuels. H₂ and ethanol are strategically important as it has low emission, is environment-benign, cleaner and a more sustainable energy system. When generated from renewable plant biomass resources, H₂ and ethanol could contribute substantially to the reduction of greenhouse gas emissions. H₂ from renewable sources might be considered as the ultimate clean and climate neutral with no greenhouse gas emission energy system. With the DOE’s mission to provide clean and renewable energy alternatives such as hydrogen and bio-ethanol, tools for enhanced energy production from renewable sources need to be developed. The proposed genome sequencing of C. cellulolyticum would provide important information regarding the metabolic and regulatory pathways responsible for cellulose degradation and energy production. Such information is critical for the design of process control strategies for the optimization of bio-energy production. Further, this information will allow more robust and comprehensive design strategies for genomics approaches such as DNA microarrays and mass spectrometry-based proteomics aimed at evaluating and monitoring the cellulose fermentation and energy production processes carried out by C. cellulolyticum. The genomic information from C. cellulolyticum would greatly add to our understanding of these important processes.

Carbon cycling.
Life on Earth depends on photosynthesis, which results in production of plant biomass having cellulose as the major component. The carbon cycle is closed primarily as a result of the action of cellulose-utilizing microorganisms present in natural environments and the guts of animals. Thus, microbial cellulose utilization is responsible for one of the largest material flows in the biosphere and is of interest in relation to analysis of carbon flux at both local and global scales. Cellulolytic Clostridia including C. cellulolyticum are important in this context because these bacteria are ubiquitous in cellulosic anaerobic environments (Leschine, 1995), and their efficiency in cellulose degradation. Understanding the mechanisms and effectiveness of cellulose degradation by C. cellulolyticum and by Clostridia in general would help understand the interactions between global carbon cycling and the biosphere. More importantly, as described above, plant biomass can be utilized as a renewable industrial raw material via microbial cellulose fermentation processes using cellulolytic bacteria such as C. cellulolyticum. Because of the CO₂-consuming character of plant growth, cellulosic plant biomass-based processes and products can be incorporated into nature’s photosynthesis-driven carbon cycle with lifecycle greenhouse gas emissions approaching zero in some cases (Tyson et al., 1993.), thus curbing the risk of global climate change from the excessive emission of greenhouse gases.

References

Doi, R. H., A. Kosugi, K. Murashima, Y. Tamaru, and S. O. Han. 2003. Cellulosomes from mesophilic bacteria. J. Bacteriol. 185:5907–5914.

Desvaux, M., E. Guedon, and H. Petitdemange. 2001. Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium. J. Bacteriol. 183:119-130.

Giallo, J., C. Gaudin, J. P. B?la?ch, E. Petitdemange, and F. Caillet-Mangin. 1983.
Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10. Appl. Environ. Microbiol. 45:843-849.

Guedon, E., S. Payot, M. Desvaux, and H. Petitdemange. 2000. Relationships between cellobiose catabolism, enzyme levels and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium. Biotechnol. Bioeng. 67:327-335.

Leschine, S. B. 1995. Cellulose degradation in anaerobic environments. Annu. Rev. Microbiol. 49:399-426.

Lynd, L. R., C. E. Wyman, and T. U. Gerngross. 1999. Biocommodity engineering. Biotechnol. Prog. 15:777-793.

Mitchell, W. J. 1998. Physiology of carbohydrate to solvent conversion by Clostridia. Adv. Microb. Physiol. 39.

Nandi, R., and S. Sengupta. 1998. Microbial production of hydrogen: an overview. Crit. Rev. Microbiol. 24:61-84.

Payot, S., E. Guedon, C. Cailliez, E. Gelhaye, and H. Petitdemange. 1998. Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth. Microbiology 144.

Schwarz, W. H. 2001. The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol. Biotechnol. 56:634-649.

Tyson, K. S., C. J. Riley, and K. K. Humphreys. 1993. Fuel cycle evaluations of biomass-ethanol and reformulated gasoline, vol. I. DOE Office of Transportation Technologies, Washington, D.C.

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