Increasing emissions of greenhouse gases from transportation sector is recognized as one of the powerful causes for global warming and associated with it climate change. In addition to these environmental problems dwindling fossil fuel resources make the search for renewable energy resources more urgent than ever. In the short term, ethanol has been trusted as an environmentally sound motor fuel alternative that can be used either in blend with gasoline or alone. Widely available and renewable lignocellulosic waste streams from agriculture, paper and wood industry, etc., as well as specially grown energy crops are recognized as the plentiful and low cost feedstock for fuel ethanol large-scale production (Demain at al. Microbiology and Molecular Biology Reviews, March, 2005; p. 124-154, vol. 69, #1). Such approach is strengthened further by the fact that cellulosic ethanol exhibits a net energy content three times higher than corn ethanol, and emits a low, net level of greenhouse gases (Wang. “Well to Wheel” life cycle analyses for cellulosic ethanol. Argonne National Laboratory). The primary impediment to widespread production of ethanol from recalcitrant lignocellulosic materials is the general absence of a low cost technology (Lynd RR; Germgross TU, Wyman CE: Biocommodity engineering. Biotechnol Prog 1999, 15:777-793).
Current approach for development of “third generation” biofuels in particular depends on the potential of corresponding enzymes hydrolases-producing by micro organisms and on a detailed understanding of their biosynthesis regulating mechanisms. In natural environments, fungi are the primary degraders of lignocellulosic biomass, excreting both hydrolytic and oxidative enzymes to deconstruct cell walls. Among them, ascomycetes, such as Trichoderma or Aspergillus, secrete enzymes that deconstruct the polysaccharides (cellulose and hemicelluloses) to simple sugars required for metabolism, and basidomycetes, such as white-rot fungi, secrete enzymes that deconstruct all components of the cell wall, including cellulose, hemicelluloses, and lignin.
Although various microorganisms have been evaluated for their ability to deconstruct lignocellulosic biomass, only few have demonstrated production levels compatible for industrial applications (Lynd et al., 2002; Demain et al., 2005; Das et al., 2007; Yu et al., 2007; Kumar et al., 2008 ). These are microscopic fungi, producing cellulases and xylanases. Basidial fungi are also very important for lignocellulose degradation, mainly for delignification.
The majority of cellulases used in biotechnology are still derived from well-characterized non-extremophilic microorganisms and there is a very little information regarding cellulases from extremophiles. An important drawback of these commonly used industrial enzymes is the lack of activity at high temperature and the tendency of these enzymes to denature at elevated temperatures or other critical conditions. Stable cellulases could be obtained either by isolating extremophilic microorganisms where such unique properties of extremophilic cellulases already exist or being reached by protein engineering (Viikari et al., 2007). Thus, thermophillic fungi and bacteria such as Allesheria terrestris, Chaetomium thermophile, Sporotrichum thermophile, Scytalidium thermophillum, Clostridium straminisolvens, Thermonospora curvata, Pyrococcus furiosus, Acidothermus cellulolyticus, and Saccharophagus degradans producing cellulase complex may be valuable sources of heat stable cellulases (Kvesitadze et al., 1999; Kato et al., 2004). Systematic studies of these fungi identify some promising candidates for industrial application.
Proposed project is focused on obtaining stable enzymes from DIBB unique extremophilic mycelial fungi collection (accounting above 2500 individual strains) for the creation of biotechnology of production of fuel-bioethanol from agricultural and industrial lignocellulosic wastes.
The overall goal of the project is development of cost effective, ecologically friendly, biotechnology of fuel bioethanol production from agricultural and industrial lignocellulosic wastes. The main objective of the project is obtaining stable, industrially robust enzymes: cellulases, xylanases and laccases for lignocellulosics effective degradation and fermentation of hydrolysis product glucose by yeasts to ethanol.
Specific approaches proposed for project realization involve:
1. Selection of producers of stable cellulases, xylanases and laccase among the diverse mycelial fungi extremophile strains collection of DIBB, AUG.
2. Selection of optimum cultivation conditions of selected fungi strains for cellulase, xylanase and laccase biosynthesis in submerged conditions.
3. Characterization of cellulases and xylanases synthesized by microscopic fungi (temperature and pH optimum of action, heat and pH stability, potential to hydrolyze of crystalline and amorphous cellulose).
4. Development of lignocellulosic substrates effective pretreatment method (physical, chemical and biological by selected basidial fungi strains, active producers of laccase).
5. Hydrolysis of different pretreated lignocellulosic substrates by selected stable cellulase and xylanase preparations (time and deepness of hydrolysis, optimum enzyme-substrate ratio, glucose yield).
6. Fermentation of hydrolyzed glucose to ethanol by selected yeasts strains.
Multidisciplinary project will combine studies in microbiology, biochemistry and biotechnology and will be completed with the selected fungal stable cellulases, xylanases and laccases for the creation of biotechnology of production of fuel-bioethanol from agricultural and industrial lignocellulosic wastes.
The expected results of the project are:
· Producers of industrially important, stable cellulases, xylanases and laccase among the mycelial fungi extremophilic strains collection of DIBB, AUG, isolated from extreme environment of diverse soil climatic zones of southern slopes of Caucasus will be selected.
· Physiology of growth and development of selected fungi strains optimal for cellulase, xylanase and laccase biosynthesis in submerged conditions will be established.
· Industrially important characteristics of cellulases and xylanases such as: temperature and pH optimum of action, heat and pH stability, hydrolysis of crystalline and amorphous cellulose will be determined. To accelerate the process of lignocelluolse hydrolysis possible activators and inhibitors of enzymes action will be revealed.
· Effective pretreatment method of lignocellulosic substrates with the aim of delignification for further enzymatic degradation will be developed.
· Optimum parameters for exhausted enzymatic hydrolysis of delignified cellulosic wastes with maximum glucose yield will be determined.
· Effective yeasts strains will be selected and fermentation conditions of hydrolyzate to ethanol will be designed.
The proposed project has theoretical significance from point of view of identification of novel extremophilic mycelial fungi producers of lignocellulose deconstruction enzymes from diverse geographical region, characterized by multiply repeated 15 soil-climatic zones and extreme environments. Taking into consideration that stability of the enzymes with desired properties are main limiting factors in fuel bioethanol production from lignocellulosic wastes, project is of great practical importance. Stable enzymes from extremophilic fungi effectively degrading lignocellulose have high potential for commercialization for which substantial effort will be made to establish business contacts with research teams and companies involved in cellulosics-based fuel ethanol R&D and production.
