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Global challenges encountered today within energy sector are attributable to increasing emissions of green house gases in parallel with the worldwide growing demand for energy. Replacement of fossil raw materials by renewable ones would provide at least one feasible solution for relieving the problem. Within the transport sector, the use of bioethanol as fuel is increasing. However, so far bioethanol has predominantly been produced by 1st generation technology from sugar cane or starch derived from maize. In Europe instead of sugar cane, grain and sugar beet have been favoured as raw materials. 2nd generation biofuels would be much more energy efficient and contribute significantly more to reduced CO2 emissions.

Production of 2nd generation biofuels is generally based on lignocellulosic biomass originating from agricultural and forestry wastes and energy crops. For degrading pretreated lignocellulosic raw materials to fermentable sugars, novel tailormade enzyme systems would be needed. Enzymatic hydrolysis of biomass is known to provide higher yields of sugars under mild reaction conditions than the traditional chemical hydrolysis with strong acids.

Besides easily fermentable hexoses (C6 sugars), a significant portion (15-40%) of sugars in lignocellulosic biomasses consists of pentoses (C5 sugars).  Yeast and bacterial strains capable of fermenting C5 sugars (e.g. xylose, arabinose) have recently been developed. However, for facilitating economically feasible biofuel production, more robust strains with increased rate of pentose fermentation should be developed.

Main NEMO objectives  

The project seeks technologies with highest impact on bioethanol production and will focus on

1) Development of high-performance enzymes and enzyme mixtures for efficient hydrolysis of lignocellulosic raw materials of European interest

2) Generation of stress tolerant yeasts for efficient fermentation of C6 and C5 sugars derived from lignocellulosic materials to ethanol

NEMO Work Packages

WP 1. Raw materials
WP 1 will focus on developing optimized pretreatment methods of selected raw materials of European interest (straw, energy crops and soft wood). Pretreated material will be supplied in large amounts with reproducible quality. The methods applied will be based on steam explosion and/or acidic and chemical treatments. 

WP 2. Efficient novel enzymes
New enzymes active on the pre-treated raw materials and residues remaining undigested in pretreatment will be screened from culture collections, genomic databases, and  cDNA and metagenomic libraries. More thermotolerant enzymes and enzymes with higher specific activity and/or reduced end-product inhibition, and/or showing less nonproductive binding than the commercially available enzymes will be identified and/or engineered.

WP 3. Optimized enzyme mixtures
WP 3 aims at generating optimized enzyme mixtures for the selected raw materials and process concepts to enable efficient hydrolysis of the pretreated raw materials.  Emphasis will be focused on reduction of the enzyme amount needed.

WP 4. New strains through mutagenesis and screening
Robust broad-substrate range, pentose-fermenting yeast strains will be generated through mutagenesis, and through evolutionary engineering. Target will be Saccharomyces strains with comparison to non-S. cerevisiae species. Mutants obtained will be analyzed and candidate genes and strains for further metabolic engineering selected.

WP 5. Targeted strain engineering 
Rational metabolic engineering approaches will be applied in WP 5 for improving stress tolerance and yields and rates of C6 and C5 co-fermentation of yeasts. Physiological systems biology analyses and metabolic modeling will be used  as tools and several genetic alterations will be combined into a single strain.

WP 6. Process assessment

WP 6 is designed to provide information to WPs 1-5 and supply data for process economic calculations for WP 7. The improved enzymes, microorganism and processes will be compared to the baseline conditions. Novel process regimes (temperature, feeding strategies, etc) for improved yield and productivity will also be generated. The best enzyme, microorganism and process combinations identified will be evaluated at pilot scale.

WP 7. Process calculations
The process options developed will be evaluated based on process calculations and models of the techno-economic feasibility. The impact of various process options on energy efficiency and Green House Gas emissions of the process regimes will be addressed.