Work packages

WP 1. Raw material supply

Of the various pretreatment options, hydrothermal pretreatments have been most extensively studied. Pretreatments disrupt the plant cell wall and improve enzymatic access to the polysaccharides. The capital and operating costs for lignocellulose pretreatment, such as steam explosion, are estimated to be about 20% of the total production costs of ethanol. In this project, the previously developed IBUS pre-treatment technology (Inbicon A/S) will be the main method of pretreatment. It is based on hydrothermal treatment, resulting in high yield and excellent enzymatic digestibility of all carbohydrates. The method can be run in pilot and demonstration scales from 10-4000 kg/hour.

Within Europe straw is presently one of the most available feedstock for lignocellulosic bioethanol. The focus of this project is to develop conversion technologies mainly for agricultural by products or dedicated crops; available in major parts of Europe. The suitability of the pretreatment technology on dedicated energy crops will be optimized and evaluated. Based on these results, the relevance of forest residues as potential lignocellulosic raw materials will be estimated.

WP 2. New enzymes

The high cost of cellulases is still considered a key barrier to economic production of cellulosic ethanol. Despite the declared reductions in enzyme price there is still an obvious need for further development on a wider basis. Several approaches have been applied to improve cellulase performance and decrease the amount of enzymes needed to saccharify lignocellulosic substrates; improving individual cellulase components or complementing or replacing the set of cellulases by novel proteins. Presently, the relevant commercial enzymes are only able to act at a temperature ranges close to 40-50°C. Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes needed, enhanced stability allowing improved hydrolysis performance, better inhibitor tolerance and increased flexibility with respect to process configurations, all leading to improvement of the overall economy of the process.

Lignin has been shown to bind cellulases and reduce their service life and therefore, reducing their non-specific binding of enzymes to lignin is a new challenge. A further approach to improve the performance of enzymes is to add enzymes active on other polymers in the matrix, such as hemicellulases, or to add other rate-limiting enzymes enhancing the hydrolysis. Enzymes acting on the polymer layers interlinked with each other; i.e. lignin, hemicellulose and cellulose, can improve the solubilisation of the carbohydrate polymers.

A special target in this project is to improve the performance of individual key cellulases by further improving the specific activity of thermostable key cellulases and by reducing their non-specific binding to lignin. Targeted modifications in the cellulose binding domains of cellulases would expectedly reduce this undesirable phenomenon. The focus will be on the development of more efficient enzyme mixtures for liquefaction and saccharification.

WP 3. Development of hydrolysis technology

One of the single most important process parameters with regard to process economy and efficiency is the overall substrate consistency level. Low carbohydrate substrate levels not only increase the capital cost due to equipment size but they also result in excessive energy requirement with regard to heating, cooling and distillation. Until recently, carbohydrate levels of 10% or less were the standard applied. When aiming at an ethanol concentration above 5% the dry matter consistency has to be over 20%. A new approach to be further studied in this project is based on gravity mixing of biomass. Gravity mixing enables efficient hydrolysis and processing of biomass up to a dry matter level of 40%, and at the same time the efficient mixing improves the enzyme efficiency. New integrated high temperature (HT) liquefaction technology based on enhanced accessibility of the substrate and improved enzymes will be developed. The main aim is to minimize the amount of externally added enzymes by optimizing the hydrolysis conditions for various raw materials.

WP 4. Development of fermenting organisms

Significant improvements have already been achieved to increase the ethanol yield by efficient fermentation of pentose sugars to ethanol using the tools of metabolic engineering. The traditional yeast, Saccharomyces cerevisiae, is considered most competitive due to its long standing industrial tradition, good productivity and tolerance for high ethanol concentration. Stable xylose-fermenting S. cerevisiae strains have been obtained by two major routes. The present published yields and rates are, however, still lower than with traditional glucose fermenting yeasts. The theoretical improvement of ethanol yield through fermentation of all available sugars may be up to 30%.

Consolidated bioprocessing (CBP) refers to a conversion processes where the production of enzymes, hydrolysis of cellulose (and hemicellulose) and fermentation is carried out basically in one step. The projected bioethanol production costs by CBP could be essentially lower than with traditional methods. In spite of this great promise, no micro-organisms with the required properties (substrate hydrolysis and product formation) are currently available. There are two approaches to develop these organisms; to add the key cellulolytic enzyme genes to a good ethanol producer, such as yeast, or to improve the product (ethanol) producing capabilities of cellulolytic organisms, such as anaerobic Clostridia, which have been most extensively studied. An interesting organism for the consolidated process is Fusarium oxysporum, known to produce several enzymes acting on cellulose and hemicellulose and to be able to ferment sugars (glucose and xylose) into ethanol with reasonably good yield. F. oxysporum has been shown to produce ethanol with a yield of about 1.8 mole ethanol/mole of glucose and 1 mole ethanol/mole of xylose. The primary aim is to improve the ethanol production of selected F. oxysporum strains using xylose fermenting yeast s trains as reference. The aim is also to optimize the enzyme production stage as part of the consolidated bioprocess.

WP5. Consolidated bioprocessing

The technology development for a high consistency process (at 20% or higher dry matter content) will include several factors, such as product inhibition, transport phenomena and water activity. In this project the principle of gravity mixing will be used for development of the hydrolysis as well as fermentation. The prehydrolysis (preferably at high temperature) allows to reduce rapidly the viscosity of the high solid content substrate and enables better mixing for the inocculation of the fermenting organism.

A continuous liquefaction and saccharification reactor is based on integration of liquefaction, saccharification and fermentation and will be used for pilot scale testing of consolidated bioprocessing combined with externally added liquefying enzymes. The novel process technology to be developed aims at extending the service life of the enzymes, maintaining a high cell count and productivity of the micro-organisms and reducing the distillation cost. The project will also provide solutions for entire new process concepts.

WP 6. Pilot-scale testing and feasibility

The process concept will expectedly result in decreased ethanol production costs, decreased energy demand in the process, i.e. distillation and evaporation, and in an increased yield of the solid fuel (lignin) co-product. An important part is the possibility to integrate the system into a combined heat and power (CHP) plant. The economic evaluation will estimate the benefits of this new concept and its integrated use. Based on results in pilot-scale, a feasibility study will enable a full technical and economical evaluation of the concept. The feasibility study also allows to compare different process designs and will point out areas of improvement and technical bottlenecks in different process designs.

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