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Concerted action : on process development (biochemical conversion), and enzymatic hydrolysis

Obiettivo

The work to be supported by this coordination activity represents a continuation, although at a much reduced level, of work funded under the previous biomass energy programme. The objective is to increase the cost effectiveness of biological processes for converting biomass into fuels, chemical feedstock, organic fertilizers and fibre (pulp), for paper and fabric.
Clostridium thermocellum cellulase consists of a high molecular weight, multienzyme complex, termed cellulosome. Catalytic subunits of the cellulosome comprise various beta-glycanases (cellulases, xylanases, mannanases), which are anchored to a large scaffolding component termed CipA. Each of the catalytic components comprises a noncatalytic anchored domain, termed dockerin domain, which binds to a complementary domains of CipA, termed cohesin domains (intr=eraction of type I). CipA is anchored to bacterial cell surfaces by means of a specialised dockerin domain, which binds to surface proteins harbouring a complementary, specific cohesin domain (interactions of type II). We study indevidual catlaytic subunits as well as interactions of type I and II, with a view to reconstitute artificail protein complexes. These may help to understand the high specific activity of the cellulosome in degrading cellulose and open the way for the design of multienzyme complexes not necessarily involved in cellulos hydrolysis.

A pilot plant for enzymatic hydrolysis of cellulosic biomass has been developed. It consists of 2 reactors working in series. The first reactor works with soluble cellulase while the second one is a tubular fixed bed reactor filled with cellulase immobilized in hydrogels of poly-2-hydroxyethyl methacrylate.

The importance of inter-phase mass transfer resistances has been studied and it was found that, in the whole range of adopted experimental conditions, transport of substrate and product outside the pellets do not play an important role. The use of the immobilized enzyme reactor produces effluent streams from the plant which are rich in glucose.

The concerted action on enzymatic hydrolysis has brought together activities in lignocellulose hydrolysis catalyzed by hemicellulases and cellulases from bacteria, actinomycetes and fungi with studies on pretreatment and fermentation from 20 laboratories sited in 8 of the member states.

Research concerned the development of antibodies against a reiterated conserved sequence element present in several endoglucanases and xylanase from Clostridium thermocellum.
The overall studies have culminated in the description of the 3-dimensional structure of the endoglucanase CelD; establishment of the alpha carbon skeleton, the signal peptide, a beta-sheet containing domain, an alpha-helix core and several loops forming the catalytic site.

A study is also taking place of the dissociation and reassociation of the cellulase within the multicomponent cellulosome. The cellulosome was isolated from the rest of the extracellular cellulase system by affinity chromatography on a column of Whatman cellulose CC41.
Isolated in this way, the cellulosome contained more than 95% of the carboxymethylcellulase activity applied to the column and could effect extensive solubilization of crystalline cellulose. As a result the way may now be open for the fractionation and identification of the individual components of the cellulase complex.

A clostridial cellulase is also being investigated (from C cellulolyticum). So far 3 genes corresponding to endoglucanases have been identified, one of which celCCA coding for EGCCA, has been sequenced.

Work on fungal systems involved the design of methodologies for gene cloning and expression in Trichoderma reesei CL847, a hypercellulolytic mutant strain.
The cellulase system of C cellulolyticum is also under investigation. This bacterium, is being used to model cellulose breakdown. It has been found that very slight variations in the reducing sugar concentrations affect both bacterial growth and production of cellulase. The possibility of incre asing the stability and yield of products of cellulase activity was researched using a biphasic system prepared with water and a water immiscible organic solvent. Results showed that the amount of reducing groups produced at 45 C during 48 h of hydrolysis of wheat straw (32 to 48 mesh fraction) in the presence of 0.03 g/l of enzyme was increased working with organic solvents (75% volume for volume butyl acetate).

The effect of growth conditions on phenol oxidase activities and on cellulolytic activities have been studied using Trametes trogii Berk 202 grown on synthetic media plus wheat straw as a carbon source in submerged culture. The mode of attack on the substrate by this fungus has been examined using transmission electron microscopy. Different micromorphological features have been found, according to the composition and the amount of nutritive supplementation of the culture medium. A correlation between the degree of lignin breakdown and cellulose susceptibility to enzymatic hydrolisis with foreign enzymes has also been shown.

The enzymatic hydrolysis of a model biomass, wheat straw, was studied at 45 C either in buffer or a 2-phase system. The reaction medium was prepared with buffer solution (50 mM sodium acetate/acetic acid, pH 4.8) with different amounts of butyl acetate added.

Experiments carried out during the 48 h operation clarified that a substancial enhancement (up to 63%) of the reaction yield was obtainable when operating in the 2-phase system. This mainly depended upon the optimization of the following parameters: reaction temperature (45 C); optimal combination of cellulases from Aspergillus niger and Trichoderma viride in the enzyme composite, (namely 25% weight for weight of the first one); biomass loading (25 g/l); biomass composition and/or biomass structure at the end of pretreatment; volumetric percentage of butyl acetate in the reaction media (75% volume for volume).
In this project there are two common themes:

1) The isolation, identification and genetic improvement of relevant microorganisms (anaerobic bacteria, fungi, actinomycetes and yeasts) using methods which include isolation from the wild, mutation, selection and genetic manipulation as well as the isolation study and use of specific enzymes (cellulases, hemi-cellulases and lignases isolated from them). The objectives are to improve specificity, improve yield, improve temperature and pH stability and reduce end-product inhibition. This work has relevance to the production of sugar solutions for use as feedstock for both biological and chemical conversion to alcohols, acids and organic polymers; delignification of paper pulp; improved composting and anaerobic digestion for fertilizer and biogas energy through dissolvation of cellulose; reduction in organic pollution of ground water; disposal of sewage sludge, etc as well as aspects of animal nutrition (ruminants), etc.

2) Design of improved bioreactors and fermentation systems, including membrane and high solids reactors, immobilised and entrapped cell/enzyme systems, thermophilic (high temperature) fermentations, etc. The objectives are related to those in part 1; that is the improvement of volumetric productivity and cost of product recovery for biological conversion systems aimed at recovery of energy or valuable products from fermentation, or enzyme conversion, products of biological metabolism of products of lignocellulose substrate.

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Meccanismo di finanziamento

CSC - Cost-sharing contracts

Coordinatore

CPL SCIENTIFIC PUBLISHING SERVICES LTD
Contributo UE
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Indirizzo
Kingfisher court 43, hambridge road
RG14 5SJ Near newbury
Regno Unito

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Partecipanti (17)