title: The analysis of saccharification in biomass using an automated high-throughput method.
abstract: The recalcitrance of the cell wall to enzymatic hydrolysis represents one of the greatest challenges for using biomass to replace the petroleum as a feedstock for fuels and chemicals. Cell walls are complex in architecture and composition, posing a biochemical challenge for the development of efficient enzymes to release the sugars from the polysaccharide components. The complex composition of the polymers that constitute the cell wall requires a mixture of enzymes to hydrolyze the different glycosidic bonds present in biomass. The improvement of the properties of biomass, in turn, requires the screening of large populations of plants in order to identify markers associated with saccharification potential or pinpoint the genes that regulate recalcitrance. The improvement of both, enzymes and biomass together, requires the capacity to deal with large numbers of variables in a combinatorial approach. We have developed a high-throughput system that allows the determination of cellulolytic activity in a 96-well plate format by automatically handling biomass materials, carrying out hydrolytic reactions, and determining the release of reducing sugars. This platform consists of a purpose-made robot that grinds, formats, and dispenses precise amounts of solids into 96-well plates, and a liquid-handling station specifically designed to carry out pretreatments, hydrolysis, and the determination of released reducing sugar equivalents using a colorimetric assay. These modules can be used individually or in combination according to the function needed. Here we show some examples of the capabilities of the platforms in terms of enzyme and biomass evaluation, as well as combining the robot with off-line analytical tools.
authors.affiliation: CNAP, Department of Biology, University of York, Heslington, York, United Kingdom.
authors.forename: Caragh
authors.initials: C
authors.lastname: Whitehead
authors.forename: Leonardo D
authors.initials: LD
authors.lastname: Gomez
authors.forename: Simon J
authors.initials: SJ
authors.lastname: McQueen-Mason
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D018533:Biomass; D002473:Cell Wall / Q000378:metabolism; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism; D009005:Monosaccharides / Q000378:metabolism*; D006109:Poaceae / Q000378:metabolism*
chemical_list: D009005:Monosaccharides; C036909:lignocellulose; D002482:Cellulose; D008031:Lignin; D002480:Cellulase