title: High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration.
abstract: The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.
authors.affiliation: Technology Division, Joint BioEnergy Institute, Emeryville, CA, United States. rbharad@sandia.gov
authors.forename: Rajiv
authors.initials: R
authors.lastname: Bharadwaj
authors.forename: April
authors.initials: A
authors.lastname: Wong
authors.forename: Bernhard
authors.initials: B
authors.lastname: Knierim
authors.forename: Seema
authors.initials: S
authors.lastname: Singh
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Holmes
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
mesh_terms: D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D050260:Carbohydrate Metabolism / Q000187:drug effects; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000648:ultrastructure; D004553:Electric Conductivity; D006868:Hydrolysis / Q000187:drug effects; D052578:Ionic Liquids / Q000494:pharmacology; D007700:Kinetics; D008031:Lignin / Q000378:metabolism*; D008904:Miniaturization; D008956:Models, Chemical; D013997:Time Factors
chemical_list: D052578:Ionic Liquids; C036909:lignocellulose; D002482:Cellulose; D008031:Lignin; D002480:Cellulase