Development and validation of an automated microplate method for hydrolysis of AFEX treated lignocellulosics using enzyme mixtures and synergistic additives

Shishir Chundawat, Venkatesh Balan, Bruce E. Dale

Research output: Chapter in Book/Report/Conference proceedingConference contribution


Some of the biggest disadvantages of the standard enzymatic hydrolysis protocols for cellulosic enzyme systems are; throughput time, reagent usage, labor intensive and non-realistic substrates (i.e. filter paper, purified xylans, chromogenic substrates). Recent research has shown that choice of an appropriate enzyme preparation depends more on the characteristics of the substrate rather than on standard enzyme-activities measured. Therefore, the need for a rapid hydrolysis assay based on lignocellulosic substrates becomes apparent. The automated microplate method is a rapid hydrolytic assay technique (essentially a scaled down version of the NREL LAP 009 protocol) developed at our laboratory to screen hydrolysis of lignocellulosic biomass using multi-enzyme systems. The microplate assay of lignocellulosic biomass is a better assessment of enzymatic activity compared to other assays (i.e. filter paper activity) for biomass conversion. The critical bottlenecks that need to addressed in the development of a suitable microplate technique are; reproducible substrate delivery, minimizing evaporation losses, rapid sugar assay and high throughput. The most suitable method for delivery of substrate was through suspension of biomass in slurry, dispensed using a robotic liquid handling workstation. Delivery parameters were optimized for varying substrate slurry concentrations. The optimization and statistical analysis results are shown for avicel and ammonia fiber expansion (AFEX) treated corn stover hydrolyzed using a mixture of cellulase and beta-glucosidase. We also screened several commercially available cellulases, xylanases and beta-glucosidases under a wide range of total enzyme concentrations for the hydrolysis of AFEX pretreated biomass.

Original languageEnglish (US)
Title of host publication233rd ACS National Meeting, Abstracts of Scientific Papers
Publication statusPublished - Dec 28 2007
Externally publishedYes
Event233rd ACS National Meeting - Chicago, IL, United States
Duration: Mar 25 2007Mar 29 2007


Other233rd ACS National Meeting
CountryUnited States
CityChicago, IL


All Science Journal Classification (ASJC) codes

  • Chemistry(all)

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