Biological conversion assay for determining plant feedstock quality

Authors: LEE, SCOTT - University Of Massachusetts; WARNICK, THOMAS - University Of Massachusetts; PATTATHILL, SIVAKUMAR - University Of Georgia; ALVEO-MAUROSA, JESUS - University Of Massachusetts; SERAPIGLIA, MICHELL - Cornell University; YOUNG, NAOMI - University Of Massachusetts; SCHNELL, DANNY - University Of Massachusetts; SMART, LAWRENCE - Cornell University; HAHN, MICHAEL - University Of Georgia; Pedersen, Jeffrey; MCCORMICK FORD, HEATHER - University Of Georgia; BROWN, VIRGINIA - University Of Georgia

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2012
Publication Date: 8/5/2012
Citation: Lee, S.J., Warnick, T.A., Pattathill, S., Alveo-Maurosa, J.G., Serapiglia, M.J., Young, N.F., Schnell, D.J., Smart, L.B., Hahn, M.G., Pedersen, J.F., Mccormick Ford, H., Brown, V. 2012. Biological conversion assay for determining plant feedstock quality. Biotechnology for Biofuels. 5:5.

Interpretive Summary: A distinct and promising approach to cellulosic biofuels production is consolidated bioprocessing technologies for conversion of biomass to fuel. Consolidated bioprocessing could lead to a significant reduction in processing costs, greater than any other potential improvement, such as reducing enzyme loading, eliminating pre-treatment, or processes associated with converting sugars to ethanol. The recently discovered anaerobic forest soil bacterium Clostridium phytofermentans may serve as a real agent of change. It produces ethanol as the major fermentation byproduct during growth on all substrates tested, including cellulose, hemicellulose, pectin, starch, switchgrass, corn stover and pulp wastes. Here we report an assay that provides the ability to measure the impact of pretreatment, conversion processes, and microbial and plant genetic diversity on digestibility, and thereby determine the potential effects of numerous variables in biofuel production. The assay was shown to detect significant differences in ethanol produced from a set of sorghum lines previously shown to differ in lignin content, digestibility, and ethanol yields using sacchariciation-based conversion assays. Compositional analysis of the sorghum biomass before and after inoculation suggested differences in xylan metabolism is in part responsible for differences in ethanol yields. Additionally, natural genetic variation for Clostridium phytofermentans conversion efficiency in Brachypodium distachyon and shrub willow was demonstrated. The use of C. phytofermentans takes into consideration specific organismal interactions, which will be critical in single stage fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of the mechanisms underlying plant feedstock quality.

Technical Abstract: A strategy to develop renewable sources of energy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon economic feasibility including development of energy crops amenable to facile deconstruction. Reliable high throughput assays to characterize feedstock quality are needed to measure the effects of pretreatment and processing as well as plant and microbial genetic diversity that influences bioconversion efficiency. We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the microbe’s ability to convert biomass directly into ethanol with little to no pretreatment. Plant samples were added to an anaerobic minimal media and inoculated with C. phytofermentans, incubated for three days, after which the supernatant was analyzed for ethanol concentration. The assay detected significant differences in supernatant ethanol among wild type sorghum and brown midrib mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested differences in xylan metabolism is in part responsible for differences in ethanol yields. Additionally, we characterize natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow. Results agree with previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be critical in single stage fermentation (SSF) or consolidated bioprocessing (CBP). The ability to detect such phenotypic variation facilitates the genetic analysis the mechanisms underlying plant feedstock quality.