Location: Bioenergy Research Unit
Title: Identifying enzyme resistant xylo-oligomers from processing switchgrass to bioethanol Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 9, 2011
Publication Date: June 9, 2011
Citation: Bowman, M.J., Dien, B.S., Hector, R.E., Sarath, G., Cotta, M.A. 2011. Identifying enzyme resistant xylo-oligomers from processing switchgrass to bioethanol [abstract]. American Society for Mass Spectrometry. p. 66. Technical Abstract: Introduction: Switchgrass (SG) is a potential renewable biomass source for conversion to liquid biofuels. Efficient biochemical conversion requires rational strategies for pretreatment and enzymatic saccharification to fermentable sugars. The major carbohydrates present in SG are hemicellulose (e.g. xylan) and cellulose; commercial constraints demand efficient conversion of both to monosaccharides. Developing more effective enzyme formulations for saccharification of xylan is impeded by the scarcity of analytical methods for fully analyzing the residual xylo-oligomers. The presence of xylo-oligomers following enzymatic saccharification represents a conversion loss and, furthermore, their presence is also known to inhibit the cellulases used for hydrolyzing cellulose. Therefore, it is expected that analysis of residual oligosaccharide sequences will reveal structures resistant to hydrolysis that may also interfere with cellulose hydrolysis. Methods: Replicate samples of dilute ammonia-pretreated SG were enzymatically depolymerized for 72 hours by the action of a combination of industrial enzymes to determine a baseline for the presence of oligosaccharides that remain resistant to hydrolysis by enzyme cocktails. The enzymes used are susceptible to product inhibition; therefore, simultaneous saccharification and fermentation (SSF) of pretreated SG, using either a standard or a xylose-utilizing engineered strain of Saccharomyces cerevisiae, was employed to mitigate product inhibition. Residual soluble oligosaccharides were analyzed by hydrophilic interaction liquid chromatography (2.1 mm x 15 mm column) mass spectrometry (HILIC-MS) with a ThermoFinneganDecaXP**plus ion trap mass spectrometer. Prior to analysis, anthranilic acid (AA) was incorporated into sugars by reductive amination to facilitate identification of reducing sugar components. Preliminary data: Quadruplicate samples of ammonia-pretreated SG were run under three conditions: enzyme-only digestion; SSF with a standard S. cerevisiae strain, and SSF with a xylose-utilizing strain of S. cerevisiae. Enzyme–only and SSF sample supernatants were analyzed by standard methods (mixed-mode high performance liquid chromatography (HPLC) with refractive index detection) to determine monosaccharide release and, for the SSF experiments, ethanol yields. All sample data was consistent with expected yields for the respective experiments demonstrating that samples are representative of valid biomass conversion protocols. Therefore, sample supernatants were dried and labeled under standard reductive amination conditions. Analysis of reducing-end labeled residual soluble oligosaccharides by HILIC-LC-MS revealed that the dominant compositions present are (Pent)-AA, (Pent)2-AA, (Pent)4-AA, (Pent)5-AA, and (Pent)6-AA. Under the chromatography conditions used, (Pent)2-AA, (Pent)4-AA, and (Pent)6-AA appear as single peaks, whereas (Pent)5-AA occurs as two well-defined peaks. (Pent)-AA and (Pent)2-AA share retention times with AA-labeled linear xylo-oligosaccharides, while larger d.p. oligomers do not co-elute with the corresponding linear xylo-oligosaccharides. This indicates that these are likely arabinose-containing oligosaccharides. Aside from the expected dominance of (Pent)2-AA signal, one isomer of (Pent)5-AA has the strongest signals in both ultraviolet (UV) and MS monitoring. On-line tandem MS allows for distinction in the isomeric forms of (Pent)5-AA; however, the isobaric nature of arabinose and xylose leads to ambiguous structural assignments and warrants further study. Samples that were treated with the enzyme cocktail show only the same pentose-based oligomers, as well as (Hex)1-3-AA due to product inhibition of the cellulolytic portion of the enzyme cocktail. Detailed structural characterization work of putative arabino-xylo-oligosaccharides is ongoing. The structures will serve as standards for future work in variations of pretreatment and/or enzyme treatment for maximal, rapid depolymerization of these structures. Novel aspects: Characterization of enzyme hydrolysis-resistant oligosaccharides will allow for targeted approaches to enzymatic depolymerization of switchgrass biomass for biofuel production.