Lignocellulosic plant biomass is comprised mostly of cell walls, which are a complex composite of proteins, lignin, and polysaccharides; the latter holds promise as raw material for biofuel production. The most abundant polysaccharide in the majority of tissues is cellulose, which exists as unbranched chains containing up to 15,000 β-(1,4)-linked glucose molecules . By contrast, the shorter hemicelluloses are chemically and physically more complex . The most abundant forms exist as glucan chains much shorter than cellulose or β-(1,4)-linked xylose, both with diverse side-chain substitutions of arabinose, galactose, fucose, xylose, or glucuronic acid. Biological conversion relies on an organism, such as a unicellular fungus or bacterium, which will convert these simple sugars to high-energy chemicals such as ethanol or butanol. Unlike seed starch or the soluble sugars found in phloem sap, the fermentable sugars found in cell walls are recalcitrant to extraction. The composition and interaction between the polysaccharides and lignin strongly influence their amenability for conversion to renewable fuels. Whereas lignification has extensive merits for the plant, it has adverse effects on the digestibility by ruminant and biofuel-generating microbes. For example, up to 50% of the variation in in vitro digestibility of commercial maize hybrids can be attributed to differences in their lignin content . Lignin is composed ofthree monolignols: p-coumaryl, coniferyl, and sinapyl alcohols, which polymerize to form p-hydroxyphenyl, guaiacyl, and syringyl phenylpropanoid units, respectively . The biosynthesis of alcohol monomers occurs in a specialized branch of phenylpropanoid metabolism, through which successive reductions, hydroxylations and methylations can occur. Crosslinking lignin with polysaccharides in the secondary cell walls of vascular tissue increases hydrophobicity, and thus gives these functional tissues the capacity to efficiently conduct water . Concurrently, the polysaccharides are less accessible to enzymatic digestion or mechanical penetration by potential pathogens . The pathway for lignin biosynthesis is well conserved among vascular plants, and involves at least 10 gene families, including CAD (cinnamyl alcohol dehydrogenase) and COMT . Each step in the lignin pathway has been perturbed in various species, resulting in changes in lignin content, composition, and, in many cases, digestibility [7, 8].
Genetic diversity of plant cell-wall properties within species is evident in the decades of plant breeding for improved feed and forage quality in crops such as maize, sorghum, and alfalfa [9, 10]. The merits of animal feed have been tested frequently in vivo, either by evaluating animal performance in response to a particular feeding regimen, or by estimating digestibility in vivo using livestock with fistulae . With the latter approach, the gastrointestinal tract of a surgically prepared animal is sampled to measure the remaining biomass. An equivalent in vitro method was developed using rumen fluid inoculum from fistulated cows . Digestibility is estimated through analysis of the organic matter lost from the simulated ruminant gut conditions after 4 days of incubation. The throughput of this approach is considerably higher than in vivo, and begins to meet the needs of traditional plant-breeding efforts and genetics research. High-throughput assays to estimate feed and forage quality parameters also include compositional measurements of cellulose , total lignin and monomer content [14, 15], and hemicellulose content and composition [16, 17], which also serve as valid measurements of biofuel feedstock quality. Although the parallels between digestibility and amenability to conversion to biofuels might be apparent, industry standards for biofuel feedstock quality are still needed.
Regardless of species, all new crop varieties must meet certain standards for industrial-processing efficiency and consumer-market quality. Beyond the expectation of high biomass yield with few inputs on marginal land, conversion quality standards for energy crops have yet to be defined by the biofuels industry. Recently, several methods, including some high-throughout platforms, have been established that treat plant samples with hydrolytic enzyme cocktails, such as fungal cellulase and xylanase/xylosidase, then assay for total sugars as a measurement of digestibility [18–22]. This approach can be taken one step further, using translational assays that mimic industrial simultaneous saccharification and fermentation (SSF) paradigms, in which the liberated sugars from the polymers can then be fermented by Saccharomyces cerevisiae to measure total ethanol yield [23, 24].
A distinct and promising approach to cellulosic biofuel production is consolidated bioprocessing (CBP) technology for conversion of biomass to fuel. CBP could lead to a significant reduction in processing costs, greater than the reductions gained from any other potential improvement, such as reducing enzyme loading, eliminating pre-treatment, or improving the processes associated with converting sugars to ethanol . The recently discovered anaerobic forest soil bacterium Clostridium phytofermentans may further enhance the efficiency of CBP. This organism produces ethanol as its major fermentation byproduct during growth on all substrates tested, including cellulose, hemicellulose, pectin, and starch , as well as switchgrass, corn stover, and pulp wastes ([Warnick and Leschine, unpublished data). Unlike S. cerevisiae, of which only engineered strains are capable of limited pentose utilization ,C. phytofermentans directly converts a wide array of fermentable components of biomass to ethanol, including cellulose, pectin, polygalacturonic acid, starch, xylan, arabinose, cellobiose, fructose, galactose, gentiobiose, glucose, lactose, maltose, mannose, ribose, and xylose . Without the addition of exogenous cellulases and xylanases, CPB using C. phytofermentans can yield approximately 70% of the yield of SSF using engineered S. cerevisiae .
In this paper, we report on an assay that provides the ability to measure the influence of variation in biomass composition, pre-treatment methods, and conversion processes on digestibility, and thereby determine the potential effects of numerous variables in biofuel production. In addition to sorghum, feedstock quality was evaluated for cultivars of shrub willow (Salix spp.) and Brachypodium distachyon accessions to demonstrate the applicability of this assay for a wide range of feedstocks, from woody crops to herbaceous grasses. The C. phytofermentans bioassay provides a direct and quantitative means of assessing feedstock quality, both in terms of digestibility and conversion.