Cellulosic sugars are the primary currency of the bio economy. Lignocellulose is the most abundant source of biomass on the planet and represents an enormous storehouse of sugars. Monosaccharides from biopolymers of lignocellulosic feedstock can be fermented into a wide variety of biofuels and biochemicals including ethanol, organic acids (e.g., lactic acid), solvents (e.g., acetone, butanol), 5-hydroxymethyl furfural, levulinic acid and lubricants
. Hemicellulose can also be used to produce ethanol, xylite, furfural, nylons and plant gums such as thickeners, adhesives and stabilizers
. Canada, with its enormous forests and huge annual amounts of agriculture residues, is a huge producer of cellulosic biomass. The weight of straw produced from cereal crops equals or exceeds that of grain, and represents a significant opportunity as a biorefinery feedstock to contribute to environmental, social and economic sustainability
. Similarly, recent work has shown that alfalfa is 2 ~ 3 times more efficient than corn or soybean as a biomass source owing to its high biomass yield, perennial nature, fixation of aerial nitrogen, and production of valuable co-products, making it a model forage species for biofuel research
. However, cellulosic material is remarkably recalcitrant; making the release of fermentable sugars with current hydrolysis options commercially cost prohibitive
A crucial step in the bioconversion of lignocellulosic feedstock to biofuels and biomaterials is to maximize the saccharification of cellulose and hemicellulose components to fermentable sugars in a manner that is cost effective. One of the challenges is the high cost of enzymes involved in the saccharification of the lignocellulose and the loss of some of the hemicellulose sugars during pre-treatment. Most acidic and alkaline pretreatments (e.g., dilute acid, steam explosion, ammonia recycle percolation) remove a significant fraction of hemicellulose and/or lignin, thereby enhancing enzyme accessibility. While pretreatments like ammonia fiber expansion (AFEX) do not physically extract hemicellulose or lignin as separate fractions, they do modify the cell wall ultra-structure through mechanisms that are currently not well understood
. It is reasonable to assume that varying types of pretreated biomass would require specific mixtures of enzymes that were tailor-made for efficient hydrolysis
. Similarly, in order to minimize costs pertaining to enzyme production, identification of major enzymes and optimization of their relative ratios could reduce enzyme usage without sacrificing the rate or yield of substrated during hydrolysis
. Likewise, plant cell walls are a primary source of nutritional energy for ruminants, but with many forages less than 50% of the cell wall fraction is digested
. Substantial benefits would be realized if a greater percentage of this potential energy was made available for fermentation in the rumen through an increase in the digestibility of the plant cell wall fraction.
This study was designed to examine the ability of key fungal enzymes from Aspergillis niger and Thielavia terrestris, in combination with commercial enzymes (Accellerase 1500 and Accellerase XC) or mixed rumen enzymes to further enhance the breakdown of alkaline peroxide (AP) pretreated alfalfa hay and barley straw. AP pretreatment was selected as it accomplishes a degree of delignification, with relatively low environmental impact and without the need for special reaction chambers
. The process causes selective removal of lignin and xylan through a combination of lignin oxidation and de-acetylation and also decreases cellulose crystallinity, enhancing the susceptibility of plant cell walls to enzymatic degradation
. However, conservation of acetyl and feruloyl ester linkages and only partial lignin oxidation has been reported at the low concentrations (≤2.0%) used in this study
. Therefore, we utilized a selection of purified auxiliary enzymes (i.e., esterase (AXE16A_ASPNG, AXE16B_ASPNG, FAE 1a); pectinase (PGA28A_ASPNG); α-arabinofuranosidase (ABF54B_ASPNG); endoglucanase GH7 (EGL7A_THITE); endoxylanase (XYN11A_THITE); β-glucosidase (E-BGLUC) and β-xylosidase (E-BXSRB) to explore their ability to enhance the activity of commercial enzyme and rumen enzyme mixtures. A similar approach using a combination of statistical design, robotic dispensing of substrate slurry and high throughput micro plate techniques to assess enzymatic hydrolysis at comparable protein to biomass loads and reaction volumes has been reported earlier
. The high throughput micro assay adopted in this study is based on Chundawat et al.
, a procedure which has been standardized for solid delivery in biomass slurries, mass transfer related parameters, reproducibility and validity through comparision to conventional National Renewable Energy Laboratory protocols