Biomass, chemicals and enzymes
Corn plants, either in the immature state to be used for ensiling (CS) or as mature WCP were obtained from Michigan State University Farms (East Lansing, MI, USA). The corn hybrid used was NK 49-E3 (Syngenta, Basel, Switzerland) which is a typical CS hybrid used in the Great Lakes Region. The corn plants used in this study were planted on 8 May 2008 and harvested on 19 September 2008 for ensilation. The WCP were harvested after the plant reached physiological maturity, which occurred approximately 6 weeks after harvest for ensilation. WCP was harvested as stover and grain separately (moisture content < 15% DWB). WCP-based samples were milled using a Wiley mill (Christy and Morris, Chelmsford, UK) (10 mm sieve attachment) followed by mixing of the grain and stover fractions at a mass ratio of 1:1 (w/w). Ensiling was accomplished by sealing 500 g immature entire corn plant samples in plastic bags using a commercial grade vacuum seal food machine (CG-15; Cabela, Sidney, NE, USA). The sealed bags were stored at 21°C for 30 days to imitate a typical on-farm ensiling process. We also evaluated the effect of a commercially available microbial inoculant product (Silo-King, Agri-King Inc., Fulton, IL, USA) on ensiled corn digestibility at a 0X, 1X and 10X loading (X = the manufacturer recommended loading rate of 0.0015 g/g). The inoculant product is composed of lactic acid-producing organisms, such as Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecium, and is used by farmers to enhance the feed quality of ensiled corn . The CS samples were frozen using liquid nitrogen, milled using a laboratory blender (Hamilton Beach, Washington, NC, USA), and passed through a 10 mm screen sieve. The milled CS samples were stored in sealed Ziploc Storage Bags (SC Johnson, Racine, Wisconsin, USA) at -20°C for long-term storage. The moisture content of CS was between 63 and 67% (DWB). The CS samples were dried to < 10% moisture (DWB) using a 50°C oven, to allow suitable adjustment of the water loadings used during pretreatment.
Avicel PH101 and soluble starch S5160599 (lot #054261) were purchased from Fluka (Tokyo, Japan) and Fisher Scientific (USA), respectively. Commercial enzymes used for degrading cellulose were Spezyme CP™ (88 mg/ml) and Accellerase 1000™ (84 mg/ml; lot #1600844643) (both gifts from Genencor Division, Danisco US Inc., Rochester, NY, USA), The enzymes used for degrading starch were Novozyme 188™ (149 mg/ml) (Sigma-Aldrich (Sigma, St. Louis, MO, USA) and Stargen 001™ (62 mg/ml; lot #4900851951) (gift from Genencor Division). The enzyme used for degrading hemicellulose was Multifect Xylanase™ (35 mg/ml) (gift from Genencor Division). The concentrations of these enzymes were estimated using a Kjeldahl-based method (Dairy One Feed Stock Analyzing Co., Ithaca, NJ, USA).
Crude protein, starch, crude fat and water-soluble carbohydrate content of CS (0X, 1X and 10X) and WCP were determined at the Forage Testing Laboratory (Dairy One Inc.). In addition, acid and neutral detergent fiber values were determined for WCP. Polysaccharide (cellulose, xylan and arabinan), Klason lignin, extractive and ash content were determined based on the standard National Renewable Energy Laboratory protocols . Glucan content refers to total cellulose and starch composition of the substrate. WCP was composed of 49.2% starch and 15.5% cellulose (total glucan 64.7%).
AFEX pretreatment was carried out as described previously . After charging liquid ammonia into the reactor containing the biomass at the appropriate moisture content, the reactor temperature was raised rapidly to the desired level and held constant for 5 min. Subsequently, ammonia was rapidly released through the exhaust valve. The treated biomass was removed from the reactor and air-dried overnight in a fume hood to remove residual ammonia. AFEX was carried out on CS at different moisture loadings (20 to 200% DWB), temperatures (50°C to 130°C) and ammonia loadings (0.1-3 g ammonia per gram dry weight of biomass). WCP, starch and Avicel samples were pretreated with AFEX at 90°C for 5 min reaction time (total residence time in the reactor after injection of ammonia was ~ 25-30 min), 60% moisture (DWB) and 1:1 (w/w) ammonia to biomass loading.
AFEX-treated substrates were used without washing with water before hydrolysis. Enzymatic hydrolysis of substrates was carried out based on the National Renewable Energy Laboratory (NREL) protocol  at a total volume of 15 ml using screw-capped vials. The substrate was hydrolyzed in 50 mM sodium citrate buffer (pH 4.8) at various enzyme loadings (as mg protein per gram cellulose, starch or xylan). Tetracycline (40 mg/L) and cycloheximide (30 mg/L) were added to prevent microbial growth. Hydrolysis was conducted at 50°C with mild agitation (150 rpm). Sampling was carried out at 12, 24, 72 and 168 h.
High solid loading-based enzymatic hydrolysis
High solid loading hydrolysis was based on 6% glucan (cellulose + starch) loading for each substrate. The pretreated substrate was hydrolyzed in fed-batch mode in two stages (3% glucan loading for each stage) separated by a 24 h time interval. The hydrolysis was carried out in a 2000 ml conical flask (500 ml reaction volume) with 50 mM sodium citrate buffer (pH 4.8) and incubated at 50°C with shaking at 250 rpm. After 24 h, a second batch of solids and appropriate quantity of enzymes were added to the flasks and incubated under identical conditions for an additional 48 h. Tetracycline at 40 mg/L was added to avoid microbial growth during hydrolysis. The hydrolyzates were centrifuged at 10,000 rpm (10, 100 × g) for 30 min, and the supernatants were sterilized by filtration for subsequent ethanol fermentation.
Separation and quantification of monomeric sugars was conducted using a high performance liquid chromatography (HPLC) machine equipped with an automatic sampler ( LC2010; Shimadzu Scientific Instruments, Columbia, MD, USA) and refractive index detector (Waters RI Detector, 410; Waters Corporation, Milford, MA, USA). For acidic-based hydrolyzates, a HPX-87H Aminex column (Bio-Rad, Hercules, CA, USA) maintained at 65°C using a 5 mM sulfuric acid-based mobile phase (flow rate of 0.6 mL/min) was used for monosaccharide analysis, and a HPX-87P Aminex column maintained at 85°C using water as the mobile phase (0.6 ml/min) was used for analysis of enzymatic hydrolyzates. The concentrations of glucose, xylose and ethanol in the fermentation broths were simultaneously estimated using the HPX-87H column.
Fermentation culture and media
Genetically engineered S. cerevisiae 424A (LNH-ST) was obtained from Dr Nancy Ho (Purdue University, West Lafayette, IN, USA). This strain contains xylose-metabolizing genes integrated into the host chromosome . This strain was cultured routinely in YEPX (1% yeast extract, 2% peptone and 2% xylose) medium at 30°C with shaking at 150 rpm. The culture was maintained on YEPX-agar plates at 4°C for regular use.
The seed culture was prepared by inoculating YEP-glucose medium with cells from the plate culture followed by incubation at 30°C with agitation at 150 rpm. After 48 h, the cells were harvested by centrifugation. The supernatant was discarded and cells were transferred to 100 ml of fresh fermentation medium in 250 ml Erlenmeyer flasks. The flasks were closed with rubber stoppers pierced with a thin surgical needle to allow release of the carbon dioxide formed during fermentation. The inoculated flasks were incubated at 30°C with agitation (100 rpm) in a temperature-controlled orbital shaker. The culture growth was monitored by measurement of optical density at 600 nm. The initial OD600 of all cultures was about 0.1. During fermentation, 1 ml culture samples were removed at regular time intervals and analyzed for glucose, xylose and ethanol. The metabolic ethanol yield (Yp/s) was calculated as the mass of ethanol produced per unit mass of sugar utilized during fermentation. The theoretical yield of ethanol for glucose or xylose is 0.51 g ethanol per gram sugar. Volumetric ethanol productivity (Qv) of fermentation was calculated as the amount of ethanol (g/L) produced per unit time (h) of fermentation.