Four hybrid varieties of corn stover were investigated. The Pioneer varieties 33A14, 34M95 and 33B51 were harvested from the Kramer farm in Wray, Colorado. The fourth type of corn stover, INL, was harvested in 2008 by Dr. Neal Yancey at Idaho National Laboratory (INL) from a farm near Hugoton, Kansas, following a test of harvesting methods. The compositional analysis results for the four corn stover varieties are shown in Table . Each corn stover lot was tub ground at the farm and then further milled at NREL through a Mitts & Merrill rotary knife mill (model 10 × 12, Saginaw, MI, USA) to pass a 1/2-inch (12.7 mm) rejection screen. All Kramer corn stover lots were stored in a dry place immediately after harvest. INL corn stover was left on the field for six months post harvest then stored in a dry place. Therefore, the INL corn stover lot had a lower sucrose content than the Kramer corn stover lots. The INL corn stover also had the lowest acetyl content and the highest lignin content of the group, as shown in Table .
Compositional analysis of corn stover feedstocks
Deacetylation of each corn stover lot was performed by dilute alkali extraction in a Recirculating Atmospheric Pressure Impregnation (RAPI) system as described by Tucker [27
]. Approximately 10 kg of dry (approximately 93 wt %) 1/2-inch milled corn stover was loaded into a Hastelloy C-276 wire mesh (40 mesh screen) basket and immersed in the recirculating bath containing 120 L of 0.4 wt % NaOH (0.1 M) solution at 70°C for three hours. The initial pH was approximately 12. After extraction, the excess alkali solution was drained and a liquid sample was retained for pH, acetate and sugar concentration analysis. The solid sample was weighed and measured for percent total solids.
Acid impregnation was performed using the same system described [27
]. Approximately 120 L of warm (48° to 50°C), 0.5 wt % H2
solution was prepared in a recirculation tank. A Hastelloy C-276 wire mesh (40 mesh screen) basket was loaded with 10 kg of 1/2-inch milled corn stover (approximately 93% solids) and immersed into the warm (48° to 50°C) recirculating dilute sulfuric acid bath for two hours. After acid impregnation, the feedstock in the basket was drained of excess acid solution to approximately 16% to approximately 18% solids and loaded into the mold of a hydraulic dewatering press, where the acid impregnated feedstock was pressed to approximately 45% solids.
The NREL 4-L steam gun reactor was used for all pretreatment experiments. The NREL steam gun is a 4-L steam-explosion reactor equipped with a jacket, a 4-in (10-cm) ball valve at the top for loading biomass, a 2-in (5-cm) ball valve at the bottom for discharging the contents of the reactor, two steam-injection ports near the top and bottom, and K-type thermocouples inserted near the top and bottom for reactor-temperature measurements. The reactor is made of Hastelloy C-22 TM to resist corrosion. The reactor temperature is controlled at the desired value using a pressure-control valve to regulate the steam-supply pressure. At the beginning of each experiment the reactor is preheated to the desired operating temperature by injecting steam into the jacket and cycling steam repeatedly through the reactor. The reactor is then loaded with the desired amount of pre-processed feedstock and quickly heated (approximately 5 to 10 sec) via direct steam injection to the reaction temperature. After the desired pretreatment time the steam is shut off and the bottom ball valve is quickly opened (< 1 second), explosively discharging the pretreated solids into a 55 gallon nylon HotFill (CDF Corporation, Plymouth, MA, USA) bag inside a 200-L flash tank. The bag is removed from the flash tank, labeled, sealed and stored at 4°C until ready for analysis.
The steam explosion reactor was pre-warmed to pretreatment temperature (150°C) and then loaded with 750 g of acid impregnated corn stover (approximately 45% solids). Direct steam injection brings the feedstock quickly to the reaction temperature (150°C) in less than 15 seconds. Pretreatments were carried out at the conditions of 150°C, 0.5 wt % H2SO4, and at 5, 10, and 20 minutes residence times.
Novozymes (Raleigh, NC, USA) cellulase, Cellic CTec2, and hemicellulase, Cellic HTec2, were used in the current study. The Novozymes enzyme preparations are a proprietary mixture of various enzyme activities. The enzyme loadings were calculated on a protein basis for 20 mg CTec2 (16FPU) and 2 mg HTec2 per gram of cellulose. The protein concentrations of the two commercial enzyme preparations were measured by the BCA assay (Thermo Scientific Pierce Biotechnology, Rockford, IL, USA) calibrated versus standard curves derived from bovine serum albumin standards supplied with the kits.
For washed solids enzymatic hydrolysis, the pretreated slurries were repeatedly washed with DI water in centrifuge bottles at 10,000 rpm by centrifugation and decanting until the xylose concentration was measured below < 0.01 g/L by a Yellow Springs Instrument 7100 MBS (Yellow Springs, OH, USA) calibrated versus supplied standards; usually six cycles were required. Enzymatic hydrolysis under dilute conditions (approximately 2% solids) was performed in 125 mL Erlenmeyer flasks containing 2.5 mL 50 mM citrate buffer, pH 4.8, 2.5 mg tetracycline, and enzyme, at a glucan loading of 1.0% (w/v). Enzymatic hydrolysis conditions were 150 rpm at 50°C.
Hydrolyzate preparation for fermentability test
The pretreated corn stover samples were subjected to whole slurry enzymatic hydrolysis to prepare the hydrolyzate for the fermentability test. The unwashed slurry was adjusted to a pH of 5.0 by successive additions of small amounts of concentrated ammonium hydroxide (29 wt %) and mixing. No citrate buffer was added because of the inhibition of citrate ions to growth and fermentation. Saccharifications were carried out in 125 mL wide-mouth polypropylene roller bottles (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 60 g of pH-adjusted slurries at 25% total solids. The roller bottle saccharification reactors employ gravitational tumbling as the mixing mechanism to homogenize solids by horizontally rotating the reaction vessels at 4 rpm on a three-deck roller apparatus for mini bottles (Wheaton Industries Inc., Millville, NJ, USA) [28
]. The roller apparatus was placed in a general purpose incubator (Model 1545, VWR International, LLC, West Chester, PA, USA) for temperature control at 48.5°C. The enzymatic hydrolysis will take a total of 7 days/168 hours. After hydrolysis, the produced slurry is centrifuged and the separated liquor are saved and refrigerated for fermentability test as described below.
Hydrolyzate fermentablility test
The liquid phase separated from whole slurry enzymatic hydrolysis as described in the last sections were subjected to the fermentability test. Samples were analyzed using two assays developed for toxicity analysis: growth and mini-fermentation assays using Zymomonas mobilis 8b as the biocatalyst. Toxicities were also tested for previously generated hydrolyzate liquors (after acid pretreatment with the biomass separated in the absence of saccharification enzymes). Two hydrolyzate liquor samples were also tested for toxicity for comparison purposes. The vertical reactor (VT) samples were generated from corn stover pretreated at 190°C, 3 wt % sulfuric acid for one minute in a pilot scale Sunds (Metso USA, Norcross, GA, USA) one metric ton/day vertical reactor. The conditions used to generate the horizontal reactor (HT) samples were of lower severity, 158°C, 2% sulfuric acid for five minutes in a pilot scale Metso (Metso, Norcross, GA) 200 kg/day horizontal screw reactor.
Z. mobilis 8b was used for the evaluation of hydrolyzate toxicity. It was revived from frozen glycerol stocks for approximately 6 to 8 hrs in 10 mL of RMG (2% glucose) at 33°C prior to inoculating overnight seed cultures in RMG8X2 (8% glucose, 2% xylose) using Blank volume shake flasks filled to 80% capacity at 33°C at 120 rpm. When the glucose concentration decreased from approximately 80 g/L to approximately 20 to 40 g/L, cells were spun down at 3,840 × g for 10 minutes at room temperature and re-suspended in RMG (2% glucose, 10 g/L yeast extract, 2 g/L K2HPO4) at a 10-fold concentration and used as inocula for Bioscreen C growth assays or fermentation studies.
For growth assays, samples were diluted to 10 wt % of the starting slurry material from pretreatment with the addition of 10 g/L yeast extract, 2 g/L K2
, sugars and water. Depending upon the initial concentrations of sugars present, samples were normalized to the same concentrations for glucose and xylose. In our case, concentrations of glucose and xylose are normalized to 45 g/L and 31 g/L, respectively. Pure sugar fermentations at the same concentrations were conducted as controls. Bioscreen C assays were carried out as described previously [24
]. Cells were inoculated into Bioscreen C wells containing a total volume of 300 μL and incubated without shaking at 33°C at an initial cell density of OD = 0.05 (approximately 5 × 106
cells/mL). Turbidity measurements (OD420-580 nm
) were taken every 10 minutes for up to 48 hours.
Mini-fermentation assays were conducted with whole slurries conducted at higher biomass loadings in miniature vials vented with 18 gauge needles capped with 0.2 μM filters, using a total 4 mL volume. Yeast extract, K2HPO4, water, glucose and xylose were added, while normalizing the total glucose and xylose to the same concentration for each sample. Mini-fermentations were conducted by inoculating log phase cultures of Z. mobilis 8b at an initial cell OD of OD600 nm 1 (approximately 1 × 108 cells/mL). Samples were taken at 0, 24, 48, and 72 hours for OD600 nm readings and HPLC analysis. Fermentation assays were conducted at 33°C at 120 rpm.
Chemical analysis and yield calculations
Pretreatment and enzymatic saccharification liquors and fermentation samples were analyzed using HPLC according to standard NREL laboratory analytical procedures (LAPs) [29
]. Solid residues were also analyzed according to standard NREL LAPs [29
]. Sugar yields from high solids enzymatic hydrolysis were calculated using the equations developed by Zhu et al.
In the liquor phase of pretreated slurry, acetate is present in two forms: 1) free acetate/acetic acid, released from the xylan; and 2) acetyl groups covalently bound to the dissolved xylan oligomers. Free acetate is measured by direct injection on a Shodex SP0810 acid column (Kawasaki, Japan), while total acetate is measured with the same column after a 4% acid hydrolysis of the filtered pretreatment liquid that contains all solubilized compounds. The 4% acid hydrolysis at 121°C for 1 h hydrolyzes the remaining acetyl groups covalently bound to the xylooligomers.