Populus trichocarpa woody biomass used from this study was generously provided through the BioEnergy Science Center (BESC) from Pacific Northwest National Laboratory. The composition of the raw Poplar as determined by following NREL LAP (version 08-03-2012) was 47.0% glucan, 16.9% xylan, and 21.2% acid-insoluble lignin.31 The biomass was air-dried and knife milled using a laboratory mill (Model 4, Arthur H. Thomas Company, Philadelphia, PA) to pass through a 1 mm internal sieve size. Novozymes® generously provided Cellic® Ctec 2 cellulase with a protein content estimated using a Pierce BCA analysis kit of 250 mg/ml. The National Renewable Energy Laboratory (NREL) generously provided the D5A yeast strain, a Saccharomyces cerevisiae variant.
CELF Pretreatment: Prior to CELF pretreatment, milled Poplar wood chips were soaked overnight at 4°C in a 1:1 ratio (weight basis) of THF to water containing 0.5 wt% H2SO4. A dry biomass loading of 7.5 wt% of the total working mass was CELF pretreated in a 1 L Hastelloy Parr autoclave reactor (236HC Series, Parr Instruments Co., Moline, IL) equipped with a double stacked pitch blade impeller rotating at 200 rpm. A series of pretreatments were carried out at 160°C for 15 minutes, i.e., conditions that resulted in maximum sugar recovery for CELF followed by enzymatic hydrolysis (not published). Temperature inside the reactor was measured by an in-line thermocouple (Omega, K-type), and all reactions were maintained within ± 2°C using a 4 kW fluidized sand bath (Model SBL-2D, Techne, Princeton, NJ). At the end of each pretreatment, the reactor was cooled by submerging quickly in a large room temperature water bath. The solids were then separated from the liquor by vacuum filtration at room temperature through glass fiber filter paper (Fisher Scientific, Pittsburgh, PA). The mass and density of the liquid fractions were measured to calculate yields and close mass balances. The solids collected were washed with water until clear water ran through the solids which were then hydraulically pressed to reduce the moisture content to 61%.
Enzymatic hydrolysis of CELF pretreated Poplar: Batch enzymatic hydrolysis was performed by following the standard NREL protocol32 using 125 mL flasks with a total working volume of 50 mL. For each, CELF pretreated biomass was loaded to a 1 wt%, glucan loading. In addition, 50 mM of citrate buffer (pH 4.5) along with millipore water and 0.02% sodium azide as an antimicrobial agent were added to 50 mL to reach a final pH of 4.8. Triplicates were run with the appropriate amount of substrate. The flasks were placed in a 50°C incubator shaker (Infors HT Multitron, MD) and allowed to equilibrate for 1 h at 150 rpm. Appropriate amounts of Cellic® Ctec2 enzyme were added to the flasks at target protein loadings, and the flasks were then returned to the incubator shaker. 1 mL samples were taken at the times reported, centrifuged (Centrifuge 5424, Eppendorf, Germany) at 15000 rpm for 10 min, diluted, and analyzed for sugar concentration in the broth.
Fractal modeling of enzymatic hydrolysis kinetics: The following fractal model based on first order breakdown of glucan to form glucose with a rate coefficient kt that varied with time raised to the -h power was used to describe cellulose hydrolysis over hydrolysis time:
\(\frac{dC}{dt}=-{k}_{t}C\) , where \({k}_{t}=k{t}^{-h}\) 1
The k, kt, and h parameters in Eq. 1 were fit to enzymatic hydrolysis data by MATLAB.
Seed inoculum preparation: Saccharomyces cerevisiae (D5A) yeast were grown in 10 mg/mL of yeast extract (Becton, Dickinson and Company, Redlands, CA), 20 mg/mL of peptone (Becton, Dickinson and Company, Redlands CA), and 50 mg/mL of glucose to reach exponential growth and then stored in glycerol (~ 14 wt%). When needed, the frozen stock was thawed and grown overnight in 10 mg/mL yeast extract, 20 mg/mL peptone, and 50 mg/mL glucose in a 250 mL baffled flask in a 37°C, 130 rpm shaking incubator (Infors HT Multitron, MD). The inoculum was then centrifuged and resuspended in sterile deionized (DI) water to reach an optical density (O.D.) of 0.5 as determined by (SpectraMax ABS Plus, Molecular Devices, CA) at 600 nm.
Pure sugar fermentations and growth: Pure sugar fermentations by D5A were carried out in 125 mL flasks at glucose concentrations specified in the Results and Discussion section. The appropriate glucose amount was dissolved in Millipore water (Milli-Q, EMD Millipore, Darmstadt, Germany) and added to a flask and bubble trap assembly. Duplicates and a substrate blank were sterilized at 121°C for 35 min in an autoclave (HA-300MII, Hirayama Manufacturing Corporation, Japan) and cooled in a laminar flow hood (Baker and Baker Ruskinn, Sanford, ME) to prevent contamination followed by adding water to adjust for evaporative losses. 50 mM citrate buffer (pH 4.5) was added to reach a final pH of 4.8 in 50 mL, and 40 mg/L of tetracycline along with the seed inoculum were used in 48 h fermentations shaking at 130 rpm and 37°C. 0.75 mL samples were taken every 2 h until the stationary phase was reached. These samples were centrifuged at 15000 rpm for 10 min, diluted, and analyzed for ethanol and sugar concentrations.
Simultaneous Saccharification and Fermentation (SSF): Batch SSF experiments were performed in 125 mL flasks with a total working volume of 25 mL containing CELF pretreated biomass corresponding to desired glucan loadings to which were added 50 mM citrate buffer (pH 4.5) to reach a final pH of 4.8 in 25 mL followed by 40 mg/L tetracycline (Sigma Aldrich, St. Louis, MO) as an antimicrobial agent. Cellic® Ctec2 cocktail was loaded at 15 mg-protein per g-glucan-in-raw poplar, and the yeast inoculum was added next. Flasks with attached bubble traps were loaded with Millipore water and the appropriate amount of substrate. Duplicates with substrate as well as substrate blanks were sterilized at 121°C for 35 min in an autoclave (HA-300MII, Hirayama Manufacturing Corporation, Japan). Next, the flasks were cooled in a laminar flow hood (Baker and Baker Ruskinn, Sanford, ME) to prevent contamination and reweighed to determine how much water to add to restore the intended solids loading. After adding buffer, antimicrobial agent, enzyme cocktail, and yeast inoculum, SSF was carried out for 10 days at 37°C at 130 rpm in an incubator shaker (Infors HT Multitron, MD). 1 mL samples were taken periodically, centrifuged at 15,000 rpm for 10 min, diluted, and analyzed for sugar and metabolite concentrations in the liquid.
Measuring sugar and ethanol concentrations: Liquid samples along with appropriate calibration standards were analyzed by HPLC (Waters Alliance 2695 system, Waters, Milford MA) equipped with a Bio-Rad Aminex® HPX-87H column (BIO-RAD, Hercules CA) and Waters 2414 RI detector with a 5 mM sulfuric acid eluent flow rate of 0.6 ml min− 1. Chromatograms were integrated by the Empower® 2 software package (Waters Co., Milford MA).
Model equations: Percent glucan conversion to glucose via enzymatic hydrolysis of a 1 wt% glucan loading was calculated as follows:
\(Percent glucan conversion to glucose=\frac{{C}_{Glucose}\times 0.9\times WV}{{M}_{G}}\times 100\) 2
In which,
\({C}_{Glucose}\) is the concentration of glucose in the enzymatic hydrolysis liquid at a given time, mg/ml,
\(WV\) is the working volume in the flask, ml (50 ml),
\({M}_{G}\) is the mass of glucan initially added, g.
At lower solid loadings, i.e., < 5 wt%, the density of the solvent phase was assumed to be the same as the density of water. As the insoluble solid fraction increased, the density of the liquid fraction first increased due to increasing sugar concentrations and then dropped slightly due to increasing ethanol concentration. The fluid density was measured directly.
The ethanol yield as a percent of the theoretical maximum was calculated as follows:
with
\({V}_{L}=({M}_{W}+{M}_{DS})/\rho\) 4
\({V}_{L}=({M}_{W}+{V}_{W}\times \left({C}_{Eth}+{C}_{Glucose}+{C}_{Glycerol}+etc.\right))/\rho\) 5
In which \({C}_{Eth}\) is the ethanol concentration in the fermentation broth, mg/ml,
\({C}_{Glucose}\) is the ethanol concentration in the fermentation broth, mg/ml,
\({C}_{Glycerol}\) is the ethanol concentration in the fermentation broth, mg/ml,
\({V}_{L}\) is the volume of liquid fraction in the fermentation medium, ml,
\({M}_{G}\) is the mass of glucan initially added, g,
\({M}_{W}\) is the mass of water initially added, g,
\({M}_{DS}\) is the mass of dissolved solids in the fermentation medium at any given time point, g, and
\(\rho\) is the density of the medium, g/ml.