Aim of the study and sourcing of biomass
The aim of this research has been to compare the effects of steam explosion pretreatment on enzymatic saccharification, and simultaneous saccharification and fermentation of rice straw and rice husk. Field grown rice (Oryza sativa, cv. KhangDan18) straw and husk was harvested at maturity in spring 2012 at Ba Vi, Hanoi, Vietnam. The biomass was fumigated and then air-dried under ambient conditions (approx. 34 °C, 84 % RH) at the Agricultural Genetics Institute (Hanoi, Vietnam). Before steam explosion pretreatment, the moisture content of the straw was determined to be 9.01 % and the husk 9.98 % (w/w).
Steam explosion of rice straw and husk
Both rice straw and husk were steam exploded into hot water (70–80 °C) using a Cambi™ Steam Explosion facility with a 35 L reactor (Cambi AS, Asker, Norway). The rice straw was cut into lengths of 2–3 cm prior to loading the reactor. No size reduction was needed for the husk. The reactor was charged with 500 g of feedstock, sealed and heated with steam to the desired temperature (180–230 °C) at which it was retained for 10 min. After this time, the contents of the heating chamber were exploded into 3.5 L of hot water. The pretreated slurry was collected and fractionated into solid and liquid phases by centrifuging through a 100 µm nylon mesh. The insoluble residue was washed extensively to remove any water-soluble material. Both fractions were quantified, and samples stored at −40 °C. Portions of this material were freeze-dried for carbohydrate analysis.
Carbohydrate composition of solids
Freeze-dried solids were acid hydrolysed (72 % (w/w) H2SO4, 3 h, RT followed by dilution to 98 g/L H2SO4, and heating for 2.5 h, 100 °C) to convert polymeric sugars into their monomeric constituents [20] . The hydrolysed samples were cooled on ice (>10 min) and then centrifuged (2500 rpm, 2 min). To a 1 mL aliquot from each hydrolysed sample 100 µL ribose internal standard (30 mg/mL) was added. Samples were neutralised with CaCO3 (2.5 mL, 2 mol/L). The precipitated salt was removed by centrifugation (3000 rpm, 10 min). Filter plates (AcroPrep™ 0.2 µm GHP Membrane 96 Well Filter Plates, VWR International Ltd, Lutterworth, UK) were used to filter portions of each sample (1 mL) prior to HPLC by centrifugation at 500 rpm for 10 min. Deep well collection plates were sealed with pierceable lids (Starlab (UK) Ltd, Milton Keynes, UK) and loaded directly onto Series 200 LC instrument (Perkin Elmer, Seer Green, UK) equipped with a refractive index detector and employing an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) with matching guard columns.
Small scale saccharification
Small scale saccharification was carried out in 25 mL universal vials (Sterilin, Newport, Gwent, UK), hydrolysing 1 g DM equivalent of wet pretreated solid, made to 5 % substrate concentration with sodium acetate/acetic acid buffer (8.2 g/L, pH 5.0). The buffer contained 0.01 % (w/v) thiomersal to prevent microbial contamination. Hydrolyses were conducted for 96 h at 50 °C, under continuous agitation in a Thermoshake Incubating Orbital Shaker (Gerhardt, Königswinter, Germany) after adding an appropriate amount of cellulase. The two commercially available enzyme preparations used in this study were Cellic® CTec2 assayed following Ghose [21] and Cellic® HTec2 (Novozymes, Bagsvaerd, Denmark). Hydrolysis reactions were terminated by heating the hydrolysate in sealed tubes (100 °C, 10 min) after which the samples were cooled, centrifuged (2000 rpm, 2 min 25 °C) and the supernatants recovered for analysis.
Small scale simultaneous saccharification and fermentations
Small scale simultaneous saccharification and fermentation (SSF) was conducted in 30 mL wide-necked glass vials containing 1 g (DM equivalent) of wet pretreated solid, made up to 17.9 mL with yeast nitrogen base (Formedium, Hunstanton, UK) at pH 5.0. The bottles were then autoclaved (121 °C, 15 min) to ensure sterility. The bottles were cooled to 25 °C, and 2 mL of yeast grown in Difco YM media (Fisher Scientific UK Ltd, Loughborough, UK), was added along with 100 µL Cellic® CTec2 (Novozymes, Bagsvaerd, Denmark), 20 FPU/g substrate. The yeast inoculum used was a Saccharomyces cerevisiae strain—NCYC 2826—chosen from the National Collection of Yeast Cultures (UK), selected on the basis of its high ethanol tolerance (15–20 % v/v). The inoculum had a viable cell count of 9.87 × 107 cells/mL. Bottles were incubated under continuous agitation (120 h, 25 °C) after which, a measured sample was boiled in gas tight screw cap tubes (Starlab Ltd, Milton Keynes, UK), centrifuged (13,000 rpm, 5 min) and supernatant filtered through AcroPrep™ 0.2 µm GHP Membrane 96 Well Filter Plates (VWR International Ltd, Lutterworth, UK) into a 96 deep well collection plate before analysis.
Five litre pilot scale hydrolyses in high torque bioreactor
Pilot scale hydrolyses (5 L) were conducted in a bespoke high torque bioreactor [22]. Digests were conducted at 20 % (w/v) substrate concentration, using 1 kg DW equivalent of steam exploded rice straw/husk suspended in sodium acetate/acetic acid buffer (4.1 g/L, pH 5.0). The buffer and substrate were initially heated to >85 °C for 10 min to minimise the possibility microbial contamination. The mixture was then cooled to 50 °C and an appropriate amount of Cellic® CTec2 was added—the optimum was either 6.49 or 10 FPU/g DM for straw and husk, respectively. The hydrolysate was agitated at 39 rpm for 4 days, taking 100 mL samples of the digest every 24 h. Each sample was heated in a sealed tube to (100 °C, 10 min), centrifuged (2000 rpm, 2 min) and the monomeric sugar composition determined.
Quantification of hydrolysis and fermentation products
The concentration of reducing sugars released from the substrates was quantified using a scaled dinitrosalicylic acid (DNS) method [23]. Glucose concentrations were quantified using a glucose-specific kit (GOPOD, Megazyme International Ireland, Bray, Ireland). Substrate and enzyme controls were included wherever necessary.
Terminated fermentations, after centrifugation and filtration through a 0.2 μm filter, were loaded directly onto a Series 200 LC instrument (Perkin Elmer, Seer Green, UK) equipped with a refractive index detector. The analyses were carried out using an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) with matching guard columns operating at 65 °C with ultrapure water as mobile phase at a flow rate of 0.6 mL/min as described [22].
Quantification of fermentation inhibitors
Pretreatment-derived supernatants were recentrifuged (2465×g) and 200 μL of the supernatant syringe filtered into vials (0.2 µm, Whatman International Ltd, Maidstone, UK). The concentration of the fermentation inhibitors 2-furfuraldehyde (2-FA), 5-Hydroxymethylfurfural (5-HMF) and the organic acids (formic and acetic acid) were analysed by HPLC using a Flexar LC instrument (PerkinElmer, Seer Green, Bucks., UK) equipped with refractive index and photo diode array detectors (reading at 210 nm wavelength) in series. The analyses were carried out using an Aminex HPX-87H carbohydrate analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) operating at 65 °C with 0.005 mol/L H2SO4 (Sigma-Aldrich) as mobile phase at a flow rate of 0.6 mL/min.
Statistical analysis
Unless otherwise stated, all analyses were performed in triplicate and presented as means and standard deviations. Curve fitting was conducted in Genstat v.18 (VSN International).