Cellic ctec2

Enzymatic hydrolysis was performed using commercial enzyme preparations (Cellic CTec II, Novozymes, Denmark) at a dosage of 10 FPU per gram of sample (dry weight basis), corresponding to 200 IU of β-glucosidase. The total cellulases and β-glucosidase activity determined using the Celic CTec II extract were 92 FPU mL⁻¹ and 1800 UI mL⁻¹, respectively. Each hydrolysis experiment was conducted in 50 mL Falcon tubes containing 200 mg of milled sample (dry weight basis) and 10 mL of 50 mM sodium-acetate buffer (pH 4.8) in addition to the enzyme solution. The flasks were incubated at 45 °C with rotary agitation at 120 rpm. The reaction was stopped at defined periods from 4 to 72 h by heating the flask to 100 °C for 5 min, followed by centrifugation of the material at 7000×g for 10 min. The soluble fractions were assayed for glucose using HPLC with an HPX87P column (Bio-Rad) at 45 °C using water as an eluent at an elution rate of 0.6 mL min⁻¹. The sugars were detected using a temperature-controlled infrared detector set at 45 °C. The glucan conversion level reported herein refers to the conversion of the polysaccharides to their monomers. Values (mean ± SD) for the hydrolysis of the samples were estimated from triplicate runs.

The aspen hydrolysate was generated after steam explosion pretreatment of aspen wood chips, which was performed in 40 L reactor at 175 °C, with 25% w/w total solids for 10 min. H₂SO₄ was added to a final concentration of 1% w/w. The generated slurry was enzymatically hydrolyzed using an enzyme loading of 0.1 g/of enzyme preparation per g of DM in the slurry. Cellic CTec II (Novozymes, Bagsværd, Denmark) was used as the enzyme cocktail. Hydrolysis was performed at 50 °C, at pH 5, with 8% suspended solids.
Prior to fermentations, the hydrolysate was diluted 2 times and minerals and yeast extract were added to the cultivation medium to match growth conditions of P. ostreatus in previous experiments (Section 2.3). 4 L STR bioreactors (Belach Bioteknik, Skogås, Sweden) with a working volume of 3 L were used for the cultivation. An inoculum of 5% (v/v) of the working volume was added in the bioreactor. Cultivation temperature and pH were adjusted to 28 °C and 5, respectively. The agitation speed varied from 200 to 800 rpm depending on the oxygen demand and viscosity increase in the culture. At specific time intervals, samples were withdrawn for biomass and protein estimation.

Waste wheat straw (WWS) was supplied by the Shandong Quanlin Group (Shandong, China). The large amount (69.2%) of ash contained in the WWS was washed out using tap water at 1:10 (w/v) for five times, and stored at 4 °C [6 (link)]. Commercial cellulase (Cellic® CTec.2.0) was provided from Novozymes North America (Franklinton, NC). Humic acid was purchased from Sigma-Aldrich (Shanghai, China), and metallic salts (KCl, NaCl, CaCl₂, FeCl₂·4H₂O, FeCl₃·6H₂O, AlCl₃) was analytical grade and purchased from Nanjing Reagent Company (Nanjing, China). Bradford reagent and bovine serum protein standard solution were also purchased from Sigma-Aldrich (Shanghai, China).

The branches and twigs of PM were harvested from the planting at the Guangzhou Academy of Forestry. After air-drying for 24 h, the branches and twigs were ground and sieved through a 40-mesh sieve. The resultant PM sawdust was stored at room temperature until processing. The crude endo-β-1–4-xylanase used in this study was produced from Trichoderma reesei and provided by Jiangsu Kangwei Biotechnology Co., Ltd. of China. The measured enzyme activity was 30 U/mL. Commercial cellulase (Cellic® CTec.2.0) was provided by Novozymes North America (Franklinton, NC). Bradford reagent and bovine serum protein standard solution were purchased from Sigma–Aldrich (Shanghai, China). Other chemicals, such as H₂SO₄, MgSO₄, CaCl₂, KH₂PO₄ and (NH₄)₂SO₄, were of analytical grade and were purchased from Nanjing Reagent Company (Nanjing, China).

Commercial cellulase enzyme Cellic CTec 2.0 was purchased from Novozymes China (Beijing, China), with 256 FPU/mL of filter paper activity, 4653.3 CBU/mL of cellobiase activity, and 86.3 mg/mL of protein content according to the method from National Renewable Energy Laboratory (NREL) (Adney and Baker 1996 ), Ghose (Ghose 1987 (link)), and Bradford (Bradford 1976 (link)) respectively. Glucose, xylose, and other reagents were of analytical grade and obtained from Titan Scientific Co. (Shanghai, China). Yeast extract and peptone were purchased from Oxoid (Hampshire, UK).

Corn stover used in this study was purchased from Zhengzhou (Henan, China). It was chopped by a high-speed shredder (HC-3000, Wuyi Haina Electric Appliance Co., Ltd., Jinhua, China) before sieving through 1-mm-sized screen. Then, the samples were washed for three times with water to remove the adhering dirt, stones, and metals, oven-dried at 105 °C until the weight was constant, and stored in a desiccator for long-term storage. The raw corn stover contained 35.9% glucan, 21.7% xylan, 22.1% total lignin, and 2.3% ash (Table 1, Entry 1). The other materials were proteins, non-structural sugars, organic acid, inorganics, crude fat, waxes, coloring material, etc. Specifically, there were 8.7% water extractives and 4.5% ethanol extractives, respectively, in the raw corn stover. Cellic^® CTec2, a commercial enzyme blend containing aggressive cellulases, high level of β-glucosidase, and hemicellulase [47 ], was purchased from Novozymes (Tianjin, China). The total protein concentration of the enzyme blend was determined to be 116.2 mg/mL. The filter paper activity and β-glucosidase activity were 247.1 FPU/mL (filter paper units per milliliter of enzyme solution) and 4167 CBU/mL (cellobiase units per milliliter of enzyme solution), respectively, as assayed by the published procedures [48 , 49 (link)]. The chemical reagents used in this study were of analytical grade and purchased locally.

Poplar used in this work was kindly supplied by Prof. Yong Xu from Nanjing Forestry University. The material was air dried for 3 days, followed by pulverization to obtain a powder with an 80 mesh particle size (≤ 0.178 mm), having a moisture content of 9.40%. The contents of glucan, xylan, acid insoluble lignin and acid soluble lignin in the raw poplar were 43.38%, 17.36%, 24.43%, and 3.50%, respectively, determined using a method published by the National Renewable Energy Laboratory [53 ].
Cellic CTec2 (Novozymes A/S, Bagsværd, Denmark) had an activity of 123.0 filter paper units (FPU)/mL (176.2 mg protein/mL) determined according to the International Union of Pure and Applied Chemistry standard assay [54 ]. The Novozyme 188 (β-glucosidase) was determined to be 8451 nkat/mL (187.9 mg protein/mL) as described previously [55 ]. The enzyme protein was quantified by the Lowry method using bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as the standard [56 (link)]. Xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were purchased from Megazyme (Wicklow, Ireland).