## 2 Materials and Methods
### Soil Sample Collection and Raw Material Preparation
#### 2.1.2 Oil Palm Empty Fruit Bunches Preconditioning  
The oil palm empty fruit bunches (OPEFB) used in this work was provided by Sime Darby East Oil Mills, Carey Island, Selangor, Malaysia. The fresh OPEFB sample was collected immediately after it was discarded from Sime Darby East Oil Mill's shredding units. At the laboratory, OPEFB was washed with distilled water to eliminate the unwanted elements, and thereafter dried under the sun for a day, and then in a convection oven (Protech AB, Sweden) at 60 \({}^{\circ}\)C for 12 h to remove excess moisture. The moisture content of the samples was kept constant at 12-13%. The sample was further pulverized through a cutting mill (FRITSCH GmbH, Germany) with a 10 mm screen mesh.  
Dilute acetic acid (DAA) pretreatment on the pulverized OPEFB was performed at Sime Darby Plantation Sdn. Bhd. pretreatment facility. This pretreatment process was carried out by Sime Darby in collaboration with Mitsui Engineering and Shipbuilding Co. Ltd. (Japan). In this study, the DAA pretreatment was chosen for treating OPEFB since the molasses generated from the facility was rich in dissolved sugars. This facilitates acid catalysed hydrolysis, hence leading to greater hemicellulose removal with low sugar decomposition products (furfurlands and 5-hydroxylmethylfurfural). Moreover, the presence of acetic acid in the fermentation media is also theoretically favorable as this leads to higher biohydrogen evolution based on the metabolic pathway of biohydrogen-producing bacteria. Accordingly, raw OPEFB was soaked in 0.3% (\(w/v\)) acetic acid and loaded into the pretreatment vessel. The vessel was heated until 250 \({}^{\circ}\)C and maintained for 60 min. After the pretreatment, the slurry was pressed to separate the liquid molasses (labelled as DAA molasses). The pretreated DAA OPEFB was rinsed with distilled water and stored at \(-20\) \({}^{\circ}\)C until further use.  
Ammonia fiber expansion (AFEX) pretreatment of the pulverized OPEFB was conducted at the Biomass Conversion Research Laboratory of Michigan State University, USA using a method previously described elsewhere [17]. Samples containing 50% moisture (g moisture/g biomass) were charged into a 2 L bench-top high pressure Parr reactor (PARR Instrument, USA). The reactor was gradually loaded with liquid ammonia in a 1:1 (g ammonia: g biomass) ratio, and the reactor temperature was raised to 135 \({}^{\circ}\)C (\(\sim\)40 bar) for 45 min. The ammonia was allowed to evaporate after the pressure was released.  
Enzymatic hydrolysis of the AFEX-pretreated OPEFB was performed at 50 \({}^{\circ}\)C, and pH 4.8, and shaken at 150 rpm. The glucan loading was set at 1% (\(w/v\)) in 30 mL tubular scintillation vials (Wheaton, IL, USA) with a working volume of 15 mL containing citrate buffer (0.05 M). Then, 17.7 \(\upmu\)L of enzymes Cellic CTc2(r) (activity = 85.4 g protein/L) and 15.4 \(\upmu\)L of Cellic HTC2(r) (activity = 65.2 g protein/L) (Novozymes, Denmark) were loaded to achieve 16.7 mg protein/g glucan. At the end of 48 h of enzymatic hydrolysis, the enzymatic reactions were halted by boiling denaturation within 10 min. The sugars thus produced were measured using high-performance liquid chromatography (HPLC) using established conditions, as described by [PERSON] et al. [18]. While DAA OPEFB molasses is rich in soluble sugars, most of the sugars are made of pentose (xylose). By contrast, AFEX OPEFB's enzymatic hydrolysate is relatively inhibitor-free and rich in hexose (glucose). The utilization of pentose-rich DAA OPEFB molasses and hexose-rich AFEX OPEFB hydrolysate as the fermentation substrate would create an attractive option for the development of biohydrogen commercialization.