Dr6000

All chemical reagents were of analytical grade and used without further purification. Oil palm trunk chips were purchased from a local factory. Phosphoric acid (H₃PO₄, 80%) and MB were purchased from Sigma-Aldrich, (Petaling Jaya, Malaysia).
The surface morphology of oil palm trunk activated carbon (OPTAC) was characterized using a scanning electron microscope (model JSM-5910, JEOL USA, Peabody, MA, USA). A Nicolet iS20 spectrophotometer was used for the identification of chemical functional groups present in OPTAC through FTIR-ATR analysis. Meanwhile, a Bruker D2 Phase X-ray diffractometer with Cu-Kα (λ = 0.154060 Å) radiation source operating at 40 kV and 25 mA was utilized for studying the diffraction patterns of OPTAC. A UV-vis spectrophotometer (HACH DR6000) was used for the determination of MB removal percentage.

The chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) in the wastewater and effluent were analyzed once every 24 h using a spectrophotometer (Hach DR 6000, Düsseldorf, Germany). The content of total solids (TS) was determined gravimetrically. The pH was measured with a pH meter (1000 L, VWR International, Radnor, PA, USA). Biogas flow rate was measured continuously using a digital gas flow meter (Aalborg Instruments & Controls, Inc., Orangeburg, NY, USA). Biogas composition was analyzed once every 7 days using a GMF 430 m (GasData, UK) and a gas chromatograph (GC, 7890A Agilent, Santa Clara, CA, USA) equipped with a thermal conductivity detector (TCD). The GC was fitted with two Hayesep Q columns (80/100 mesh), two molecular sieve columns (60/80 mesh) and a Porapak Q column (80/100) operating at a temperature of 70 °C. The temperature at the injection and detector ports was 150 °C and 250 °C, respectively. Helium and argon were used as the carrier gases at a flow rate of 15 mL/min. Samples for molecular profiling of the bacterial community were extracted from filling elements at the end of each variant. The molecular analysis was performed to determine the percentage of ammonia-oxidizing bacteria (AOB) in the biofilm using the fluorescent in situ hybridization (FISH) technique [20 (link)].

T, DO, pH, ORP, and EC of the water samples were determined using Hydrolab DS5 (Hach Company, USA). According to the standard methods, TN, NO₃⁻-N, NO₂⁻-N, and NH₄⁺-N were measured using DR6000 (Hach Company, USA) [22 ]. The different forms of transferable nitrogen for sediment were evaluated using the sequential extraction method (Figure 1b) and more information is shown in the Supplementary Material [23 ,24 (link)].

The activity was determined for both free and immobilized enzymes by a spectrophotometric method: 5 mg of zeolite/β-glucosidase or 100 µL of the diluted pure enzyme were incubated in 0.1 M acetate buffer (pH 5; 2 mL final volume), and 800 µL of 5 mM p-nitrophenyl-β-D-glucopyranoside (p-NPG) for 10 min at 50 °C. The reaction was stopped with 3 mL of sodium hydroxide (NaOH), and the immobilized enzyme was centrifugated at 3175× g at 4 °C for 10 min, after which aliquots of the supernatants were taken, and their absorbance at 385 nm was measured in a spectrophotometer (DR 6000 of HACH, Loveland, CO, USA). The concentration of the purified protein was determined by the Bradford method using bovine serum albumin as a standard [24 (link)].