Physicochemical characteristics of the sludge

Sludge suspensions were sampled daily from the reactors. The mixed liquor suspended solids (MLSS), sludge volume index (SVI) and effluent suspended solids (ESS) of a sludge sample were measured in accordance with the standard methods 2710 D⁴⁰. The MLSS was obtained by filtering the mixed liquor through a glass fiber filter and measuring the mass of the filter residue after drying at 105 °C for 24 h⁴⁷. Also, 100 mL of sludge suspension were transferred to 100 mL cylinders and settled for 30 min, and the volume of precipitated sludge was recorded for calculating SVI. The SVI value specifies the sludge settle ability and compressibility¹⁷. The supernatant fraction was collected from top of these cylinders as the effluent, and their effluent suspended solids (ESS) were measured. The total amount of ESS in effluents was determined by filtering the effluents through a glass fiber filter and measuring the mass of the filter residue after drying at 105 °C for 24 h⁴⁷. The ESS indicates the performance of the microbial flocculation and effluent clarification²⁷.

The micrographs of sludge flocs were taken using a scanning electron microscope (SEM) (Hitachi S-570, Japan). Sludge samples were taken out from the reactors and were fixed in phosphate buffer (pH 7.0) with 2.5% glutaraldehyde for 12 h at 4 °C. The fixed samples were then washed with buffer three times (10 min for each washing series). Then, samples were dehydrated gradually by successive immersions in ethanol solutions of increasing concentrations (50%, 70%, 80%, 90% and three rounds of 100%)⁴⁸. The dried samples were coated with gold prior to SEM analysis with an Emitech K550 sputter coater. All images were acquired digitally using Quartz PCI software (Vancouver, BC, Canada). The element components (e.g., C, O, Na, Mg, Al, S, Si, P, K, Ca and Fe) of sludge were detected using an energy dispersive X-ray (EDX) spectroscopy (JEOL JSM 5900 LV) coupled with the SEM²⁶.

The specific surface area of sludge and fly ash were measured via nitrogen adsorption/desorption isotherms. In this method, fly ash was pretreated at 105 °C overnight for contamination removal prior to analysis. The sludge samples were taken out from reactors at the 60^th and 80^th days directly, and then ground into powders after freeze-drying treatment.The Brunauer-Emmett-Teller (BET) surface area of the fly ash and sludge samples was measured using a NOVA-2200e Autosorb according to a previously established method⁴⁹.