The South Wastewater Treatment Plant of Tehran is the biggest wastewater treatment plant in the Middle East, playing a vital role in collecting and treating wastewater of Tehran’s population. Built-in 8 phases, the plant treats wastewater of 4,200,000 people, living in the central and southern districts of Tehran. There are 6 mesophilic anaerobic digesters having 53,400 m3 capacity in total, producing around 36,000 m3/day biogas. The digesters treat combined primary and waste activated sludge with a ratio of 40%/60%. All sludge samples used in this study were collected from this wastewater plant.
The thickened waste activated sludge was collected from belt thickener in this plant. After collecting, the sludge was immediately transferred to the University’s laboratory and after measurements, it was kept at 4 ℃ and low pH. Characteristics of the waste activated sludge was as follows, which are achieved from triplicate tests with standard error: pH 6.45 ± 0.00, l solid (TS) 40.10 ± 1.56 g/L, volatile solid (VS) 32.00 ± 0.91 g/L, otal suspended solids (TSS) 36.20 ± 1.03 g/L, volatile suspended solids (VSS) 29.40 ± 0.83 g/L, chemical oxygen demand (COD) 49.20 ± 1.12 g/L and SCOD 3.92 ± 0.10 g/L.
The inoculum used in this research for biochemical methane potential tests was harvested from the mesophilic digesters of the plant. Characteristics of the inoculum were as follows, which are obtained from triplicate measurements with standard error: pH 7.58 ± 0.00, TS 31.75 ± 0.93 g/L, VS 24.75 ± 0.86 g/L, TSS 27.75 ± 1.02 g/L, VSS 22.45 ± 0.79 g/L, TCOD 37.30 ± 0.63 g/L and SCOD 3.43 ± 0.15 g/L.
Fenton and FNA methodology
For conducting FNA pre-treatment, first, the pH of waste activated sludge samples was reduced to 5.5 with 1 M HCl solution. Then, 4 M nitrite salt (NaNO2) was added to the mixtures, to provide the designated FNA concentrations in the sludge environment (table.2). In the last stage, the mixtures were shacked with a shaker at 150 rpm for 5 hours. FNA concentration was computed by the equations N-HNO2 = (SN-NO2)/(Ka × 10 pH) and Ka = e–2300/(273+℃), in which ℃ is the temperature of the room (23 ℃ in this experiment), Ka is a constant which is dependent on the temperature and SN-NO2 is the nitrite salt concentration .
For conducting Fenton reaction, first, waste activated sludge samples were put into 1 L bottles, then their pH was decreased to 3 with H2SO4 solution (99%). In the next stage, iron salt (FeSO4) was added to the mixture, to produce the designated Fe+2 in the sludge environment (table.2). Then, the designated hydrogen peroxide concentrations were added to the reaction. The ratio of H2/Fe+2 was set at 1/0.067 according to the literature [20, 23–25]. The mixtures were finally shacked with a shaker at 150 rpm for 1 hour at ambient temperature, so that the Fenton reaction would be approximately terminated [20, 24].
For combined FNA and Fenton pre-treatments, first, FNA pre-treatment was applied to waste activated sludge at 5 hours exposure time (pH = 5.5), then Fenton pre-treatment was applied at 1 hour (pH = 3). The combined conditions are shown in Table2.
Biochemical methane potential tests
In order to measure the volume of methane production from waste activated sludge, 27 batch reactors in addition to blank were carried out in 1000 mL glass bottles with a working volume of 500 mL (Additional file 1). The schematic of the reactor is shown in Figure 4. The ratio of inoculum and substrate (I/S) was adjusted to 2, based upon dry volatile solid . The pH of treated waste activated sludge samples was adjusted to 7 and their temperature was increased to 37 ℃ prior to mixing with the inoculum, so as to prevent any temperature and pH shock to the inoculum. After mixing, the bottles were flushed with N2 gas for 1 minute (1 L/min), then they were put into the water bath, whose temperature was controlled at 36 ± 1 ℃ by automatic heaters. The bottles were permanently stirred by magnetic stirrers at 100 rpm, to provide adequate mixing and uniform temperature distribution. All tests were carried out in triplicate. The digestion process lasted for 44 days when biogas generation was approximately terminated.
Routine experiments on sludge quality such as TS, TSS, VS, VSS, COD and SCOD were measured according to standard methods for the examination of water and wastewater. For measuring soluble proteins before and after the treatments, Folin Phenol reagent was used according to Lowry’s method . Soluble polysaccharides were also measured with phenol and sulfuric acid, according to Dubois’s method . In order to separate soluble matter from suspended solids, 10 minutes centrifuge at 15000 rpm was first implemented, then the solution was passed through 0.45 μm pore size glass fibre filter, using Buchner funnel, and vacuum equipment.
The biogas volume was measured according to the liquid displacement method with an acidified liquid barrier (pH = 2) that was saturated with NaCl for minimizing the solubility of biogas . Methane production was measured by gas chromatography (GC), using a Thermal Conductivity Detector (TCD). The temperature of the column and TCD was set respectively at 75 ℃ and 104 ℃. In each measurement, 0.05 cc sample was injected to the equipment, and 1 minute exposure time was considered for each measurement. In this study, biogas and methane production were measured once daily and once every fifth day, respectively during the digestion process.
Key hydrolytic enzymes (Protease and cellulose) activity was measured according to well agar diffusion method in our previous study [37–39]. For carrying out the enzymatic tests, methanogenic organisms should be first eliminated so as to decline potential errors . For eliminating methanogens from the samples, heat treatment at 102 ℃ for 30 minutes and BESA (2-bromoethanesulfonic acid) were applied to the BMP reactors [41, 42]. The samples then were maintained at 37 ℃ for 72 hours before being assessed for enzymatic activity.