2.4 Analytical methods:
The sludge samples were collected periodically from the two-stage anaerobic digesters and subjected to basic characterization such as pH, temperature and TDS/ conductivity immediately after collection. The characterization was done using WP-90 conductivity/ TDS-pH/ temperature meter. Specific intracellular superoxide radicals [\(O_{2}^{-}]\) are short living reactive intermediates, so its measurement was done immediately after collection of the sample. [\(O_{2}^{-}]\) characterization from sludge samples was done using dihydroethidium (DHE) dye 23. Briefly, 1ml of the sample was collected and centrifuged at 5000 rpm for 10 minutes at 4 °C to isolate the pellet. The pellet was washed and suspended in 1ml of phosphate buffer (pH 7.4). DHE was added to a final concentration of 10µM and incubated in dark for 30 minutes at 37 °C. The fluorescent dye DHE is freely permeable across cell membranes and reacts with the intracellular superoxide radicals and produce a fluorescent product that can be measured in a fluorescent spectrometer (excitation: 485/20, emission: 590/35). Standard plot for superoxide concentration was plot using Xanthine/Xanthine Oxidase system 23. Total and soluble chemical oxygen demand (tCOD and sCOD) measurements were done using HACH COD reagent, followed by colorimetric analysis in ORION UV- spectrometer. The soluble fraction of sludge was acquired by centrifugation of samples at 5000 rpm for 10 minutes at 4 °C, followed by filtration of supernatant using a 0.45 µm Whatman filter paper. Total solids and volatile solids (TS and VS) were measured using standard APHA protocol 25. Volatile fatty acids (VFAs) and total alkalinity were measured using direct titration technique26. Sludge bioactivity was measured using TTC-DHA (Triphenyl tetrazolium chloride- dehydrogenase) activity. TTC-DHA activity involves the reduction of triphenyl tetrazolium chloride (TTC) to formazan (TF) by free electrons, that are produced as a result of oxidation reactions catalyzed by dehydrogenase enzymes27. The TTC-DHA activity was measured using the redox-sensitive tetrazolium dye, according to TTC- DHA assay28. The red formazan salt produced during the assay was deducted colorimetrically at 485 nm. Protein and carbohydrate characterization were done using Bicinchoninic Acid method29 and phenol-sulfuric acid method30 respectively, in 96-well microtiter plates. The biogas produced was measured using a GA 2000 gas analyzer.