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.