Biogas composition was analyzed using a gas chromatograph
(Varian CP 3800 GC) with a thermal conductivity detector, provided
by a column CP-Molvsieve5A (15 m 0.53 mm 15 lm)
followed by a column CP-Porabond Q (25 m 0.53 mm 10 lm).
Hydrogen (13.6 mL min1) was used as the carrier gas. The injection
port temperature was set at 150 C and the detector temperature
was 175 C. Total volatile fatty acids (TVFA) were analyzed
using a gas chromatograph (Varian CP 3800 GC) equipped with a
Nukol capillary column (30 m 0.25 mm 0.25 lm) and a flame
ionization detector. The carrier gas was helium and the temperature
of the injector was 250 C. The temperature of the oven was
set at 150 C for 3 min and thereafter increased to 180 C.
Three samples were collected for SEM analysis. Mesophilic AS
sample and sludge samples obtained from R1 and R2 at the end of
the experiment. Microbial cells in the sludge samples were fixed
using 2% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2–7.4
for 90 min. Then, samples were washed three times for 20 min
with phosphate buffered saline (PBS) at 4 C and were dehydrated
in ethanol graded series (30, 70, 90, 100% v/v). Dehydrated samples
were dried and sputter-coated with gold in high vacuum
(0.05–0.07 mbar) with a coater Blazers SCD 004. The samples
were examined using a JOEL JSM 6840 LV scanning electron
microscope.