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4.3.5 Analytical methods

The volume of biogas produced was recorded by water displacement gas meter. The
hydrogen content was measured by gas chromatography (Shimadzu GC 14A equipped with
thermal conductivity detector, TCD) fitted with a 1.5 m stainless steel column paced with
molecular sieve 58 (80/100 mesh). Argon was used as a carrier gas at a flow rate of 15 mL/min.
The temperature of the injection port, oven and detector were 100, 50, and 100°C, respectively.
0.5 mL of sampling gas was injected in triplicate (O-Thong et al., 2008; Akutsu et al., 2009).

Volatile fatty acids (VFAs) (i.e. acetic, propionic, butyric acids) and alcohol (ethanol)
were analyzed by a gas chromatography (Shimadzu, GC 8A) equipped with a flame ionization
detector (FID). A column capillary packed with nitroterephthalic acid-modified
polyethleneglycol (DB-FFAP) and with a length of 30 m was used. The chromatography was
performed using the following program: 100°C for 5 min, 100-250°C with a ramping of
10°C/min, 250°C for 12 min. The detector temperature was set at 300°C (O-Thong et al., 2008;
Prasertsan et al., 2009). Lactic acid (HLa) was analyzed by a high performance liquid
chromatography (HP1100, Hewlett-Packard GMGH) with the following operating conditions;
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Pinnacle II C18 Columns, ultraviolet (UV) detector at 210 nm, 2.5 mM of H 2SO 4 was used as a
mobile phase with a flow rate of 0.8 mL/min, and an oven temperature of 45°C (Prasertsan et al.,
2009). The liquor samples were first centrifuged at 10,000 rpm for 10 min, and were then filtered
through 0.45 µm nylon membrane. Calculation of chemical oxygen demand (COD) balance was
followed the method described by Sittijunda and Reungsang (2012).

Chemical oxygen demand (COD), pH, total solid content (TSC), Volatile solid content
(VSC), alkalinity, total Kjeldahl nitrogen (TKN), protein content, total organic nitrogen (TON),
carbohydrate content, sulfate content, oil and grease and soluble phosphorus were determined in
accordance with the procedures described in the standard methods (APHA, 1999).

4.4 Results and discussion

4.4.1 H 2-CSTR experiments

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The H 2-CSTR reactor was operated at the HRTs of 2.25 days (from 1 to 19 day of
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operating time) and 4.50 days with, (from 54 to 68 day of operating time) and without dilution
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(from 20 to 53 day and 69 to 95 day of operating time, respectively), corresponding to the
organic loading rates (OLRs) of 20 g-VS/L reactor d, 10 g-VS/L reactor d, and 5 g-VS/L reactor d,