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The sufficient amounts of macro-nutrients and buffer are playing a vital role on microbial
growth and to resist the pH in the anaerobic digester change. There are several components of
macro-nutrients such as sulphur, phosphorus, potassium, calcium, magnesium, and iron are
required for specific proteins and cofactor for enzyme activity (Batstone et al., 2002). However,
among them in this research was focused on phosphorus from Na 2HPO 4.12H 2O and potassium
obtained from the empty fruit brunch (EFB) ash as phosphorus and potassium sources. A study
from O-Thong et al. (2008) found that the optimal C/P ratio for hydrogen production by using
single substrate fermentation of POME was 559 with high hydrogen production yield of
6.33±0.14 L H 2/L-POME. Meanwhile, the reason for using of EFB ash as potassium source due
to it relatively high potassium content was 139.35 mg/kg (Udoetok, 2012) which could utilize for
potassium source and as well as to utilize of solid organic wastes generated in palm oil mill.
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Bicarbonate (HCO 3 ) is often the main buffer in anaerobic digesters to resist the pH in the
anaerobic digester change. However, digesters present at high concentration of bicarbonate
supplemented in substrate, resulting low in hydrogen and methane with both quantity and quality
in biogas generated as CO 2 was released from buffer supplemented.

This study was conducted with the aims to investigate the optimum mixing ratio of SLS
to POME, to investigate the optimum concentration of buffer and nutrients concentrations
including NaHCO 3, Na 2HPO 4.12H 2O and empty fruit brunch (EFB) ash concentrations,
respectively for biohydrogen production in the first phase, and to investigate the potential of the
sequential methane production in the second phase under thermophilic temperature (55°C).

3.3 Materials and methods

3.3.1 Anaerobic seed sludge

The anaerobic seed sludge originating employed in this research was collected from
mesophilic wastewater treatment pond of Palm Pattana Southern Border Co, Ltd., Pattani
Province; Southern Thailand was used as inoculum for hydrogen and methane production.
Hydrogen producing seed used in this work was obtained from a lab-scale continuously stirred
tank reactor (CSTR) that generated biohydrogen from co-digestion of SLS and POME. The H 2-
CSTR reactor was operated by using the mixture consisting of SLS and POME at the volumetric
mixing ratio of 1:1 under thermophilic temperature. At the same time, the sludge used for
produced methane was enriched for methanogens by using basic anaerobic (BA) medium
supplemented with sucrose 3 g/L for a week in batch reactor. Afterwards, the enriched sludge
was used as inoculum in batch tested to determining its ability to generate hydrogen and methane
from co-digestion of SLS and POME.