Potential for using enriched cultures and thermotolerant bacterial isolates for production of biohydrogen from oil palm sap and microbial community analysis

A limited number of bacteria can convert oil palm (Elaeis guineensis) sap to hydrogen with satisfactory yield and productivity. In this study, a total of 18 fermentative enriched cultures and 36 newly isolated thermotolerant bacterial strains were compared for hydrogen production from oil palm (OP) sap. The new isolates were obtained from hot springs, palm oil mill effluent and oil palm sap. The test was conducted in three steps: (i) a test for hydrogen production from mixed substrates (cellulose, starch, xylose, and glucose) and OP sap; (ii) a test for substrate concentration tolerance; and (iii) a test for thermotolerance. Five enriched candidates for each of the hydrogen producers were selected according to the criteria defined for the screening test. The hydrogen production of these selected bacterial strains from hot springs were cultivated in batch fermentation of oil palm sap at room temperature (30 ± 2 °C). Five enriched cultures, namely 81RN1, OPS, 85RN5, 89SR3-2 and 112YL1 were found to give high cumulative hydrogen formation of 1085, 1009, 994, 983 and 778 mL H2/L-OP sap, respectively, with the hydrogen content of 29.8, 29.4, 28.7, 27.1 and 27.5%, respectively. PCR–DGGE profiling showed that all these five enriched cultures consisted of species closely related to the genus Clostridium sp. based on the 16S rRNA gene. For pure cultures, the top five hydrogen producers were the isolates encoded as PS-3, PS-4, PS-5, PS-7 and PS-8 exhibiting the hydrogen production of 1973, 1774, 1335, 1170 and 1070 mL H2/L-OP sap, respectively, with the hydrogen content of 33.7, 29.6, 32.5, 31.5 and 26.4%, respectively. Identification of these high hydrogen producers using 16S rRNA sequence matching showed that the isolates PS-3 and PS-8 belonged to Clostridium beijerinckii, while the isolate PS-7 belonged to Clostridium acetobutylicum and the isolates PS-4 and PS-5 belonged to Klebsiella sp. and Klebsiella pneumoniae, respectively. Therefore, the pure culture C. beijerinckii PS-3 exhibited 1.8 folds higher hydrogen production (1973 mL H2/L-OP sap) than the enriched cultures of 81RN1 (1085 mL H2/L-OP sap).

► The selected enriched OPS culture can produce 1 L H2/L-oil palm (OP) sap. ► Predominant hydrogen producers were closely related to Clostridium sp. ► Clostridium beijerinckii PS-3 capable of utilizing oil palm sap were isolated. ► The strain PS-3 exhibited 2 folds higher hydrogen production (1.97 L H2/L-OP sap) than OPS culture.