ANALYSIS OF 16S rDNA PRIMER SETS FOR MOLECULAR TAXONOMY STUDY OF MIXED CULTURE THAT PRODUCE BIOHYDROGEN.

Molecular taxonomy study of mixed culture especially in a biohydrogen production system has become important as a basic study in understanding bacterial communities that contributed to biohydrogen production. One of the keystone methods in the molecular analysis of bacterial communities is the amplification of 16S rDNA gene via polymerase chain reaction (PCR). Various PCR primer sets for gene amplification have been applied for bacterial taxonomy analysis from different samples. The problem of being too general and unspecific was encountered when identifying biohydrogen producer bacteria from mixed culture samples using such universal primer set. In this study, we evaluated the performance of five primer sets from in-silico analysis using the SILVA SSU r132 RefNR database and PCR-DGGE for the identification of biohydrogen producing bacteria in mixed cultures from biohydrogen reactors determined. Primer set 357f/518r was the best primer set to be used in microbial community analysis of the biohydrogen producing bacteria. The primer set 357f/518r was related to hypervariable 3 (V3) region involving most bacterial genera, produced the highest number of biohydrogen producer bacteria sequence and with highest percentage of specificity (84.6%). This was followed by 357f/907r and 968f/1392r primer sets with 67.3% and 49.5% specificity, respectively. The PCR-DGGE analysis showed the presence of specific genus of biohydrogen producer bacteria such as Thermoanaerobacterium sp. and Clostridium sp. This study proposed an alternative approach in selection of primer sets during PCR analysis of mixed culture communities from biohydrogen cultivation systems. Thus, such method could also be applied for analysis of mixed culture from other environmental samples. [ABSTRACT FROM AUTHOR]