IDENTIFYING ACETICLASTIC AND HYDROGENOTROPHIC METHANOGENS IN PSYCHROPHILIC AND MESOPHILIC GRANULAR SLUDGES TREATING SYNTHETIC SEWAGE BY MEANS OF FISH AND CSLM

Aceticlastic and hydrogenotrophic methanogens were comparatively investigated by 16S rRNA-based fluorescence in situ hybridization (FISH) technique combined with Confocal Scanning Laser Microscopy (CSLM) to evaluate temperature influence on two functional groups of methanogens. Microorganisms were grown in the form of anaerobic granular sludge in two lab-scale Upflow Anaerobic Sludge Bed (UASB) reactors fed with synthetic wastewater and operated at psychrophilic and mesophilic temperature conditions. Since hydrogenotrophic and aceticlastic methanogenesis are known to play a crucial role, digestion may be effectively blocked if these processes are ceased due to unfavorable environmental temperature conditions. Mesophilic (35 ± 2C) and psychrophilic (10 ± 1C) reactors were inoculated by the same mesophilic granules taken from a full-scale UASB reactor. According to FISH results, Archaea representing methanogens were found dominating at bottom sampling ports of reactors and dominant member of Archaea was the genus aceticlastic Methanosaeta (MX825). Other aceticlastic methanogen such as Methanosarcina-like (SARCI645) species were also identified which were higher in psychrophilic granules. Members of order Methanobacteriales (MB310) constituted the major hydrogenothrophic methanogens in both reactors, whereas other hydrogenothrophic methanogens members of order Methanococcales (MC1109) and Methanogenium (MG1200) relatives were absent. Abundance of originally mesophilic Methanosaeta-related Archaea under low temperature conditions demonstrated the adaptive capacity of microorganisms to psychrophilic conditions. This might be attributed to enzymatic alterations in Methanosaeta cells originating from seed sludge, which were exposed to sub-mesophilic temperatures at start-up and then to psychrophilic conditions during gradual temperature decreases. Both sludge granules remained undisturbed and kept their rigidity even after exposure to very low temperatures. Quantification study was done using Image Pro-plus Version 4.0. Archaeal cells detected with probe ARC915 were found around 60% of the 4,6-diamidino-2-phenylindole stained cells at both reactors. Methanosaeta spp./Archaea ratios for mesophilic and psychrophilic UASB reactors were calculated as 71% and 84%, respectively. FISH and CSLM results together with quantification study showed that the microbial community of anaerobic granular sludges was dominated by Methanosaeta spp. which was identified as the major m