Yoichi Kamagata

Quantitative detection of culturable methanogenic archaea abundance in anaerobic treatment systems using the sequence-specific rRNA cleavage method.

A method based on sequence-specific cleavage of rRNA with ribonuclease H was used to detect almost all known cultivable methanogens in anaerobic biological treatment systems. To do so, a total of 40 scissor probes in different phylogeny specificities were designed or modified from previous studies, optimized for their specificities under digestion conditions with 32 methanogenic reference strains, and then applied to detect methanogens in sludge samples taken from 6 different anaerobic treatment processes. Among these processes, known aceticlastic and hydrogenotrophic groups of methanogens from the families Methanosarcinaceae, Methanosaetaceae, Methanobacteriaceae, Methanothermaceae and Methanocaldococcaceae could be successfully detected and identified down to the genus level. Within the aceticlastic methanogens, the abundances of mesophilic Methanosaeta accounted for 5.7–48.5% of the total archaeal populations in mesophilic anaerobic processes, and those of Methanosarcina represented 41.7% of the total archaeal populations in thermophilic processes. For hydrogenotrophic methanogens, members of the Methanomicrobiales, Methanobrevibacter and Methanobacterium were detected in mesophilic processes (1.2–17.2%), whereas those of Methanothermobacter, Methanothermaceae and Methanocaldococcaceae were detected in thermophilic process (2.0–4.8%). Overall results suggested that those hierarchical scissor probes developed could be effective for rapid and possibly on-site monitoring of targeted methanogens in different microbial environments.

Isolation of a Propionate-Using, Sulfate-Reducing Bacterium

Propionate is one of the most important intermediates in the anaerobic digestion process because propionate accumulation may inhibit methanogenesis, reducing the efficiency of anaerobic treatment. Subsequently, propionate degrading bacteria have received increasing attention because of their important role in maintaining digester efficiency. This report describes the isolation and characterization of a propionate-using, sulfate-reducing bacterium, named strain MUD.

Phylogenetic diversity of Archaea from terrestrial hot springs of Greece

Terrestrial hot springs are the most easily accessible sites for the investigation of subsurface microorganisms. The discovery of such organisms is significantly facilitated by the investigation of the molecular diversity of their natural habitats. To this direction, the present work presents the results on archaeal diversity investigation of four terrestrial hot springs in Greece with temperatures between 35 and 92°C. Water samples of three to five liters were collected in 2005 from the thermal springs of Lagadas (35°C), Eleftheres (41°C), Edipsos (82°C) and Polihnitos (92°C), in sterile plastic containers and filtered through polycarbonate filters of 0.2 μm pore size in less than eight hours after sampling. The filters were stored at -80°C until further analysis. DNA was extracted by the UltraClean Soil DNA kit (MoBio Laboratories, USA) and was used as template for the PCR amplification archaeal 16S rRNA genes by using a mixture of Archaea-specific forward primers (Grosskopf et al 1998, Y. Sekiguchi unpubl.) and universal 1492r (Lane 1991) using the minimum number of cycles which gave a visible band, in order to avoid some of the PCRx92s innate limitations. PCR products were purified using the Microcon kit (Millipore, USA) and cloned in the pT7 vector (EMD Biosciences, USA). After checking for true positive clones, the inserts of 174 clones were sequenced for approximately 500 bp using the 907r primer. Sequnces with !97% similarity were grouped as identical. To our knowledge these are the first data regarding prokaryotic diversity in Greek thermal springs, despite the countryx92s extensive and diverse geothermal fields (Lambrakis & Kallergis 2005). Polihnitos, one of the hottest terrestrial springs in Europe, showed the lowest archaeal diversity with only three different phylotypes but just one of them was dominating (89%) the system. Its closest relative (95% similarity) was an uncultured representative within Crenarchaeota retrieved from a Juan de Fuca Ridge sulfide black smoker chimney. Edipsos, although just 10°C cooler than Polihnitos, harboured a more diverse archaeal diversity (17 phylotypes) with the most frequently occurring phylotypes related to estuarine uncultured crenarchaeote and uncultured marine Archaea from methane bearing environments. Other phylotypes were related with low similarity, to sequences retrieved from gold mines, terrestrial springs and deep-sea hydrothermal vents. Eleftheres had even more diverse archaeal community with 18 different phylotypes. Most of these were related to other Crenarchaeota sequences from deep subsurface environments like gold mines, radioactive thermal springs and marine mud volcanoes, but with low similarities. Lagadas harboured only seven different phylotypes. Two of them dominated (81%) the system and were related with low similarities to sequences from an Austrian radioactive thermal spring and estuarine sediments. This study reveals the occurrence of exclusive uncultured Archaea from all four springs studied and shows that Polihnitos is a suitable habitat for isolation efforts of uncultured crenarchaeotes as it is probably inhabited solely by one such organism. Our results suggest that temperature is not the primer factor controlling archaeal diversity in terrestrial springs but possibly other parameters could play an important role in determining the community structure. Finally, although most of the retrieved sequences seem to belong to indigenous microorganisms from deep subsurface environments, the resemblance of some of our sequences with ones from coastal environments could be attributed to subsurface transport and/or contamination from surrounding habitats of the water flow. References Grosskopf R, Janssen PH, Liesack W (1998) Appl Environ Microbiol 64:960-969 Lambrakis N, Kallergis G (2005) Hydrogeol. J. 13:506-521 Lane DJ (1991) Willey-Interscience, New York, p 133, ISBN: 0471929069

Isolation and Characterization of an Anaerobic Bacterium Degrading 4—Chlorobutyrate

An anaerobic rod-shaped bacterium (strain K—1) was isolated from mesophilic digester sludge acclimatized with 4—chlorobutyrate as main energy and carbon source. This bacterium was mesophilic, Gram-negative and motile, and degraded 4—chlorobutyrate to butyrate, acetate and hydrogen. Cell morphology varied from singles to pairs, and occasionally long chains. When growth peaked at a pH exceeding 6.0, cells immediately lysed, but not less than pH 6.0. The optimal conditions for growth were an initial pH 6.5–6.8 and 37°C. Growth required yeast extract or clarified rumen fluid, but neither peptone nor clarified sludge fluid could be replaced. Fructose was utilized as a carbon source, but not other sugars.