MERİÇ BATIOĞLU, PAUL J SALLİS, EMİNE UBAY ÇOKGÖR, CANDAN TAMERLER BEHAR, ALPER T AKARSUBAŞI
Advances in Molecular Medicine - 2007;3(1):23-33
Fluorescence in situ hybridisation with rRNA-targeted nucleic acid probes can be used to directly identify microorganisms within complex samples in a few hours and therefore has widespread application in environmental and medical microbiology (Wagner et al., 2003). In this study, the acetoclastic methanogens present in the anaerobic sludge samples taken from a conventional single stage anaerobic digester at Hexham Sewage Treatment Works (United Kingdom) and from a lab-scale anaerobic membrane bioreactor treating brewery wastewater seeded with Hexham sewage sludge were detected and quantified by using quantitative fluorescent in situ hybridisation. Each sample was hybridised with fluorescent oligonucleotide probes for Bacteria, Archaea and the defined group of methanogens namely, acetoclastic methanogens. Specific cell counts were determined by a combination of in situ hybridization and confocal laser scanning microscopy for statistical analysis. The results of quantitative fluorescent in situ hybridisation (FISH) procedure indicated that the concentration values of Methanosaeta and Methanosarcina are very close to each other for the sample taken from full-scale anaerobic digester. Besides, Methanosaeta concentration is much higher than Methanosarcina concentration in the sample of lab-scale anaerobic membrane bioreactor. In addition, it has been determined that the archaeal cell concentrations are higher than the eubacterial cell concentrations for both of the samples. Once detected and quantified the populations of Bacteria, Archaea and particularly acetoclastic methanogens within the anaerobic digesters; statistical approach was applied to determine the variances within and between the samples. The outcomes of analytical process showed that the variances of archaeal cells are homogenous for three dual hybridisations carried out for each sample in this study. Furthermore, the distribution types were determined as poisson distribution for Archaea, Methanosaeta and Eubacteria and as negative binominal distribution for Methanosarcina, respectively. The statistical differences between the samples of the full-scale digester and the lab-scale digester seeded with the full-scale sludge were determined by means of i) plotting frequency distribution curves, ii) checking the normality with Anderson-Darling tests, iii) transforming the cell concentration for normal distribution according to the Box-Cox plots and iv) using one-way ANOVA (analysis of variance). The results of quantitative FISH and statistical analysis showed significant differences between the full-scale and lab-scale sludge samples. The combination of FISH with confocal laser scanning microscopy and statistical approach appears to be an appropriate method for quantification of acetoclastic methanogens present in anaerobic digesters.
