Quantitative analysis and mapping of micro-organisms in anaerobic digester granules using a combination of transmission electron microscopy with immunotechnology

The precise localization of microbes within the matrix of digester granules has not previously been fully elucidated. To address this problem, a dual approach was applied for the first time, combining a method that identifies microbes with another that defines the structure in which the microbes are located. Bacterial and archaea morphotypes within granules from an upflow anaerobic sludge blanket digester purifying brewery waste water were quantified and localized in situ from TEM micrographs. Cell numbers were translated to biomass using image analysis techniques. Cells in granule homogenates were counted using a haemocytometer while methanogen and acidogen strains were enumerated using antibody probes. Granules showed stratification ; an outer cortex consisting primarily of cells, and an inner medulla virtually devoid of cells. Methanothrix- and Methanobrevibacter- like cells (26·2% and 37% of the population, or 46·3% and 27·1% of the biomass, respectively) concentrated in two bands within the granule whereas Syntrophobacter- like cells (9·6% of the population or 19·0% of the biomass) were evenly distributed throughout the cortex. Haemocytometer counts indicated 6·95×10⁶ cells ml^?1 which was lower than values obtained from TEM quantification because of cell losses during granule homogenization. Only 39% of the cells counted using the haemocytometer, or from TEMs, were identified using antibody probes, suggesting that combined methodologies provide more accurate cell quantification within granules or similar uniform attached microbial associations. Moreover, errors arising from specific techniques can be detected and quantified.