Isolde Röske

Molecular characterization of the microbial community structure in two activated sludge systems for the advanced treatment of domestic effluents

Although activated sludge systems with enhanced biological phosphorus removal (EBPR) represent state-of-the-art technology for phosphate removal from wastewater it is still unknown which species of bacteria are responsible for the EBPR process. The aim of this study was to compare the bacterial composition of activated sludge from two laboratory plants with different modes of operation, anoxic/oxic- (EBPR, no nitrification) and Phoredox-system (EBPR, nitrification and denitrification) with particular emphasis on microorganisms responsible for EBPR process. In addition to fluorescence in situ hybridization (FISH), we applied further rRNA-based molecular techniques like terminal restriction-fragment length polymorphism analysis and comparative 16S rDNA analysis to yield additional information and to verify the results from FISH analysis, like e.g. for the identification of polyphosphate accumulating organisms (PAO). Despite the different modes of operation only minor differences in the bacterial composition were detected by FISH analysis based on the probes used in this study. In contrast T-RFLP analysis yielded characteristic community fingerprints for each of the investigated plants and comparative 16S rDNA analysis indicated highly diverse microbial communities in both plants suggesting substantial differences in the microbial structure. The results obtained by FISH analysis with specific probes for PAOs support the presumption that not only one specific organism is responsible for the EBPR. In our case Tetrasphaera spp. dominated the PAO community, but other possible PAOs, like Microlunatus spp. and members of the Rhodocyclus group, were also detected.

Microbial diversity and functional characterization of sediments from reservoirs of different trophic state

Sediment samples from four reservoirs of different trophic state were compared with regard to chemical gradients in the pore water, composition of microbial communities and extracellular enzyme activities. The trophic state was clearly reflected by steep vertical concentration gradients of ammonium and alkalinity in the pore water. A high concentration of these parameters indicated a high microbial in situ activity in the more eutrophic reservoirs. However, the total number of bacteria in sediments seemed hardly to be influenced by the trophic conditions in the water column. Differences in the microbial composition of the sediments became evident by comparative 16S rDNA analysis of extracted DNA and by fluorescence in situ hybridization. Although a high proportion of the cells detectable with the EUB probe could not be identified at the subdomain level, members of the beta-Proteobacteria constituted an important fraction in the sediments of the more eutrophic reservoirs, whereas gamma-subgroup Proteobacteria were most frequently detected in sediment samples from the dystrophic Muldenberg reservoir. The assessment of extracellular enzyme activities (esterases, phosphatases, glucosidases and aminopeptidases, respectively) in sediment samples of the four reservoirs revealed specific patterns of metabolic potentials in accordance with the trophic state and characteristics of the catchment.

A simple method to distinguish between polyphosphate and other phosphate fractions of activated sludge

Abstract A procedure of sequential extraction steps proposed by Psenner et al. ( Arch. Hydrobiol. Suppl. 70 ,111–155, 1984; Arch. Hydrobiol. Beih. Ergebn. Limnol. 30 ,98–103, 1988) to quantify ecologically relevant phosphate fractions in lake sediments has been applied to activated sludge from waste treatment plants for biological phosphate removal. By this method, the following phosphate fractions of technological significance may be distinguished: (a) redox-sensitive phosphate, mainly bound to Fe(OH) 3 ; (b) phosphate adsorbed to surfaces (Al 2 O 3 ), exchangeable against OH − , and alkali-soluble phosphate; (c) phosphate bound to CaCO 3 , MgCO 3 and in apatite; and (d) a fraction which hitherto has been designated as “organically bound phosphate”. By means of nuclear magnetic resonance spectroscopy and by inclusion of a hot water extraction step it became evident that fraction (d) mainly contained polyphosphate (PP). In activated sludge samples with phosphate-accumulating bacteria, the PP content amounted to 80 mg/g MLVSS. By combination of the Psenner fractionation and hot water extraction, indications of a significant proportion of PP in lake sediments have also been found.

Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4′,6′-Diamidino-2-Phenylindole) and Tetracycline Labeling

ABSTRACT Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a “Candidatus Accumulibacter”-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

Stability of enhanced biological phosphorus removal and composition of polyphosphate granules.

The influence of varying Ca- and Mg-concentration of the influent wastewater on the enhanced biological phosphorus removal was investigated in an anaerobic-aerobic bench-scale plant. The artificial enhancement of the Mg-concentration in the influent from 15 to 24 mg l(-1) and 31 mg l(-1), respectively, caused a raise of the mean P-removal efficiency from 85 to 97%. The P-elimination was very stable in time. A chemical precipitation of magnesium ammonium phosphate could be excluded. The elemental composition of polyphosphate granules was investigated by electron microscopy and energy dispersive X-ray spectroscopy. The elements Ca, Mg and K were the principal metal components of polyphosphate granules. Concerning the metal composition, different types of granules could be distinguished. The quantitative ratios of Ca, Mg and K varied in dependence on the influent concentration of these metals. A relation between the Mg/Ca-ratio of the granules and the efficiency of enhanced biological phosphorus removal can be supposed.

Evaluation of the metabolic diversity of microbial communities in four different filter layers of a constructed wetland with vertical flow by Biolog™ analysis

The community-level substrate utilization test based on direct incubation of environmental samples in Biolog EcoPlates is a suitable and sensitive tool to characterize microbial communities. The aim of this study was to investigate the influence of plant roots and soil structure on the metabolic diversity of microorganisms in a constructed wetland with vertical flow. Sediment samples were taken from different filter depths representing specific filter layers. The color development representing the substrate utilization was measured with the samples over a period of 10 days. The average well color development (AWCD) for all carbon sources was calculated as an indicator of total activity and in order to compensate the influence of the inoculums density on the color development in the plates. After transformation by dividing by the AWCD, the optical density data were analysed by principal component analysis (PCA). An analysis of the kinetic profile of the AWCD was carried out to increase the analytical power of the method. The corrected data have been successfully fit to the logistic growth equation. Three kinetic model parameters, the asymptote (K), the exponential rate of color change (p) and the time to the midpoint of the exponential portion of the curve (s), were used for statistical analysis of the physiological profile of the microbial community in the different filter layers of the constructed wetland. We found out that in the upper two horizons, which were rooted most densely, mainly easily degradable materials like specific carbohydrates were utilized, while in the lower layers, where only single roots occur, more biochemically inert compounds, e.g. 2-hydroxy benzoic acid, were utilized. Furthermore it could be shown that microorganisms in the surface layer benefited from the plant litter because they can utilize decay products of these. In the lower filter layers specialists took advantage because they had to cope with the biochemically inert materials and the lower nutrient supply.

Detection of phages carrying the Shiga toxin 1 and 2 genes in waste water and river water samples

Aims:  To evaluate the occurrence and abundance of phages that carry the stx1 and stx2 gene in water samples of different quality.

Sediment and pore water composition as a basis for the trophic evaluation of standing waters

Sediments and pore water samples from four reservoirs in eastern Germany with different trophic characteristics have been examined (Neunzehnhain I – oligotrophic; Muldenberg – oligotrophic, dystrophic; Saidenbach – mesotrophic; Quitzdorf – highly eutrophic). Several parameters were analyzed regarding the dependence on the trophic state and the possibility to use them for the trophic evaluation of standing waters. The strongest influence of the trophic state could be found in the case of the parameters SRP, alkalinity and ammonium in the pore water. For the dry sediment, it could be concluded that the most important factor affecting the trophic state is the ratios of Fe:P and Al:P, which were summarized to a metal:phosphorus sum ratio (MPS) and showed a clear threshold value for the potential to immobilize phosphorus in the sediment. Also, with the P fractions of the sediment samples, differences between the trophic levels became evident. Higher percentages of the reductively soluble, iron-bound fraction and of the biogenic fraction occurred in the reservoirs with higher trophic state, while comparitively higher percentages of the stable aluminum-bound fraction could be measured in the reservoirs with lower trophic state.

Correlation of community dynamics and process parameters as a tool for the prediction of the stability of wastewater treatment.

Wastewater treatment often suffers from instabilities and the failure of specific functions such as biological phosphorus removal by polyphosphate accumulating organisms. Since most of the microorganisms involved in water clarification are unknown it is challenging to operate the process accounting for the permanent varying abiotic parameters and the complex composition and unrevealed metabolic capacity of a wastewater microbial community. Fulfilling the demands for water quality irrespective of substrate inflow conditions may emit severe problems if the limited management resources of municipal wastewater treatment plants are regarded. We used flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring dynamics in community structure. These patterns were resolved in up to 15 subclusters, the presence and abundances of which correlated with abiotic data. The study used biostatistics to determine the kind and strength of the correlation. Samples investigated were obtained from a primary clarifier and two activated sludge basins. The stability of microbial community structure was found to be high in the basins and low in the primary clarifier. Despite major abiotic changes certain subcommunities were dominantly present (up to 80% stability), whereas others emerged only sporadically (down to 3% stability, both according to equivalence testing). Additionally, subcommunities of diagnostic value were detected showing positive correlation with substrate influxes. For instance blackwater (r(s) = 0.5) and brewery inflow (both r(s) = 0.6) were mirrored by increases in cell abundances in subclusters 1 and 6 as well as 4 and 8, respectively. Phosphate accumulation was obviously positively correlated with nitrate (r(s) = 0.4) and the presence of denitrifying organisms (Rhodacyclaceae). Various other correlations between community structure and abiotic parameters were apparent. The bacterial composition of certain subcommunities was determined by cell sorting and phylogenetic tools like T-RFLP. In essence, we developed a monitoring tool which is quick, cheap and causal in its interpretation. It will make laborious PCR based technique less obligatory as it allows reliable process monitoring and control in wastewater treatment plants.

Identification and Expression Analyses of Putative Sesquiterpene Synthase Genes in Phormidium sp. and Prevalence of geoA-Like Genes in a Drinking Water Reservoir

ABSTRACT The occurrence of taste and odor problems in drinking water supplies is a widespread phenomenon. From a Saxonian water reservoir we isolated a cyanobacterial species which was classified as Phormidium sp. Under laboratory conditions it produced an earthy-musty smell due to the synthesis of geosmin. The only genes shown to be involved in geosmin biosynthesis are cyc2 and geoA of Streptomyces. Based on the alignment of Cyc2 with a putative sesquiterpene synthase of Nostoc punctiforme, a degenerate primer pair was designed. By PCR, we could amplify two similar genes in Phormidium sp., which we named geoA1 and geoA2. Their expression was studied by reverse transcription-PCR. This revealed that both genes are expressed at 20°C and a light-dark cycle of 12 h. Expression was not detectable at the end of a 24-h dark period. To analyze the prevalence of geoA1 and geoA2 in samples from the phytobenthos, we generated PCR fragments with the same degenerate primer pair. Fifty-five different sequences that might represent geoA variants were obtained. The GC content ranged from 42% to 67%, suggesting that taxonomically very different bacteria might contain such genes.