Werner Manz

Performance of a bioreactor with submerged membranes for aerobic treatment of municipal waste water.

Aerobic treatment of municipal waste water in a membrane bioreactor was studied for 535 d. Apart from sampling, sludge was retained completely by a submerged hollow fibre membrane with a pore-size of 0.2 microm. The pilot plant comprised an anoxic zone to enable denitrification. The maximum liquid hold-up of the plant was 3.9 m3. In this study the reactor performance and the stability of the process and the membrane capacity were investigated. A stable flux of 181 m(-2)h(-1) could be realised with a mean transmembrane pressure difference of 0.3bar with air-bubbling and backflushing the membrane and cleaning it in place every two months for one or two hours. For about 140d, a flux of 271 m(-2)h(-1) was achieved, but cleaning became necessary more often. The hydraulic retention time (HRT) varied between 10.4 and 15.6h. Accordingly the volumetric loading rate was between 1.1 and 1.7kg CODm(-3)d(-1). No inoculum was used. The mixed liquor suspended solids (MLSS) concentration gradually increased to 18-20g MLSSl(-1). The feed to microorganism (F/M) ratio varied according to the operation conditions but decreased against a value of 0.07 kg COD kg(-1) MLSSd(-1). Treatment performance was very stable and on a high level. The COD was reduced by 95%. Nitrification was complete and up to 82% of the total nitrogen could be denitrified.

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy

A bstractThe phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga–Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga–Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga–Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms.

Microbiological aspects of a bioreactor with submerged membranes for aerobic treatment of municipal wastewater

An aerobic membrane bioreactor treating municipal wastewater at complete biomass retention was studied in respect of microbiological parameters over a period of 380 days. The results were compared to those obtained from a conventional activated sludge wastewater treatment plant (WWTP) treating the same wastewater. Microscopically, significant changes in the structure of the flocs and of the ratio between free suspended and aggregated cells could be observed. The presence of filamentous bacteria varied from almost not present to very high numbers. With the exception of short periods after changes in operating conditions, protozoa and metazoa were rarely present in the sludge community. The rate of oxygen consumption and the cell detectability by fluorescence in situ hybridizatio (FISH) with rRNA-targeted oligonucleotide probes were used to assess the physiological state of the bacterial cells Oxygen consumption rates of sludge samples obtained from both the conventional and membrane filtration plant wer determined without and after addition of different energy and carbon sources. In contrast to the conventional activate sludge, a pronounced increase in respiration activity upon the addition of organic substrates could be observed in th membrane filtration sludge. In situ probing with the Bacteria-specific probe EUB338 visualized 40-50% of all DAPI stainable bacteria in the membrane bioreactor, compared to 80% cells detectable by FISH in the conventional activate sludge. These results suggest that bacteria present in the highly concentrated biomass of the membrane reactor use the energy supplied for their maintenance metabolism and were not in a physiological state characteristic for growth This assumption could explain the zero net biomass production observed in the reactor.

A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.

Comparative genotoxicity testing of rhine river sediment extracts using the comet assay with permanent fish cell lines (rtg-2 and rtl-w1) and the ames test*

Goals, Scope and BackgroundImproved quality of surface waters and sediments requires advanced strategies for ecotoxicological assessment. Whilst at least in Germany assessment strategies on the basis of chemical analysis and acute toxicity data dominated the last decades, the development of more specific biological endpoints and biomarkers in ecotoxicology is required in order to arrive at a good ecological potential and good chemical status of surface waters in the European river basins until the year 2015, as required by the European Water Framework Directive. Since sediments have for long been known to function both as a sink and as a source of pollutants in aquatic systems, and since part of the particle-associated substances have frequently been demonstrated to cause mutagenic and carcinogenic effects in aquatic organisms, particularly in fish, there is, among other requirements, an urgent need to develop, standardize and implement integrated vertebrate-based test systems addressing genotoxicity into recent sediment investigation strategies. Thus, the present study was designed to compare the suitability of two commonly used test systems, the comet assay and the Ames test, for the evaluation of the ecotoxicological burden of surface and core sediment samples from the river Rhine.Methods (or Main Features)In order to determine the importance of inherent enzymatic activities, two permanent fish cell lines with different biotransformation capacities, RTL-W1 and RTG-2, were compared with respect to their capability of detecting genotoxic effects in 18 surface and core sediment samples from 9 locations along the River Rhine in the comet assay with and without exogenous bioactivation. For further comparison, as a prokaryotic mutagenicity assay, theSalmonella plate incorporation assay (Ames test) with the test strains TA98 and TA 100 with and without exogenous metabolic activation was used.Results and DiscussionWhereas all sediment extracts induced genotoxic effects in the comet assay with RTL-W1 cells, only 12 out of 18 sediment extracts revealed significant genotoxicity in the tests with the less biotransformation-competent RTG-2 cells. Exogenous bioactivation by addition of ß-naphthoflavone /phenobarbital-induced S9 from rat liver resulted in both reduction or increase of genotoxicity in samples from different sites, however, without consistent reaction patterns. In general, the responses of RTL-W1 cells indicated higher biotransformation capacity than in RTG-2 cells without S9 complementation. In Ames tests using TA98 with S9, 16 out of 18 extracts induced significant mutagenicity with induction factors up to 4. Compared to TA98, the strain TA100 proved less sensitive, with maximum induction factors of 1.3, indicating the potential presence of substances inducing frarneshift mutations, which can only be detected in the strain TA98. Chemical analyses revealed particularly high levels of hexachlorbenzene (up to 860 µg/kg) and priority PAHs (up to 4.8 mg/kg); so far, however, no correlation could be found between compounds analyzed and the corresponding biotests.ConclusionsResults document that both comet assay and Ames test are capable of detecting xenobiotic interaction with DNA in consequence of exposure to complex environmental samples. Whereas the alkaline version of the comet assay detects a broad range of interactions with the DNA, however without information about their eventual importance, the Ames test only reveals established mutations, but fails to detect transient (reparable) DNA alterations. However, even transient primary changes in the DNA structure might result in carcinogenic processes and, eventually, in implications at the population level. As a consequence, for hazard assessment purposes, a combination of both assays is required to avoid false negatives in genotoxicity evaluation. Poor correlation between data obtained by chemical analysis and results in bioassays is indicative of our limited understanding of the sources of genotoxicity. In fact, numerous studies combining chemical and biological approaches for hazard assessment of complex environmental mixtures indicate that priority pollutant concentrations are a poor indicator of toxicity.If compared to the cell line RTG-2, RTL-W1 proved more effective in detecting genotoxicity in surface sediment samples and, thus, indicated the importance of bioactivation of at least part of the compounds in superficial layers of sediments. Results further document that the common assumption may be wrong that, in comparison to deeper strata, surface layers carry a lower toxic burden in consequence of the current decrease in water pollution. This might at least in part be due to remobilization of more heavily polluted sediments from deeper layers during severe flood events followed by re-sedimentation in flood plains or upstream weirs, where they might cover less polluted younger sediment layers.Recommendations and PerspectivesFor a comprehensive assessment of genotoxicity in surface and core sediments, a combination of eukaryotic (comet assay) and prokaryotic assays (Ames test) with and without exogenous bioactivation is recommended. Since studies with organic sediments extracts simulate a worst-case scenario and fail to take into account bioavailability, there is broad consensus that whole-sediment exposure protocols represent the most realistic scenarios. Whereas more realistic solid phase exposure has frequently been applied in both microbial and invertebrate acute toxicity testing, there is an urgent need to develop corresponding whole sediment fish-based genotoxicity tests.

Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis.

A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH-LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow). River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed. Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH-LBA technique. In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix. The analysis could be performed on different levels of complexity. The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies. Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells. Consequently, application of the FISH-LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.

Ancient Fungal Life in North Pacific Eocene Oceanic Crust

We present evidence that eukaryotic life has existed in an extreme environment, inside the oceanic crust. Up to now only prokaryotes have been discovered within deep marine sediments and glass-rims of pillow basalts, no higher life forms are described as yet. This study demonstrates unique filamentous fossil structures observed within carbonate-filled vesicles of a massive lava flow unit from the upper oceanic crust in the North Pacific (ODP Site 1224). Based on morphological traits including branching, septa and central pores, the filaments are interpreted as fungi. The chemical composition of the fungal structures differs from the surrounding crystalline carbonate matrix in the deep basaltic rocks. Small open space between the fungi and the carbonate cement and undisturbed filamentous growth through different calcite crystals indicate endolithic fungal growth after the calcium carbonate filling. The presence of euhedral pyrite crystals within the carbonate cements points out anaerobic conditions in this habitat. Our results provide for the first time evidence for eukaryotic, fungal life in deep ocean basaltic rocks.

A DNA Microarray Platform Based on Direct Detection of rRNA for Characterization of Freshwater Sediment-Related Prokaryotic Communities

ABSTRACT A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of ∼5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.

In situ probing reveals Aquabacterium commune as a widespread and highly abundant bacterial species in drinking water biofilms

Abstract Drinking water biofilm communities originated from distribution systems in Hamburg, Berlin, Mainz and Stockholm were subjected to a top-to-bottom in situ analysis with rRNA-targeted, fluorescently labeled oligonucleotide probes including beta1–8, specific for drinking water bacteria within the beta-subclass of Proteobacteria . Independent from the raw water source used, between 19 and 77% of the total attached bacterial cell counts on polyethylen slides could be hybridized with probes beta1–8 and 19–53% of the bacteria were affiliated to the autochthonous species Aquabacterium commune . To investigate seasonal fluctuations of the bacterial population composition, polyethylen slides exposed during different times of the year were analyzed. Although changes in the species composition of the biofilms could be observed, A. commune was a dominant community member in all of the investigated biofilms. In situ probing with two oligonucleotide probes specific for A. commune revealed that during distinct sampling times an additional yet unknown species was present in the biofilm. Material induced population shifts were studied using glass, polyethylene low and high density and soft-PVC as biofilm substrata. The community composition on soft-PVC differed significantly from the other materials, the dominant species A. commune was replaced by other beta- Proteobacteria hybridizing to an amount of 66% with the Aquabacterium citratiphilum specific probe beta4.