The invasive cyanobacterium Cylindrospermopsis raciborskii is increasingly spreading to temperate freshwater habitats world wide and is of major concern due to its ability to produce potent toxins. It is therefore important to understand the mechanisms behind the dispersal of this species. Different hypotheses have been proposed to explain the phylogeography and mechanisms underlying the recent expansion of C. raciborskii into temperate latitudes, but there is still no conclusive evidence whether the obvious ecological success of C. raciborskii is due to selection mechanisms, physiological tolerance, climatic change or radiation after the last ice age. In this study, new isolates of C. raciborskii from Europe and Africa were genetically characterized by sequencing the ITS1, PC-IGS, nifH and rpoC1 genes and compared to corresponding sequences of C. raciborskii available in GenBank in order to test different phylogeographical hypotheses. The strains were also morphologically examined and screened for production of the hepatotoxic cylindrospermopsin (CYN). We clearly demonstrate that there are phylogenetic, morphological and toxicological differences between the isolated strains. The phylogenetic analyses revealed a clustering of the strains due to geographic origin. The ITS1 and nifH genes separated into American, European and Australian-African groups, whereas the PC-IGS and rpoC1

Cylindrospermopsis raciborskii is considered a cyanobacterium of tropical origin and an alien species to temperate waters. However, it has been detected as far north as northern Germany. While previous studies have shown that all isolated German C. raciborskii strains are hepatotoxic, little is known about the spatial occurrence and relative frequency of this species in temperate Germany. The aim of this study was to investigate the spatial distribution and relative frequency of C. raciborskii close to its northernmost distribution limit, to characterise the habitat in which it is most likely to occur in this climatic zone and to search for any other neocyanobacterial species that might be present in German waters but has so far been overlooked. One hundred forty-two water bodies in northeast Germany were sampled from June until September 2004. All cyanobacteria species were analysed qualitatively and semiquantitatively. Besides C. raciborskii, two additional neocyanobacterial species were detected: Anabaena bergii and Aphanizomenon aphanizomenoides. For both taxa, these findings represent their northernmost occurrence and their first report from German waters. Cylindrospermopsis raciborskii was present in 27%, Anabaena bergii in 9% and phanizomenon aphanizomenoides in 7% of the samples. The occurrence of each species was analysed in relation to maximum lake depth, Secchi depth, lake volume and lake surface area. All three species were present in a wide range of habitats, but C. raciborskii and Anabaena bergii occurred significantly more often in shallow, turbid waters than in deep, transparent water bodies. None of the parameters investigated were significantly correlated with the occurrence of Aphanizomenon aphanizomenoides. In conclusion, alien thermophilic cyanobacterial species are much more widely distributed in temperate Germany than previously known. The results are discussed with respect to the possible mechanisms that enable these organisms to expand northwards.

Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju and Aphanizomenon spp. are both freshwater cyanobacteria of the order Nostocales. C. raciborskii was thought confined to tropical and sub-tropical environments but spread to temperate climatic regions on all continents except Antarctica (Padisák, 1997). It is known to be widely distributed in Northern Germany reaching the northernmost margin of its distribution at latitudes of 53 - 54°N (Krienitz & Hegewald, 1996; Stüken et al., 2006). Strains of this species are detected to produce the hepatotoxin cylindrospermopsin, from which human injury has been clearly identified (Falconer & Humpage, 2006). Nostocales are characterized by trichomal structures such as vegetative cells, heterocysts and akinetes. The heterocysts can fix atmospheric nitrogen (N2) when aquatic nitrogen is depleted (Kim et al., 2005). Akinetes are non-motile, resistant cells that accumulate proteinaceous reserves in the form of cyanophycin granules (Wetzel, 2001). These akinetes can survive low temperatures, desiccation and other adverse environmental conditions. When favourable conditions return, they germinate to produce trichomes. The ability of Nostocales to form akinetes confers a distinct advantage in environmental adaptation and subsequent bloom formation (Kim et al., 2005; Wetzel, 2001). C. raciborskii akinetes are more commonly observed in subtropical and temperate populations late in the population cycle as a component of seasonal population dynamics (Kravchuk et al., 2006). Particular set of physico-chemical conditions stimulate akinetes to germinate (Moore et al., 2005). Light has been implicated as well as temperature and nutrients as triggering factors (Huber, 1985). According to Moore (2004) germination of akinetes occurs at temperatures between 15 °C and 30 °C in tropical Australian strains of C. raciborskii. Mischke (2003) found filaments of this species at temperatures of about 17 oC in temperate lakes with those in the Scharmützelsee region in Germany. We therefore, tried to find out at what temperature the akinetes germinate in temperate region hypothesizing it should be below 15 oC. Since Aphanizomenon species like A. gracile and A. flosaquae are the most abundant natural Nostocales in that habitat, we compare it to the invaded C. raciborskii. To figure out the germination temperature for both species, field observations are complemented by experiments at different temperatures.

The aim of this study was to clarify the phylogenetic position of the three heterocystous cyanobacteria species Anabaena bergii, Aphanizomenon ovalisporum and Aphanizomenon aphanizomenoides within the order Nostocales. We determined and phylogentically analysed 16S rRNA gene and cpcBA-IGS sequences of four A. bergii, three A. ovalisporum, one A. aphanizomenoides and seven Aphanizomenon sp. strains isolated from Spain, Germany, Israel and Senegal and complemented the analyses with 2 morphometric descriptions of these strains. The phylogenetic clustering did not follow the current botanical classification. All three species clustered separately from the majority of Anabaena and Aphanizomenon strains. A. bergii and A. ovalisporum clustered close to Nodularia, whereas the position of the cluster containing the A. aphanizomenoides strain varied between the trees and the different tree constructing methods used. In addition to A. aphanizomenoides, this cluster contained the two Anabaena species A. kisseleviana and A. oumina. All three species had highly similar DNA sequences at the two fragments analysed and thus, based on evolutionary distances, might be assigned to a single species. Further, our results contradict the previously formulated suggestion that A. bergii and A. ovalisporum are 3 morphotypes of a single species. Instead, A. bergii and A. ovalisporum consistently formed separate clusters, which were less than 96.6 % similar to each other based on 16S rRNA gene sequence analysis. Our results support the idea that the taxonomy of heterocystous cyanobacteria should be revised, but also emphasize the importance of detailed morphological information when molecular data of new strains is used for taxonomy.

Reactive multicomponent transport modeling was used to investigate and quantify the factors that affect redox zonation and the fate of the pharmaceutical residue phenazone during artificial recharge of groundwater at an infiltration site in Berlin, Germany. The calibrated model and the corresponding sensitivity analysis demonstrated that temporal and spatial redox zonation at the study site was driven by seasonally changing, temperature-dependent organic matter degradation rates. Breakthrough of phenazone at monitoring wells occurred primarily during the warmer summer months, when anaerobic conditions developed. Assuming a redoxsensitive phenazone degradation behavior the model results provided an excellent agreement between simulated and measured phenazone concentrations. Therefore, the fate of phenazone was shown to be indirectly controlled by the infiltration water temperature through its effect on the aquifer’s redox conditions. Other factors such as variable residence times appeared to be of less importance.

The spatial and temporal evolution of the seepage water chemistry below an artificial recharge pond was investigated to identify the impact of dynamic changes in water saturation and seasonal temperature variations. Geochemical analysis of the pond water, suction cup water and groundwater showed that during summer, nitrate and manganese reducing conditions dominate as long as saturated conditions prevail. Iron and sulphate reduction occur only locally. When the sediment below the pond becomes unsaturated, atmospheric oxygen penetrates from the pond margins leading to re-oxidation of previously formed sulphide minerals and enhanced mineralisation of sedimentary particulate organic carbon. The latter promotes the dissolution of calcite. During winter, both the saturated and the unsaturated stage were characterised by aerobic conditions. Thereby, nitrification of sedimentary bound nitrogen could now be observed because nitrate is not immediately consumed, as is the case during summer. This suggests that nitrification below the pond might be less affected by seasonal temperature changes than nitrate reduction.

As part of the EU-Life ENREM demonstration project the Department of Chemical Engineering, TU Berlin, was appointed to conduct the preliminary pilot trials in a representative site for verification of basic process design and operation criteria of the full-scale MBR demonstration plant. In addition to conception and construction of the pilot plant, this investigation consisted of two successive trial phases with distinct operation conditions. The first one was dedicated to the assessment of the “irregular sludge removal strategy” (the biomass is accumulating in the reactor, which is partly emptied when the sludge concentration reaches a given value). In the second trials phase normal operation conditions with daily sludge wastage were implemented with 28,5d SRT. The major outcome of the trials was that COD removal, enhanced biological phosphorus removal and the post-denitrification performed a similar way under both operational conditions. The denitrification rate was approximately 1 mgN/(h goTS). An influence of the anaerobic sludge loading on the post-denitrification rate was observed with higher rates (up to 4 mgN/(h goTS)) corresponding to higher organic loading. An influence of storage compounds built up in the anaerobic phase is assumed. Nitrification was better in the second phase when 4 mgN/(h goTS) were constantly reached while nitrification was unstable with an average of 2 mgN/(h goTS) in the phase of irregular sludge removal. The aerobic and anoxic reactors were enlarged during the regular sludge withdrawal phase by 23% resulting in 35d SRT. This led to a better COD removal and slightly better nitrogen removal. The enhanced SRT produced possibly a deterioration of biological P removal due to overloaded poly-P storage. A second possible reason is the massive reproduction of sludge worm Tubifex tubifex, which was observed after the plant enlargement. Different strategies to reduce the worm population were attempted. Ammonium dosing had no success. Copper dosing reduced the number of worms significantly but the population grew back after the dosing was stopped. The prolongation of SRT reduced the sludge yield from 0.23 gTS/gCOD at 28.5d to 0.18 gTS/gCOD at 35d.

Two parallel membrane bioreactors (2m³ each) were operated over a period of 2 years. Both pilots were optimised for nitrification, denitrification, and enhanced biological phosphorous elimination, treating identical municipal waste water under comparable operating conditions. The only constructional difference between the pilots was the position of the denitrification zone (pre-denitrification in pilot 1 and post-denitrification in pilot 2). Despite identical modules and conditions, the two MBRs showed different permeabilities and fouling rates. The differences were not related to the denitrification scheme. In order to find an explanation for the different membrane performances, a one-year investigation was initiated and the membrane performance as well as the operating regime and characteristics of the activated sludge were closely studied. MLSS concentrations, solid retention time, loading rates, and filtration flux were found not to be responsible for the different performance of the submerged modules. These parameters were kept identical in the two pilot plants. Instead, the non-settable fraction of the sludges (soluble and colloidal material, i.e. polysaccharides, proteins and organic colloids) was found to impact fouling and to cause the difference in membrane performance between the two MBR. This fraction was analysed by spectrophotometric and size exclusion chromatography (SEC) methods. In a second step, the origin of these substances was investigated. The results point to microbiologically produced substances such as extracellular polymeric substances (EPS) or soluble microbial product.