Cylindrospermopsis raciborskii is a filamentous freshwater cyanobacterium of tropical and subtropical origin that spread out up to northern temperate zone during the last decades. Belonging to the order Nostocales it can differentiate two types of nonvegetative cells: i) heterocysts, in which it can fix molecular nitrogen, and ii) akinetes, which are resting stages, that allow the species to pass-by unfavourable growth conditions in the sediment. In the tropics and subtropics, C. raciborskii is a perennial species, which often occurs in bloom densities, and which is prominent for the synthesis of various toxins including the potent hepatotoxin cylindrospermopsin (CYN) and neurotoxic paralytic shellfish poisoning toxins. In Europe, its northernmost populations were found in North German lakes. Here, it occurs only during the summer months and has not been observed to form mass developments. The highest documented biomass it attained was 23 % of the total phytoplankton biomass. To investigate the spatial occurrence and relative frequency of C. raciborskii, we undertook a systematic survey, sampling 142 water bodies in the lowland region of Northeast Germany from June till September 2004. The cyanobacteria species present were analysed qualitatively and semi-quantitatively. C. raciborskii was a common species. It was present in 39 of the samples, of which 62 % were new findings. Further, we detected two additional thermophilic Nostocales that have not been reported from Germany before: Anabaena bergii and Aphanizomenon aphanizomenoides. These are the most northerly reports of both species so far. A. bergii occurred in 13 and A. aphanizomenoides in 19 of the 142 water bodies sampled. To elucidate the regulation mechanisms of the population dynamics of C. raciborskii and its abiotic boundaries in the newly invaded habitats furthermore long-term data series (1993 - 2005) of two polymictic shallow lakes in the study area were analyzed. Population sizes largely varied between years without any distinct long-term trend. In the annual course, filaments of the species occurred in the pelagial at temperatures above 15 - 17 °C, i.e. akinetes must have been germinated at temperatures below 15 °C. Population growth started at high rates (1.5 - 2.8 d-1) that declined continuously over the season. Growth rates were not significantly correlated with temperature but they were significantly positive correlated with the mean photosynthetic active radiation in the mixed water column (Imix). At the time population starts growing, Imix has exceeded its annual maximum, and growth rate declines with decreasing Imix. The time of akinete germination is therefore of crucial importance for the success of the population: as earlier akinetes germinate the higher the growth rate will be, resulting in a greater population size, a higher number of akinetes produced, and vice versa. Therefore, we conclude that an earlier increase of the water temperature due to global warming is a major cause for the spreading of C. raciborskii into the temperate zone.

Cylindrospermopsin (CYN) is a potent hepatotoxin produced by different cyanobacteria of the order Nostocales, which is widely distributed in freshwaters and considered a hazard to wildlife and humans. In Europe, only Aphanizomenon flos-aquae is identified as CYN producer so far and the seasonal variability of CYN was not studied yet. Therefore, we studied the seasonal dynamics of the particulate and dissolved CYN concentrations in relation to the cyanobacterial occurrence and environmental factors in two German lakes over two years. Total CYN reached maximum concentrations of 0.34 and 1.80 µg L-1 in Melangsee and Langer See, respectively. In both lakes, the dissolved CYN fraction peaked after the particulate fraction, and reached higher values indicating that CYN is poorly decomposed and accumulates in the water. The cyanobacterial community was very diverse in both lakes, including potentially CYN producing species such as Cylindrospermopsis raciborskii, Anabaena bergii and Aphanizomenon flos-aquae. However, these species could be excluded as the major CYN producers. The stronges significant correlation was found for Aphanizomenon gracile and CYN, strongly indicating that the species is the main CYN producer in Langer See. CYN was also correlated with Planktothrix agardhii in Langer See and Pseudanabaena limnetica in Melangsee, but species of the order scillatoriales are not known to produce CYN. In Melangsee the CYN producer could not be identified. Different correlations of CYN with abiotic factors in the two lakes indicate the presences of further undetected CYN producers as well as different regulation mechanisms of their dynamic and the variability of CYN.

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.