Indicator bacteria and coliphages along the transects of Lake Tegel, Lake Wannsee and the artificial recharge pond Tegel The indicator microorganisms, Escherichia coli, intestinal enterococci, and coliphages were analysed in water samples from both transects for bank filtration at the Lakes Tegel and Wannsee as well as from the artificial recharge pond of Tegel. Surface water samples of both lakes were examined from May 2002 to December 2004. In addition, samples were analysed from four shallow observation wells and three pumping sites for drinking water supply along the transect Wannsee, as well as four deep and seven shallow observation wells and 2-3 pumping sites along the transect of Lake Tegel. Sampling at the artificial recharge pond of Tegel was carried out during four field surveys (August 2002-December 2002) in two shallow, three deep wells and one pumping well for drinking water supply. In addition, the microbial flora was studied in the sand filter of the recharge pond in Tegel.

The ENREM project aims at demonstrating a novel wastewater treatment process based on the technology of membrane bioreactor (MBR), set up in a configuration to enable enhanced biological elimination of nutrients. A new plant, and the related sewer system, was built in a unsewered periurban area of Berlin. The plant is to be operated over more than one year, and the process to be optimised. Performances and costs of the treatment system will be then assessed for the size 250 – 10,000pe, corresponding to semi-central schemes. The plant was started on 28 February 2006 with 8 month delay on the program identified in the LIFE proposal. As a consequence, a project extension request of 8 months will be lodged, in order to match the initial duration of 18 months for the optimisation, evaluation and dissemination phase. This delay incidentally caused also a 6 month delay for the preparation of this Interim Report. Despite these aspects, the management of the project has been achieved according to the organisation identified in the LIFE proposal. Annex 7.1 presents and discusses the key deliverables and milestones depending on the LIFE proposal and the current status. In relation to the technical content, Task 2 “Site and process definition” and Task 4 “Detailed design” were completed early 2005. Task 3 “Preliminary testing on representative site” was completed in September 2005 and enabled to validate the design, operation and start-up criteria of the MBR demonstration plant. The public tenders for the construction of the sewer network and the MBR container unit occurred during the first semester of 2005, and the construction of the scheme was completed by end 2006. In parallel, the required legal permits were acquired (for plant construction & operation, water discharge), as well as the parcel hosting the treatment unit. A relationship with the inhabitants of Margaretenhöhe was maintained in order to ensure a smooth construction phase, and a quick connection to the new sewer system. The dissemination activities covered several communication vectors (Tri-lingual website www.kompetenz-wasser.de, press-release and articles and bi-lingual KWB Newsletter, local press, scientific press, plant visits and inauguration, communication material etc). The project results will be widely communicated in the national and international medien, and a final project workshop will be organised in June 2007. The main task in 2006 will be the operation, optimisation and technical / economical evaluation of the low sewer system and the MBR demonstration plant. So far, the budget is generally in line with the expectations, or slightly below. The project finances allow the project extension of 8 months, required to achieve all announced technical outcomes of the project. After the 8 month extension the final project budget is expected to remain about 20% below the planned proposal, i.e. approx. € 600,000 savings on the total budget should be recorded, corresponding to a subvention reduction of approx. € 100,000 (also close to 20% reduction).

To evaluate the potential hazard of a harmful substance on its pathway from a source to a sensitive site, it is important to know if degradation or reversible sorption is the dominant process. While, in case of degradation, mass is removed from the system, in the case of reversible sorption the transport is only retarded. A mathematical analytical concept is outlined, which can be applied to evaluate data from field experiments, from technical and semitechnical facilities and from the laboratory. The concept is exemplified on a series of enclosure experiments performed with phages within a project investigating processes during bank filtration. Results show that parameters are not constant in the experiments and indicate a significant influence of redox conditions on both retardation and deactivation rates. On the other hand, an influence of the clogging layer could not be detected.

Four international projects subsidised by the European Commission should push forward the development and application of membrane bioreactor processes in the municipal wastewater sector.

Extracellular polymeric substances (EPS) are considered as the major cause of membrane fouling in membranebioreactors. Recent studies have revealed a linear relationship between fouling rate (increase of filtration resistanceover time) and polysaccharide (PS) concentration [1]. Several factors like the type of wastewater, sludge loading rate, sludge age, MLSS concentration, and mechanical stress are known to influence the concentration of dissolved EPS. Also, unsteady states like intermittent feeding or shifts in the oxygen supply have been identified as additionalfactors leading to an increase in EPS formation or to a change in its fouling propensity. However, no systematicinvestigation to quantify such influences has been undertaken so far. This study is aimed at determining the influenceof discontinuous excess sludge withdrawal often applied in small decentralised plants and resulting unsteady loadingrates on EPS formation and filtration resistance. The general trend of increased fouling rate at higher PS concentrationsis confirmed but data show a larger scatter which could be due to a change in the PS structure and hence their fouling potential. The levels of dissolved oxygen and nitrate which also fluctuate more strongly in MBRs withirregular sludge wastage appear to have a profound impact on EPS uptake rate and thereby on EPS concentration.

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.