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Här presenteras resultaten från forskningsprojektet ”Eutrofiering och närsalter iBottniska viken - Ett ekosystem i förändring”. Projektet är ett av fyra syntesprojektsom genomförts inom forskningssatsningen Avloppsvatten och övergödning.

Med de fyra syntesarbeten ville Naturvårdsverket och Havs- och vattenmyndighetenfå sammanfattad och analyserad kunskapsläge och kunskapsbehovinom områdena avloppsvatten och övergödning. Det övergripande syftet medsynteserna var att bidra till policyutveckling inom hållbar vattenhantering så attvi uppnår miljömålen på lång sikt och att miljöns tillstånd förbättras. Utlysningenvar inriktad på tre områden varav ett var om övergödning i kust och hav i Bottniskaviken.

Projektet har finansierats med medel från Naturvårdsverkets miljöforskningsanslag.

Rapporten har skrivits av Agneta Andersson, Siv Huseby, Joakim Ahlgren,Karolina Eriksson och Sonia Brugel från Umeå universitet.

Rapporten har granskats för vetenskaplig kvalitet av Jens Fölster (SLU) samtför praktisk relevans av Lars Åkesson (Havs- och vattenmyndigheten), Anneli Sedin(Länsstyrelsen i Västerbotten) samt Kerstin Rosén Nilsson (Naturvårdsverket).

Författarna svarar för rapportens innehåll.

Intensified land use can disturb water quality, potentially increasing the abundance of bacterial pathogens, threatening public access to clean water. This threat involves both direct contamination of faecal bacteria as well as indirect factors, such as disturbed water chemistry and microbiota, which can lead to contamination. While direct contamination has been well described, the impact of indirect factors is less explored, despite the potential of severe downstream consequences on water supply. To assess direct and indirect downstream effects of buildings, farms, pastures and fields on potential water sources, we studied five Swedish lakes and their inflows. We analysed a total of 160 samples in a gradient of anthropogenic activity spanning four time points, including faecal and water-quality indicators. Through species distribution modelling, Random Forest and network analysis using 16S rRNA amplicon sequencing data, our findings highlight that land use indirectly impacts lakes via inflows. Land use impacted approximately one third of inflow microbiota taxa, in turn impacting ∌20–50 % of lake taxa. Indirect effects via inflows were also suggested by causal links between e.g. water colour and lake bacterial taxa, where this influenced the abundance of several freshwater bacteria, such as Polynucleobacter and Limnohabitans. However, it was not possible to identify direct effects on the lakes based on analysis of physiochemical- or microbial parameters. To avoid potential downstream consequences on water supply, it is thus important to consider possible indirect effects from upstream land use and inflows, even when no direct effects can be observed on lakes. Legionella (a genus containing bacterial pathogens) illustrated potential consequences, since the genus was particularly abundant in inflows and was shown to increase by the presence of pastures, fields, and farms. The approach presented here could be used to assess the suitability of lakes as alternative raw water sources or help to mitigate contaminations in important water catchments. Continued broad investigations of stressors on the microbial network can identify indirect effects, avoid enrichment of pathogens, and help secure water accessibility.

Climate change is projected to cause alterations in northern coastal systems, including humification and intensified nutrient loads, which can lead to ecosystem imbalances and establishment of new bacterial species. Several potential pathogens, such as different species of Legionella, hide in the environment between infections, some by living inside protozoan host cells. Knowledge about the occurrence of Legionella in natural waters is missing, which disable risk assessments of exposure. We performed a study of the species diversity of Legionella in the northern Baltic Sea (Gulf of Bothnia) during early summer to map their occurrence and to identify possible environmental drivers. We detected Legionella and potential protozoan hosts along gradients of the Gulf of Bothnia. We also for the first time present third generation full-length 16S rRNA amplicon sequencing (Nanopore) to resolve environmental species classification of Legionella, with a method suitable to study all bacteria. Our data show that full length 16S rRNA sequences is sufficient to resolve Legionella while the standard short Illumina sequences did not capture the entire diversity. For accurate species classification of Legionella, harmonization between the Nanopore classification methods is still needed and the bias toward the well-studied Legionella pneumophila need to be resolved. Different Legionella species occurred both in the Bothnian Sea and in the Bothnian Bay and their abundance were linked to humic substances and low salinity. The relative abundance of Legionella was higher in the humic-rich northern waters of the Bothnian Bay. The link between Legionella species and humic substances may be indirect via promotion of the heterotrophic microbial food web, allowing Legionella species and similar bacteria to establish. Humic substances are rich in iron, which has been shown crucial for growth of Legionella species and other pathogens. Considering climate change projections in this regional area, with increased humification and freshwater inflow, this bacterial niche containing potential pathogens might become more widespread in the future Baltic Sea. This study demonstrates the significance of DNA sequencing to monitor public health relevant bacteria like Legionella species in the environment. Including sequencing of bacteria and protozoa in the environmental monitoring programs could be used to identify ecosystem imbalances, which enable appropriate responses to emerging diseases.

The hidden presence of opportunistic bacterial pathogens in the environment evokes concerns about emerging diseases, especially in the light of climate change. The co-evolution of bacteria and their predators (protozoa) has led to bacterial defence strategies of which some contribute to the ability of bacteria to cause disease. To increase our understanding of the interplay between bacteria, protozoa, land use, and climate scenarios in Nordic brackish and freshwater, four studies were designed. The first study explored the co-occurrence patterns between predation resistant bacteria (PRB) and bacterivorous protozoa in a coastal area in the northern Baltic Sea. The results showed higher PRB diversity in the bays and freshwater inlets, than in the offshore waters. Further, genotype specific interactions between protozoa and bacteria were identified. The second study focused on Legionella species diversity and their association with humic substances and low salinity, potentially facilitated through the promotion of the heterotrophic microbial food web or by iron availability. The third study examined the impact of intensified land use on bacterial taxa abundance and community composition in lake inflows, demonstrating indirect downstream effects on water quality. Factors such as pastures, fields, farms, aluminium, iron, and humic substances were linked to increased Legionella abundance. The fourth study exposed aquatic organisms to climate change scenarios, causing eutrophication or brownification with elevated iron levels. Pseudomonas aeruginosa were found to be especially persistent to iron, likely linked to the same mechanism that enables survival in protozoan cells. This trait was shared with other observed intracellular pathogens and uncultured species, who showed elevated resilience to brownification and ability to survive outside host cells. These findings identified complex relationships, which improve our understanding of the intricate dynamics that shape aquatic ecosystems, and highlight the importance of considering multiple factors in managing water resources and maintaining ecosystem health. Human activities including intensified land use can have far-reaching consequences, jeopardizing the pristine nature of water bodies and escalate the presence of environmental and opportunistic bacterial pathogens.

Bacteria are major utilizers of dissolved organic matter in aquatic systems. In coastal areas bacteria are supplied with a mixture of food sources, spanning from refractory terrestrial dissolved organic matter to labile marine autochthonous organic matter. Climate scenarios indicate that in northern coastal areas, the inflow of terrestrial organic matter will increase, and autochthonous production will decrease, thus bacteria will experience a change in the food source composition. How bacteria will cope with such changes is not known. Here, we tested the ability of an isolated bacterium from the northern Baltic Sea coast, Pseudomonas sp., to adapt to varying substrates.

We performed a 7-months chemostat experiment, where three different substrates were provided: glucose, representing labile autochthonous organic carbon, sodium benzoate representing refractory organic matter, and acetate – a labile but low energy food source. Growth rate has been pointed out as a key factor for fast adaptation, and since protozoan grazers speed-up the growth rate we added a ciliate to half of the incubations. The results show that the isolated Pseudomonas is adapted to utilize both labile and ring-structured refractive substrates. The growth rate was the highest on the benzoate substrate, and the production increased over time indicating that adaptation did occur. Further, our findings indicate that predation can cause Pseudomonas to change their phenotype to resist and promote survival in various carbon substrates. Genome sequencing reveals different mutations in the genome of adapted populations compared to the native populations, suggesting the adaptation of Pseudomonas sp. to changing environment.

Our knowledge of aquatic fungal communities, their assembly, distributions and ecological roles in marine ecosystems is scarce. Hence, we aimed to investigate fungal metacommunities of coastal habitats in a subarctic zone (northern Baltic Sea, Sweden). Using a novel joint species distribution model and network approach, we quantified the importance of biotic associations contributing to the assembly of mycoplankton, further, detected potential biotic interactions between fungi–algae pairs, respectively. Our long-read metabarcoding approach identified 493 fungal taxa, of which a dominant fraction (44.4%) was assigned as early-diverging fungi (i.e. Cryptomycota and Chytridiomycota). Alpha diversity of mycoplankton declined and community compositions changed along inlet–bay–offshore transects. The distributions of most fungi were rather influenced by environmental factors than by spatial drivers, and the influence of biotic associations was pronounced when environmental filtering was weak. We found great number of co-occurrences (120) among the dominant fungal groups, and the 25 associations between fungal and algal OTUs suggested potential host–parasite and/or saprotroph links, supporting a Cryptomycota-based mycoloop pathway. We emphasize that the contribution of biotic associations to mycoplankton assembly are important to consider in future studies as it helps to improve predictions of species distributions in aquatic ecosystems.

The co-existence of bacteria and protozoa in aquatic environments has led to the evolution of predation defense mechanisms by the bacteria. Some of the predation-resistant bacteria (PRB) are also pathogenic to humans and other mammals. The links between PRB and protozoa in natural aquatic systems are poorly known, but they are important in predicting outbreaks and determining the long-term consequences of a contamination event. To elucidate co-occurrence patterns between PRB (16S rRNA) and bacterivorous protozoa (18S rRNA), we performed a field study in a coastal area in the northern Baltic Sea. Interactions between bacteria and protozoa were explored by using two complementary statistical tools. We found co-occurrence patterns between specific PRB and protozoa, such as Legionella and Ciliophora, and we also found that the interactions are genotype-specific as, for example, Rickettsia. The PRB sequence diversity was larger in bays and freshwater inlets compared to offshore sites, indicating local adaptions. Considering the PRB diversity in the freshwater in combination with the large spring floods in the area, freshwater influxes should be considered a potential source of PRB in the coastal northern Baltic Sea. These findings are relevant for the knowledge of survival and dispersal of potential pathogens in the environment.