Im Rahmen dieser Studie wurde die chemolithoautotrophe, Nitrit-oxidierende Bakteriengemeinschaft im Belebungsbecken einer kommunalen Kläranlage und im Biofiltersystem mariner Aquakulturanlagen untersucht. Die Zellaktivität der autotrophen Bakteriengemeinschaft ist über die Assimilation von 13C-CO2, welches den Kulturen in Form von stabilen Isotopen zugefügt wurde, ermittelt worden. Der Einbau von 13C in die Zellbestandteile der autotrophen Nitrit-oxidierenden Bakteriengemeinschaft wurde über die Fettsäureanalytik (FAME-SIP) detektierbar und kann über den Markierungsgrad dargestellt werden. Die Assimilierungen des 13C ermöglichen Rückschlüsse auf die metabolische Zellaktivität der Nitritoxidanten. Der chemotaxonomische Ansatz konnte mit molekularbiologischen Untersuchungen ergänzt werden. Hierzu wurden neu entwickelte spezifische Primer und Sonden für die Gattung Nitrospira eingesetzt. Die Primer sind für die spezifische Amplifikation der 16S rRNA verwendet worden, der direkte Nachweis der Organismen erfolgte über die Fluoreszenz in situ Hybridisierung (FISH) mittels Oligonukleotidsonden.

I Sverige finns det ungefär 25 000 riskklassade förorenade områden samt ännu fler områden som potentiellt är förorenade. Naturvårdsverket har tagit fram generella riktvärden för olika förorenade ämnen. Halter över riktvärdena antyder att risken för negativa effekter på människan eller miljön bedöms vara stor och att efterbehandling av området är aktuellt. Ett vanligt, men kostsamt efterbehandlingsalternativ är urschaktning och bortforsling av jord innehållandes halter av ämnen över riktvärdena. Ett intresse finns för att utveckla lätt tillämpbara metoder för platsspecifik riskbedömning av mark i syfte att skydda markmiljön och dess funktioner. Mikroorganismer som ingår i kvävets kretslopp utför nyckelfunktioner i jorden. Flera studier indikerar att dessa mikroorganismers abundans och aktivitet påverkas av flera olika markföroreningar. Inom forskningsprojektet APPLICERA har ett lysimeterexperiment genomförts där två jordar, med olika egenskaper med avseende på bland annat kornstorleksfördelning och pH (svagt basisk sandy loam respektive sur sand), förorenats med koppar respektive PAH:er i varierande halter. Halterna motsvarar riktvärden för känslig markanvändning (KM), mindre känslig markanvändning (MKM) och 3xMKM. Provtagning av jorden har skett vid tre tillfällen under en period av 16 månader. I detta examensarbete har nitrifierande mikroorganismers potential för att användas som indikatorer för förändrad markfunktion i förorenad jord undersökts. Detta har gjorts genom att kvantifiera ammoniakoxiderande arkéer (AOA), ammoniakoxiderande bakterier (AOB) samt de nitritoxiderande bakteriesläktena Nitrospira och Nitrobacter i jord som provtagits vid de olika tidpunkterna i lysimeterexperimentet. Mätning av potentiell ammoniakoxidation (PAO) har skett för jord från det sista provtagningstillfället. Resultaten visade att abundansen av mikroorganismerna generellt var större i den svagt basiska jorden klassificerad som sandy loam än i den sura jorden klassificerad som sand. Med tiden minskade AOA:s abundans för den högsta koncentrationen av koppar och PAH, särskilt utpräglad var minskningen i den sura jorden. Även Nitrospira minskade i abundans i PAH-förorenad jord för båda jordtyperna, dock kunde en minskning i abundans i kopparförorenad jord statistisk endast säkerställas för den sura jorden. AOB:s och Nitrobacters abundans visade inte på någon tydlig uppåt- eller nedåtgående trend varken över tid eller för ökande föroreningshalt. Skillnaden i kvantitet av mikroorganismer mellan jordtyperna antyder att jordens kemiska och fysiska egenskaper påverkade mikroorganismernas abundans såväl som föroreningarnas biotillgänglighet. PAO minskade med ökande kopparhalt för båda jordtyper, däremot sågs en minskning i PAO med ökande PAH-halt endast i den sura jorden. Resultaten antyder att abundansen av AOA och Nitrospiras verkar ha störst potential att utgöra generella indikatorer över ändrad markfunktion, dock lämpligen med komplettering av förbättrade mätningar av PAO för att få mer information om kvävets kretslopp i förorenad jord. / In Sweden there are around 25,000 contaminated areas classified as a risk for human health or the environment, and even more potentially contaminated areas. The Swedish Environmental Protection Agency has developed generic guideline values for hazardous substances.  Contamination levels exceeding the guideline values indicate a large risk for human health or the environment and that there is a need to remediate the site. A frequently used and expensive aftertreatment is excavation and removal of contaminated soil. Therefore, there is an interest to develop easy applicable methods for site-specific risk assessment with the purpose to protect the soil environment and its functions. Microorganisms involved in the nitrogen cycle perform key functions in soil. Several studies indicate that the abundance and activity of N-cycling microorganisms are sensitive to various soil pollution. Within the research project APPLICERA, a lysimeter experiment was conducted with two different soil types (acidic sandy soil and slightly alkaline sandy loam) and different contamination levels of copper and PAH. The contamination levels corresponded to generic guideline values for sensitive land-use (KM), less sensitive land-use (MKM) and 3xMKM. Soil samples were taken at three times during a period of 16 months. The aim of this master thesis was to investigate nitrifying microorganism potential as indicators for altered soil functioning in contaminated soil. Ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria genera Nitrospira and Nitrobacter has been quantified in soil samples with the different treatments. Measurements of the potential ammonia oxidation (PAO) have been performed in soil from the last sample taking.  The abundance of the nitrifying microorganisms was in general higher in the slightly alkaline sandy loam soil. Over time there was a decrease in AOA abundance for the highest concentration of copper and PAH, most pronounced in the acidic sandy soil. Also, a decrease in the abundance of Nitrospira in soil contaminated with PAHs could be seen for both soil types. In copper contaminated soil a distinct decrease in the abundance of Nitrospira could only be seen in the acidic sandy soil. The abundance of AOB and Nitrobacter did not show any obvious pattern for different contamination levels or over time. The difference in abundance of nitrifying microorganisms between the soil types suggests that the soil’s chemical and physical characteristics as well as the bioavailability of the contaminant affected their abundance. A significant decrease in PAO could be seen for the highest contamination level of copper for both soil types, but for the PAH contaminated soil PAO decreased only in the acidic sandy soil. These results suggest that the abundance of AOA and Nitrospira seems to have greatest potential as general indicators for altered soil functioning. However, improved activity measurements are needed to gain greater insight into the soil N-cycle functioning in contaminated soils.

The Upper Mississippi River (UMR) basin contributes over 50,000 metric tons of nitrogen (N) to the Gulf of Mexico each year, resulting in a “dead zone” inhospitable to aquatic life. Land-applied N (fertilizer) in the corn-belt is attributed with a majority of the N-load reaching the Gulf and is difficult to treat as run-off is considered a non-point source of pollution (i.e. not from a pipe). One solution to this “grand challenge” of intercepting N pollution is utilizing filter-feeding organisms native to the UMR. Freshwater mussel (order Unionidae) assemblages collectively filter over 14 billion gallons of water, remove tons of biomass from overlying water, and sequester tons of N each day. Our previous research showed mussel excretions increased the sediment porewater concentrations of ammonium by 160%, and indirectly increased nitrate and nitrite by 40%, presumably from microbial degradation of ammonium. In response, the goal of this research was to characterize how mussels influenced microbial communities (microbiome) to determine the fate of N in UMR sediment. First, we used qPCR and non-targeted amplicon sequencing within sediment layers to identify the N-cycling microbiome and characterized microbial community changes attributable to freshwater mussels. qPCR identified that anaerobic ammonium oxidizing (anammox) bacteria were increased by a factor of 2.2 at 3 cm below the water-sediment interface when mussels were present. Amplicon sequencing of sediment at depths relevant to mussel burrowing (3 and 5 cm) showed that mussel presence reduced microbial species richness and diversity and indicated that sediment below mussels harbored distinct microbial communities. Furthermore, mussels increased the abundance of ammonia oxidizing bacteria (family Nitrosomonadaceae), nitrite oxidizing bacteria (genus Nitrospira), but decreased the abundance of ammonia oxidizing archaea (genus Candidatus Nitrososphaera), and microorganisms which couple denitrification with methane oxidation. These findings suggested that mussels may enhance microbial niches at the interface of oxic and anoxic conditions, presumably through excretion of N and burrowing activity. In response, we performed metagenomic shotgun sequencing to identify which genes of the microbiome were most impacted by mussels. We hypothesized that genes responsible for ammonia and nitrite oxidation would be greater in the sediment with mussel assemblages. We found the largest abundance of N-cycling genes were responsible for nitrate reduction and nitrite oxidation, which is corroborated by the high concentration of nitrates in UMR water. Linear discriminant analysis statistical analyses showed nitrification genes were most impacted by mussels, and this presented an opposing effect on genes responsible for producing nitrous oxide, a potent greenhouse gas. Further investigation showed an increased abundance of a novel organism capable of completely oxidizing ammonia to nitrate (Candidatus Nitrospira inopinata) and coexisted with metabolically flexible Nitrospira (sp. moscoviensis), likely enhancing both carbon and N-cycling. We demonstrated that native mussels harbor a unique niche for N-cycling microorganisms with large metabolic potentials to degrade mussel excretion products. Our findings suggest the ecosystem services of mussels extend beyond water filtration, and includes enhanced biogeochemical cycling of carbon, N, and reduces the potential for a potent microbially-produced greenhouse gas. Ultimately, this research could be used to advocate for mussel habitat restoration in the UMR to lessen the impacts of non-point pollution.

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