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The ecological roles of planktonic archaeal and bacterial ammonia oxidizers in the oceanic nitrogen cycle

Final Activity Report Summary - ERAMMON (The ecological roles of planktonic archaeal and bacterial ammonia oxidizers in the oceanic nitrogen cycle)

The nitrogen (N) cycle has a complex, central role within the oceanic biogeochemical cycles. In the marine environment, prokaryotic plankton are the major drivers of the nitrogen cycling processes. Recently, it has been shown that apart from Bacteria belonging to the ?- and ?-Proteobacteria, some Archaea (Crenarchaeota) are also capable of performing ammonia oxidation, a key process in the marine nitrogen cycle. However, the physiological ecology and the precise contribution of both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to the nitrification remains almost unknown.

To address this question, the project aimed to gain insights into nitrification in the marine environments by assessment of richness, distribution and dynamics abundance of AOA in relation to AOB and the influence of environmental factors such as the oxygenated surface seawaters. To achieve this, a highly integrated approach was employed including water chemistry, quantitative RT-PCR gene analysis and epifluorescence microscopy.

The gene encoding the active site of the ammonia monooxygenase (amoA) has been exploited as a molecular marker for studying AOB and AOA diversity in natural environments. Using molecular techniques (quantitative RT-PCR) both the archaeal and bacterial (?- and ?-AOB) nitrification amoA gene was detected and quantified in coastal surface waters of two semi-enclosed eutrophic shelf seas (NW Black Sea and eastern Irish Sea) over an annual cycle.

Autotrophic AOB and AOA amoA gene were detected in all seawater samples, implying their ubiquity in the coastal system. However, ammonia-oxidizing members of archaeal and the bacterial domain (?-AOB and ?-AOB) exhibited a substantially different pattern of distribution. In the turbid but well oxygenated brackish NW Black Sea waters, ?-AOB and AOA amoA genes were more abundant than ?-AOB amoA genes. In contrast, a higher ?-AOB and AOA amoA genes abundance was measured in the homogenous more saline surface waters of the Irish Sea basin. Further, the number of expressed crenarchaeal versus bacterial amoA genes was always higher and suggested that Archaea contribute significantly more to nitrification in the coastal Black Sea and Irish Sea than Bacteria. The key differences between environmental conditions of the studied marine coastal systems were temperature, salinity, NH4+ and NO3- concentration. In general, higher levels of amoA gene expression coincided with highest NH4+ concentration. Although salinity and inorganic nitrogen species was the main environmental variables that were quantifiable, co-variation of other key parameters make them difficult to discern as the single driving factor.

We confirmed our results using molecular tools by exploiting a modified Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) method. We could show that Nitrosococcus-like bacteria estimated from Nscoc128 hybridized cell counts seemed to predominate over Nitrosomonas- and Nitrospira-like bacteria (detected by ?-AOB specific oligonucleotide probes Nso190 and Nso1225 respectively) and shared the same general patterns as AOA in the Danube-Black Sea mixing zone waters.

The project has also included quantification of the nitrite reductase nirK gene in both the coastal study sites. Recent genetic investigations have revealed the presence of a denitrification functional nirK gene in both AOA and AOB and by inference suggests they may be associated with denitrifiers, contributing significantly to greenhouse warming by reduction of NO2- to N2O and NO. By using RT-PCR analysis we found that nirK genes were present and co-existed with amoA genes in coastal surface waters of the Black Sea and Irish Sea. Considering the AOA predominance in almost all analysed seawater samples, our results suggest that AOA may also be responsible for both nitrification and denitrification in coastal environments such as the NW Black Sea and eastern Irish Sea. However, whether marine AOA are capable of denitrification remains an important area for future study.

To our best knowledge, these are the first publishable data on nitrification and the active microorganisms at both NW Black Sea and Irish Sea; such analyses have never been performed before for these marine systems. This project provides increasingly convincing evidence for the importance of both archaeal and bacterial ammonia-oxidizers in the nitrogen cycling processes such as nitrification and denitrification.