Abstract
We investigated the N-utilizing bacterial community in anoxic brackish groundwater of the low and intermediate level nuclear waste repository cave in Olkiluoto, Finland, at 100 m depth using 15N-based stable isotope probing (SIP) and enrichment with 14∕15N-ammonium or 14∕15N-nitrate complemented with methane. Twenty-eight days of incubation at 12°C increased the concentration of bacterial 16S rRNA and nitrate reductase (narG) gene copies in the substrate amended microcosms simultaneously with a radical drop in the overall bacterial diversity and OTU richness. Hydrogenophaga/Malikia were enriched in all substrate amended microcosms and Methylobacter in the ammonium and ammonium+methane supplemented microcosms. Sulfuricurvum was especially abundant in the nitrate+methane treatment and the unamended incubation control. Membrane-bound nitrate reductase genes (narG) from Polarimonas sp. were detected in the original groundwater, while Burkholderia, Methylibium, and Pseudomonas narG genes were enriched due to substrate supplements. Identified amoA genes belonged to Nitrosomonas sp. 15N-SIP revealed that Burkholderiales and Rhizobiales clades belonging to the minority groups in the original groundwater used 15N from ammonium and nitrate as N source indicating an important ecological function of these bacteria, despite their low number, in the groundwater N cycle in Olkiluoto bedrock system.
Highlights
Nitrogen (N) is one of the basic elements of all life forms and is essential for the production of amino and nucleic acids (Bothe et al, 2007)
The bacterial community of groundwater from 100 m depth in the VLJ-cave situated in the crystalline bedrock of the Fennoscandian Shield was affected by ammonia and nitrate supplementation
Known Nitrate-reducing bacteria (NRB) belonging to ε-proteobacterial Sulfuricurvum and β-proteobacterial Hydrogenophaga/Malikia clades were greatly enriched when groundwater microcosms were supplemented with ammonium or nitrate with or without methane
Summary
Nitrogen (N) is one of the basic elements of all life forms and is essential for the production of amino and nucleic acids (Bothe et al, 2007). Use of explosives in e.g., construction into bedrock environments and mining may increase the concentration of N compounds in these environments, due to under detonated explosives (Beller et al, 2004). Use of pesticides, such as atrazine, has been shown to be a source of N pollution in karst aquifers that are in connection to agricultural sites (Iker et al, 2010). Despite the generally low metabolic activity of deep groundwater microorganisms (e.g., D’Hondt et al, 2002; Rajala et al, 2015a) these N compounds may affect the activity and growth of deep bedrock microbial communities. Nitrate is generally present only at low concentrations in pristine anoxic crystalline bedrock groundwater because it has been used as an alternative electron acceptor to oxygen and reduced to Microbial nitrogen cycling in groundwater
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