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Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO

Bacillus vireti is a nitrate-ammonifying bacterium and a partial denitrifier, reducing NO3 (-) , NO2 (-) , NO and N2 O with NarG, NrfA, CbaA and NosZ respectively. Growth is optimized through successive use of the electron acceptors O2 and NO3 (-) , followed by NO2 (-) , NO and N2 O. Fermentation takes place simultaneously with anaerobic respiration. When grown in batch culture with 5 mM initial NO3 (-) , transcription of nrfA was high and most NO3 (-) was reduced to NH4 (+) . With 20 mM initial NO3 (-) , nrfA transcription was lower and more than 50% of the nitrate was recovered as NO, N2 O and N2 . Analysis of gene transcription patterns and corresponding gas kinetics indicated that O2 and NO2 (-) or NO are main controllers of nrfA, nirB, cbaA and nosZ transcription. This was corroborated by analyses of putative binding regions for specific transcriptional regulators. Furthermore, we demonstrate that N2 O reduction in B. vireti supports growth. The high nosZ transcription but low N2 O production seen at 5 mM NO3 (-) implies that this organism can use N2 O reductase to scavenge N2 O from other organisms in the soil, thus possibly acting as a net sink for N2 O.

Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O.

Several Gram-positive bacteria carry genes for anaerobic reduction of NO3(-) via NO2(-) to NH4(+) or gaseous nitrogen compounds, but the processes are understudied for these organisms. Here, we present results from a whole-genome analysis of the soil bacterium Bacillus vireti and a phenotypic characterization of intermediate and end-products, formed under anoxic conditions in the presence of NO3(-). Bacillus vireti has a versatile metabolism. It produces acetate, formate, succinate and lactate from fermentation and performs dissimilatory nitrate reduction via NO2(-) to ammonium (DNRA) using NrfA, while NirB may detoxify NO2(-) in the cytoplasm. Moreover, it produces NO from an unknown source and reduces it via N2O to N2 using two enzymes connected to denitrification: an unusual NO reductase, qCuA Nor encoded by cbaA, and a z-type N2O reductase, encoded by nosZ. In batch cultures, B. vireti reduced all NO3(-) to NO2(-) before the NO2(-) was reduced further. The quantities of all products varied with the initial NO3(-) concentration. With 5 mM NO3(-) , 90% was reduced to NH4 (+) while with ≥ 20 mM NO3(-), 50% was reduced to NO, N2O and N2. This organism is thus an aggressive NO2(-) accumulator and may act as a net source and sink of NO and N2O.

Denitrifiers differ in how they handle the transition from oxic to anoxic respiration, with consequences for NO and N2O emissions. To enable stringent comparisons we defined parameters to describe denitrification regulatory phenotype (DRP) based on accumulation of NO2(-) , NO and N2O, oxic/anoxic growth and transcription of functional genes. Eight Thauera strains were divided into two distinct DRP types. Four strains were characterized by a rapid, complete onset (RCO) of all denitrification genes and no detectable nitrite accumulation. The others showed progressive onset (PO) of the different denitrification genes. The PO group accumulated nitrite, and no transcription of nirS (encoding nitrite reductase) was detected until all available nitrate (2 mM) was consumed. Addition of a new portion of nitrate to an actively denitrifying culture of a PO strain (T. terpenica) resulted in a transient halt in nitrite reduction, indicating that the electron flow was redirected to nitrate reductase. All eight strains controlled NO at nano-molar concentrations, possibly reflecting the importance of strict control for survival. Transient N2O accumulation differed by two orders of magnitude between strains, indicating that control of N2O is less essential. No correlation was seen between phylogeny (based on 16S rRNA and functional genes) and DRP.

summary The growth of ten species of ectomycorrhizal fungi was measured in liquid media containing different organic and inorganic nitrogen sources. All fungi grew well on ammonium. Growth on nitrate was generally lower, although there was considerable variation between different isolates of the same species. Suillus variegatus, Piloderma croceum, Paxillus involutus, Hebeloma crustuliniforme and unidentified pink and white isolates often grew as well on organic nitrogen sources as on ammonium. Growth of the other species was more variable. Isolates of Thelephora terrestris and Lactarius rufus varied in their ability to use bovine serum albumen (BSA) but two Laccaria species were poor at using the protein as a nitrogen source. The ability of mycorrhizal and non-mycorrhizal Pinus contorta Dougl. ex Loud, plants to utilize BSA was also examined. Non-mycorrhizal plants and mycorrhizal plants infected with either T. terrestris or the unidentified pink ectomycorrhizal symbiont were supplied either with ammonium or with BSA. Growth of plants supplied with BSA was significantly increased by mycorrhizal infection with the pink symbiont and not significantly different from that of plants supplied with ammonium, but non-mycorrhizal plants were unable to use the protein as a nitrogen source and had significantly lower yields and nitrogen contents than infected plants. In contrast, mycorrhizal infection with T. terrestris had no effect on growth or nitrogen contents of plants supplied with protein. The results are discussed in relation to possible physiological differences between ectomycorrhizal fungi occurring at different successional stages of forest development.