Ground emissions are largely responsible for the increase of the potent

Ground emissions are largely responsible for the increase of the potent greenhouse gas nitrous oxide (N2O) in the atmosphere and are generally attributed to the activity of nitrifying BMS-536924 and denitrifying bacteria. that they are not capable of denitrification. In 15N-labeling experiments we provide evidence that both ammonium and nitrite contribute equally via hybrid N2O formation to the N2O produced by under all conditions tested. Our results suggest that archaea may contribute to N2O production in terrestrial ecosystems however they are not capable of nitrifier-denitrification and thus do not produce increasing amounts of the greenhouse gas when oxygen becomes limiting. 2008 Smith (2012) already in the year 2000 total N2O emissions accounted for 15.8?Tg N2O-N?12 months?1 in which 5.6-6.5?Tg N2O-N?12 months?1 could be assigned to an anthropogenic source and 4.3-5.8?Tg N2O-N?12 months?1 to a land or coastal biological source. The main processes responsible for gaseous nitrogen emissions from ground are microbial transformations of ammonium nitrite nitrate and to a lesser extent chemodenitrification (Colliver and Stephenson 2000 Baggs 2008 2011 Campbell EN76 was managed at 37?°C in fresh water medium according to Tourna (2011). The AOA SCM1 was incubated at 28?°C in SCM medium according to K?nneke (2005). and cultures were supplied with 1?mM ammonium and in addition with 0.1?mM pyruvate and 0.1?mM oxaloacetate respectively. The media of and cultures were buffered with HEPES to a pH of 7.5. The AOB ATCC 25196T (supplied by Jim Prosser Aberdeen) was cultivated at 28?°C in Skinner and Walker (S+W) medium (Skinner and Walker 1961 containing 1?mM ammonium and phenol red (0.5?mg) as pH indicator at a pH of 7.5-8. The pH was regularly adjusted by adding Na2CO3. Cultures were inoculated with 10% volumes of culture. Growth was followed via photometric determination of ammonium consumption and nitrite production using a salicylic acid assay (Kandeler and Gerber 1988 or a Grie? reagent system (Promega Madison WI USA) for the latter. Screenings for contaminations were carried out regularly using light microscopy and PCR. Late exponential cultures were used to inoculate cultures for the determination of N2O production (10% inoculum) which have been set up in serum BMS-536924 bottles (122?ml total; 20-30?ml medium; sealed with butyl rubber stoppers). DNA extraction Nucleic acids were extracted based on a altered protocol of Griffiths (2000) using 2-ml Lysing Matrix E tubes (MP biomedicals Eschwege Germany) made up of a mixture of silica ceramic and glass beads in combination with the BIO101/Savant FastPrepFP120A Instrument (Qbiogene Illkirch France) for bead beating. Briefly 1 of culture was harvested and the cell pellet was dissolved in 0.5?ml SDS extraction buffer CD247 (0.7?M NaCl 0.1 Na2SO3 0.1 Tris/HCl (pH 7.5) 0.05 EDTA (pH8) 1 SDS). The further extraction was performed as explained in the study by Nicol (2005) with a DNA precipitation over night at 4?°C. Quantitative PCR Archaeal genes were quantified using the primers Cren771F and Cren957R (Ochsenreiter gene of was used with an efficiency of 101% and a slope of ?3.3. The qPCR was performed in a realplex cycler (Mastercycler ep realplex Eppendorf Vienna Austria) with the following PCR conditions: 95?°C for 15?min 40 cycles of 30?s at 95?°C 30 at 54?°C and 30?s at 72?°C followed by a melting curve analysis BMS-536924 at the end of the run to indicate the amplification of specific products. qPCR data were generated from impartial DNA extractions of quadruplicate cultures with BMS-536924 duplicated PCR amplifications. N2O quantification Cultures for the quantification of N2O were set up in replicates (3-5 cultures BMS-536924 each) in serum bottles made up of 20?ml new water medium. In addition one blank with medium only and another one with lifeless cells (autoclaved culture) as inoculum were BMS-536924 prepared. Production of N2O was tested under one fully aerated condition with 21% oxygen in the headspace and three oxygen limited conditions. To achieve this reduced pressure was applied for 30?s followed by flushing with sterile filtered N2 (0% oxygen in headspace). To achieve a concentration of 10% and 3% oxygen in the gaseous phase a defined amount of N2 was replaced by sterile filtered air flow. Initial oxygen concentrations in the aqueous phase of the cultures (37?°C) were measured with an oxygen microsensor (Presens Regensburg Germany). Initial O2 concentrations in and cultures (28?°C) were calculated according to Henry’s legislation. Oxygen concentrations measured in the aqueous phase revealed that this aimed gaseous O2 concentrations were.