Enrichment and characterization of ammonia-oxidizing archaea from the open ocean : phylogeny, physiology and stable isotope fractionation

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 5 (2011): 1796–1808, doi:10.1038/ismej.2011.58.Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the north Pacific Ocean that have been maintained in laboratory culture for over three years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S rRNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98-99% identity) to those recovered in DNA and cDNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonium (NH4 +) to nitrite (NO2 -). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (15εNH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal 15εNH3 ranged from 13- 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by 15εNH3 is similar.This work was supported by a Woods Hole Oceanographic Institution (WHOI) Postdoctoral Scholar fellowship to AES and the WHOI Ocean Life Institute

Influence of low light and a light: Dark cycle on NO3- uptake, intracellular NO3-, and nitrogen isotope fractionation by marine phytoplankton

The nitrogen isotope enrichment factor (epsilon) of four species of marine phytoplankton grown in batch cultures was determined during growth in continuous saturating light, continuous low light, and a 12:12-h light:dark cycle, with nitrate as a nitrogen source. The low growth rate that resulted from low irradiance caused an increased accumulation of the intracellular nitrate pool and/or a reduction in cell volume and was correlated to a species-specific increase in the measured epsilon value, compared with the saturating light conditions. The largest response was in the diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle, which showed a nearly 3-fold increase between high and low light conditions (6.2-15.2parts per thousand). The smallest response was in T. pseudonana (Hustedt) Hasle et Heimdal, which showed no change in the epsilon value of approximately 5parts per thousand in both high and low light conditions. There was significant but smaller increases in the epsilon value for the diatom T. rotula Meunier (2.7-5.6parts per thousand) and the prymnesiophyte Emiliania huxleyi (Lohm.) Hay et Mohler (4.5-9.4parts per thousand) between high and low light levels. In the light:dark experiments, all three diatoms but not the prymnesiophyte exhibited an increase in epsilon. This increase was linked to the ability of diatoms to assimilate nitrate at night. The results of the these experiments suggest that the light regime influences the relative uptake, assimilation, and efflux rates of nitrate and results in differences in the expression of the isotope effect by the enzyme nitrate reductase. Therefore, variations in nitrate isotope fractionation in nature can be more accurately interpreted when the light regime and species composition are taken into consideration

The mechanism of isotope fractionation during algal nitrate assimilation as illuminated by the N-15/N-14 of intracellular nitrate

The N-15/N-14 of nitrate in the external medium and intracellular pool of the cultured marine diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle was measured during nitrate assimilation under low light, a 12:12-h light:dark cycle, low temperature, or low iron conditions. The N-15/N-14 of the nitrate in the medium and the particulate matter both followed the predicted Rayleigh fractionation model, and the intracellular nitrate always had a higher N-15/N-14 than did the medium nitrate. When the experiments were compared, the results showed a negative correlation between the isotope fractionation factor and the difference in the N-15/N-14 between the two pools of nitrate. These observations imply that the variations in the isotope effect result from variations in the degree to which the fractionation by nitrate reductase is expressed outside the cell, which is, in turn, controlled by the rate of nitrate efflux relative to nitrate reduction. The low iron and low temperature experiments showed relatively small isotope effects but a large intracellular-medium difference in nitrate N-15/N-14, consistent with a relative rate of efflux (compared with influx) that is small and similar to fast-growing cells. In contrast, large isotope effects and small intracellular-medium differences in nitrate N-15/N-14 were observed for low light and light:dark cycle grown cells and are explained by higher relative rates of nitrate efflux under these growth conditions

Nitrogen isotope fractionation in 12 species of marine phytoplankton during growth on nitrate

The nitrogen isotopic composition of 12 species of marine phytoplankton were determined by isotope ratio mass spectrometry in order to investigate isotope fractionation associated with growth on nitrate. The species, representing diatoms, coccolithophores, dinoflagellates, green algae, and cyanobacteria, were grown in batch cultures in artificial seawater under the same laboratory conditions of constant light and temperature. The species (with isotope fractionation values in parenthesis) were: Thalassiosira weissflogii (6.2 +/- 0.4parts per thousand); Chaetoceros simplex (2.7 +/- 0.3parts per thousand); Ditylum brightwellii (3.3 +/- 0.4parts per thousand); Skeletonema costatum (2.7 +/- 0.3parts per thousand); Phaeodactylum tricornutum (4.8 +/- 0.3parts per thousand); Emiliania huxleyi (4.5 +/- 0.2parts per thousand), Isochrysis galbana (3.2 +/- 0.4parts per thousand); Pavlova lutheri, (3.6 +/- 0.5parts per thousand); Amphidinium carterae (2.2 +/- 0.3parts per thousand); Prorocentrum minimum (2.5 +/- 0.3parts per thousand); Dunaliella tertiolecta (2.2 +/- 0.2parts per thousand); and Synechococcus sp. (5.4 +/- 0.6parts per thousand). There was no relationship between isotope fractionation and organism group, nor was there a direct effect of cell size or growth rate on the degree of isotope fractionation among all the groups, Overall, the results show that isotope fractionation during growth on nitrate is lower than values obtained from field samples (i.e. 4 to 9parts per thousand). These results indicate that there is no simple mechanism for describing differences in isotope fractionation between groups of phytoplankton, and that a physiological understanding of isotope fractionation during uptake and assimilation of nitrate is needed to properly understand the delta(15)N signal generated by phytoplankton in the ocean

The Contamination of Commercial 15N2 Gas Stocks with 15N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

We report on the contamination of commercial 15-nitrogen (N-15) N-2 gas stocks with N-15-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. N-15(2) gas is used to estimate N-2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled N-15(2) into organic matter. However, the microbial assimilation of bioavailable N-15-labeled N-2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N-2 fixation rates. N-15(2) gas procured from three major suppliers was analyzed for the presence of these N-15-contaminants. Substantial concentrations of N-15-contaminants were detected in four Sigma-Aldrich N-15(2) lecture bottles from two discrete batch syntheses. Per mole of N-15(2) gas, 34 to 1900 mmoles of N-15-ammonium, 1.8 to 420 mmoles of (15)Nnitrate/nitrite, and 0.01 nmoles N L-1 d(-1), to 530 nmoles N L-1 d(-1), contingent on experimental conditions. These rates are comparable to, or greater than, N-2 fixation rates commonly detected in field assays. These results indicate that past reports of N-2 fixation should be interpreted with caution, and demonstrate that the purity of commercial N-15(2) gas must be ensured prior to use in future N-2 fixation rate determinations