Nitrite oxidation in geothermal springs: evidence of an upper temperature limit for thermophilic nitrite-oxidizing bacteria of 60 - 65 °C
Creators: name: Edwards, Tara 
Publisher: name: University of Nevada, Las Vegas
Year: 2012

description: Despite growing evidence of the importance of nitrification in terrestrial geothermal environments, little is known about nitrite oxidation in these environments. In order to further our knowledge, this study combined cultivation-dependent and -independent approaches with measurements of nitrogen speciation along the outflow channels of two Great Basin geothermal springs. Enrichment cultures were inoculated with sediment slurries from sites ranging in temperature from 42 - 87 °C at the sources and along the outflows of >15 hot springs. While attempts to enrich nitrite-oxidizing bacteria (NOB) from sites greater than or equal to 61 °C were unsuccessful, NOB were enriched from five hot springs located in U.S. Great Basin, southwestern China, and Armenia at sites °C. All enrichments analyzed contained organisms with greater than or equal to 97% 16S rRNA gene identity to Nitrospira calida, regardless of origin, demonstrating the wide geographic range of this organism. In addition, enrichments from Armenia contained organisms with greater than or equal to 97% 16S rRNA gene identity to Nitrospira moscoviensis. Physiological properties were similar for all enrichments, with an upper temperature limit between 60 - 65 °C and a temperature optimum of 45 - 50 °C While the rates of nitrite oxidation were significantly different for the Great Basin enrichments when compared to the Tengchong and Armenian enrichments at 50 °C (5.4 ± 2.2, 11.4 ± 3.3, and 11.6 ± 1.7 for Great Basin, Tengchong, and Armenian, respectively), the much higher rates observed for the Tengchong and Armenian enrichments may be attributed to improved cultivation conditions for the primary enrichments. 
Patterns of nitrogen speciation in water samples collected along the outflow channels of two springs (Sandy's Spring West and Rick's Hot Creek) within the Great Boiling Spring (GBS) geothermal system suggested ammonia oxidation activity at greater than or equal to 75.1 °C in both springs. In contrast, nitrite oxidation activity did not appear to be present at greater than or equal to 65 °C in either spring. "Candidatus Nitrosocaldus yellowstonii" 16S rRNA gene copy numbers were abundant in sediment samples from the outflow of both springs at less than or equal to 79.6 °C, butNitrospira16S rRNA gene sequences were only abundant at less than or equal to 57.9 °C. Thus, an apparent difference in the upper temperature limit for ammonia oxidation and nitrite oxidation exists within the GBS system, decoupling the two steps of nitrification and leading to accumulation of nitrite above ~60 °C. In addition, 16S rRNA sequences belonging to known NOB were absent from 557,076 pyrotag sequences obtained from hot springs located in the U.S. Great Basin, and Tengchong, China at temperatures greater than or equal to 55 °C and no significant matches for entire NOB genome sequences were found in the ~250 Mbp of metagenomic data from GBS environmental samples at 77 to 85 °C. In addition, no significant matches for entire NOB genome sequences were found in the 557 Mbp of metagenomic data from Yellowstone National Park environmental samples obtained from 16 springs at temperatures ranging from 52.9 to 90 °C. This study presents evidence that the upper temperature limit for nitrite oxidation in geothermal systems worldwide may be similar to the upper temperature limit observed within the GBS system and demonstrates the wide geographic range of Nitrospira spp. in geothermal environments. Finally, we propose that the temperature-driven decoupling of ammonia oxidation and nitrite oxidation leads to a high temperature nitrite shunt in the nitrogen cycle whereby nitrite produced by ammonia oxidation is used directly by denitrifiers in geothermal ecosystems. We propose that the high temperature nitrite shunt is complete at temperatures exceeding ~65 °C, with greater flow of nitrogen through nitrate with decreasing temperature below ~65 °C.