The cyanobacterium Mastigocladus fulfills the nitrogen demand of a terrestrial hot spring microbial mat

Abstract

Cyanobacteria from Subsection V (Stigonematales) are important components of microbial mats in non-acidic terrestrial hot springs. Despite their diazotrophic nature (N2 fixers), their impact on the nitrogen cycle in such extreme ecosystems remains unknown. Here, we surveyed the identity and activity of diazotrophic cyanobacteria in the neutral hot spring of Porcelana (Northern Patagonia, Chile) during 2009 and 2011-2013. We used 16S rRNA and the nifH gene to analyze the distribution and diversity of diazotrophic cyanobacteria. Our results demonstrate the dominance of the heterocystous genus Mastigocladus (Stigonematales) along the entire temperature gradient of the hot spring (69-38 °C). In situ nitrogenase activity (acetylene reduction), nitrogen fixation rates (cellular uptake of (15)N2) and nifH transcription levels in the microbial mats showed that nitrogen fixation and nifH mRNA expression were light-dependent. Nitrogen fixation activities were detected at temperatures ranging from 58 °C to 46 °C, with maximum daily rates of 600 nmol C2H4 cm(-2) per day and 94.1 nmol N cm(-2) per day. These activity patterns strongly suggest a heterocystous cyanobacterial origin and reveal a correlation between nitrogenase activity and nifH gene expression during diurnal cycles in thermal microbial mats. N and C fixation in the mats contributed ~3 g N m(-2) per year and 27 g C m(-2) per year, suggesting that these vital demands are fully met by the diazotrophic and photoautotrophic capacities of the cyanobacteria in the Porcelana hot spring.

Figure 1

(a) Location of the Porcelana hot spring in northern Patagonia, Chile (X Region, Comau fjord). (b) The pigmented microbial mat was formed throughout the temperature gradient; the sampling sites are indicated by red triangles. The gray bar represents the mat extension (~10 m) within the thermophilic temperature gradient.

Figure 2

Comparison of the interannual cyanobacterial diversity at different temperatures in the Porcelana hot spring based on the 16S rRNA gene and DGGE. (a) Bray–Curtis dissimilarity index dendrogram. (b) Relative abundance of 16S rRNA-DGGE bands (phylotypes) for each temperature and year investigated.

Figure 3

Relative abundance and interannual diazotrophic bacterial diversity in the Porcelana hot spring based on the nifH marker gene and clone libraries. (a) Bray–Curtis dissimilarity index dendrogram. (b) Relative abundance of the nifH gene (OTUs) determined using clone libraries obtained for each temperature and year investigated.

Figure 4

Nitrogen fixation assessed by the ARA and ¹⁵N₂ uptake analysis for the different temperatures and years investigated. ARA measurements (black bars) were conducted during the 4 years, whereas ¹⁵N₂ uptake measurements (gray bars) were performed in 2012 and 2013.

Figure 5

Diel cycles in nitrogenase activity (NA) and nifH gene expression in the Porcelana hot spring. (a) Diel cycles at different temperatures in 2012. The bars represent ARA and the dotted line represents the number of nifH gene transcripts at 47 °C. (b) Diel cycles at different temperatures in 2013. The bars and the dashed line represent activities at 58 °C and 48 °C. Error bars indicate the s.d. The top bar represents the light (white) and night (black) periods; the latter is also illustrated by gray shading.