The high availability of electron donors occurring in coastal upwelling ecosystems with marked oxyclines favours chemoautotrophy, in turn leading to high N₂O and CH₄ cycling associated with aerobic NH₄⁺ (AAO) and CH₄ oxidation (AMO). This is the case of the highly productive coastal upwelling area off central Chile (36° S), where we evaluated the importance of total chemolithoautotrophic vs. photoautotrophic production, the specific contributions of AAO and AMO to chemosynthesis and their role in gas cycling. Chemolithoautotrophy was studied at a time-series station during monthly (2007–2009) and seasonal cruises (January 2008, September 2008, January 2009) and was assessed in terms of the natural C isotopic ratio of particulate organic carbon (δ¹³POC), total and specific (associated with AAO and AMO) dark carbon assimilation (CA), and N₂O and CH₄ cycling experiments. At the oxycline, δ¹³POC averaged −22.2‰; this was significantly lighter compared to the surface (−19.7‰) and bottom layers (−20.7‰). Total integrated dark CA in the whole water column fluctuated between 19.4 and 2.924 mg C m⁻² d⁻¹, was higher during active upwelling, and contributed 0.7 to 49.7% of the total integrated autotrophic CA (photo plus chemoautotrophy), which ranged from 135 to 7.626 mg C m⁻² d⁻¹, and averaged 20.3% for the whole sampling period. Dark CA was reduced by 27 to 48% after adding a specific AAO inhibitor (ATU) and by 24 to 76% with GC7, a specific archaea inhibitor. This indicates that AAO and AMO microbes (most of them archaea) were performing dark CA through the oxidation of NH₄⁺ and CH₄. Net N₂O cycling rates varied between 8.88 and 43 nM d⁻¹, whereas net CH₄ cycling rates ranged from −0.41 to −26.8 nM d⁻¹. The addition of both ATU and GC7 reduced N₂O accumulation and increased CH₄ consumption, suggesting that AAO and AMO were responsible, in part, for the cycling of these gases. These findings show that chemically driven chemolithoautotrophy (with NH₄⁺ and CH₄ acting as electron donors) could be more important than previously thought in upwelling ecosystems, raising new questions concerning its relevance in the future ocean.

Composition, carbon and nitrogen uptake, and gene transcription of microbial mat communities in Porcelana neutral hot spring (Northern Chilean Patagonia) were analyzed using metagenomics, metatranscriptomics and isotopically labeled carbon (H¹³CO₃) and nitrogen (¹⁵NH₄Cl and K¹⁵NO₃) assimilation rates. The microbial mat community included 31 phyla, of which only Cyanobacteria and Chloroflexi were dominant. At 58^∘C both phyla co-occurred, with similar contributions in relative abundances in metagenomes and total transcriptional activity. At 66^∘C, filamentous anoxygenic phototrophic Chloroflexi were >90% responsible for the total transcriptional activity recovered, while Cyanobacteria contributed most metagenomics and metatranscriptomics reads at 48^∘C. According to such reads, phototrophy was carried out both through oxygenic photosynthesis by Cyanobacteria (mostly Mastigocladus) and anoxygenic phototrophy due mainly to Chloroflexi. Inorganic carbon assimilation through the Calvin–Benson cycle was almost exclusively due to Mastigocladus, which was the main primary producer at lower temperatures. Two other CO₂ fixation pathways were active at certain times and temperatures as indicated by transcripts: 3-hydroxypropionate (3-HP) bi-cycle due to Chloroflexi and 3-hydroxypropionate-4-hydroxybutyrate (HH) cycle carried out by Thaumarchaeota. The active transcription of the genes involved in these C-fixation pathways correlated with high in situ determined carbon fixation rates. In situ measurements of ammonia assimilation and nitrogen fixation (exclusively attributed to Cyanobacteria and mostly to Mastigocladus sp.) showed these were the most important nitrogen acquisition pathways at 58 and 48^∘C. At 66^∘C ammonia oxidation genes were actively transcribed (mostly due to Thaumarchaeota). Reads indicated that denitrification was present as a nitrogen sink at all temperatures and that dissimilatory nitrate reduction to ammonia (DNRA) contributed very little. The combination of metagenomic and metatranscriptomic analysis with in situ assimilation rates, allowed the reconstruction of day and night carbon and nitrogen assimilation pathways together with the contribution of keystone microorganisms in this natural hot spring microbial mat.

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). Insitu nitrogenase activity (acetylene reduction), nitrogen fixation rates (cellular uptake of 15N2) 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.