Manabu Nishizawa, Miho Hirai, Shingo Hirao, Akiko Makabe, Tohru Kikuchi, Naohiro Yoshida, Keisuke Koba, Takuro Nunoura, Ken Takai, Masanori Kaneko, Takazo Shibuya, Jun-ichiro Ishibashi, Junichi Miyazaki, and Toshiro Yamanaka
We report here the concurrence and interaction among forms of nitrogen metabolism in thermophilic microbial mat communities that developed in an ammonium-abundant subsurface geothermal stream. First, the physical and chemical conditions of the stream water at several representative microbial mat habitats (including upper, middle and downstream sites) were characterized. A thermodynamic calculation using these physical and chemical conditions predicted that nitrification consisting of ammonia and nitrite oxidations would provide one of the largest energy yields of chemolithotrophic metabolisms. Second, near-complete prokaryotic 16S rRNA gene clone analysis was conducted for representative microbial mat communities at the upper, middle and downstream sites. The results indicated a dynamic shift in the 16S rRNA gene phylotype composition through physical and chemical variations of the stream water. The predominant prokaryotic components varied from phylotypes related to hydrogeno (H 2 )- and thio (S)-trophic Aquificales , thermophilic methanotrophs and putative ammonia-oxidizing Archaea (AOA) located upstream (72 °C) to the phylotypes affiliated with putative AOA and nitrite-oxidizing bacteria (NOB) located at the middle and downstream sites (65 and 57 °C, respectively). In addition, the potential in situ metabolic activities of different forms of nitrogen metabolism were estimated through laboratory experiments using bulk microbial mat communities. Finally, the compositional and isotopic variation in nitrogen compounds was investigated in the stream water flowing over the microbial mats and in the interstitial water inside the mats. Although the stream water was characterized by a gradual decrease in the total ammonia concentration (ΣNH 3 : the sum of ammonia and ammonium concentrations) and a gradual increase in the total concentration of nitrite and nitrate (NO 2 − + NO 3 − ), the total inorganic nitrogen concentration (TIN: the sum of ΣNH 3 , NO 2 − and NO 3 − concentrations) was nearly constant (250 μM) throughout the stream. Based on the level of detectable dissolved molecular oxygen (O 2 ) of the stream water (⩾38 μM) along with metabolic measurements, it was predicted that nitrification by thermophilic AOA and NOB components in the microbial mats that were exposed to the stream water would constrain the concentrations and isotopic ratios of ΣNH 3 , NO 2 − and NO 3 − of the stream water. The δ 15 N value of ΣNH 3 increased from 0‰ to 7‰ with decreasing concentration, which was consistent with the previously reported isotopic fractionation for microbial ΣNH 3 oxidation. In contrast, the δ 15 N value of NO 2 − was 22‰ lighter than that of NO 3 − in the steam water at the same site, indicating an inverse isotopic fractionation for microbial NO 2 − oxidation. The variation in concentrations and δ 15 N values of ΣNH 3 , NO 2 − and NO 3 − was largely explained using a two-step nitrification model, and the apparent nitrogen isotopic fractionations of ΣNH 3 oxidation and NO 2 − oxidation were estimated to be 0.986 and 1.020, respectively. In the interstitial water within the microbial mats, the compositional and isotopic properties of TIN at the downstream site indicated potential denitrification by the anaerobic microbial components. The geochemically deduced transition of microbial nitrogen metabolism was substantiated through cultivation-independent microbiological analyses.