Nitrogen Fixation in Thermophilic Chemosynthetic Microbial Communities Depending on Hydrogen, Sulfate, and Carbon Dioxide

Abstract

The activity of nitrogen fixation measured by acetylene reduction was examined in chemosynthetic microbial mats at 72-75°C in slightly-alkaline sulfidic hot springs in Nakabusa, Japan. Nitrogenase activity markedly varied from sampling to sampling. Nitrogenase activity did not correlate with methane production, but was detected in samples showing methane production levels less than the maximum amount, indicating a possible redox dependency of nitrogenase activity. Nitrogenase activity was not affected by 2-bromo-ethane sulfonate, an inhibitor of methanogenesis. However, it was inhibited by the addition of molybdate, an inhibitor of sulfate reduction and sulfur disproportionation, suggesting the involvement of sulfate-reducing or sulfur-disproportionating organisms. Nitrogenase activity was affected by different O₂ concentrations in the gas phase, again supporting the hypothesis of a redox potential dependency, and was decreased by the dispersion of mats with a homogenizer. The loss of activity that occurred from dispersion was partially recovered by the addition of H₂, sulfate, and carbon dioxide. These results suggested that the observed activity of nitrogen fixation was related to chemoautotrophic sulfate reducers, and fixation may be active in a limited range of ambient redox potential. Since thermophilic chemosynthetic communities may resemble ancient microbial communities before the appearance of photosynthesis, the present results may be useful when considering the ancient nitrogen cycle on earth.

Keywords: chemoautotrophic bacteria; microbial mats; nitrogen fixation; sulfate reduction; thermophilic bacteria.

Fig. 1

Photo images of two sampling sites (A, C) and typical pale tan colored microbial mats (B) and streamers (D) at Nakabusa hot springs. A, Microbial mats developed on a concrete wall that hot spring water runs down (Wall Site); B, typical pale tan colored microbial mats at 72–75°C at the Wall Site; C, a hot spring water stream in which pale tan colored streamers are observed (Stream Site); D, typical pale tan colored streamers at 75°C at the Stream Site.

Fig. 2

Bacterial community composition at the phylum level in microbial mats at the Wall Site (A) and streamers at the Stream Site (B) from 16S rRNA amplicon gene sequence results (V4 region). Less abundant OTUs (<0.2% read abundance for each) are shown all combined as “Others.”

Fig. 3

In situ nitrogenase activity of microbial mats and streamers A, Ethylene production after the in situ incubation of microbial mats and streamers. One to 17 were different vials containing microbial mats collected at the Wall Site (WS), and 18 to 20 were streamers collected at the Stream Site (SS). One to three and 18 to 20, four to 11, and 12 to 17 were collected on July 22, August 4, and November 8, 2016, respectively. B, A plot of ethylene production shown in A (–17) versus methane production.

Fig. 4

Nitrogenase activity after a pre-incubation under air or an argon atmosphere. Microbial mats were pre-incubated at approximately 50°C for 10 h under air or an argon atmosphere. After pre-cultivation, acetylene-reducing activity was measured under argon gas phase with the addition of 0% or 5% O₂ without (A) or with (B) molybdate (20 mM). Three experimental results under each condition are shown in individual bars in a 20-mL vial. Note that the vertical scale differs between Figs. 4A and 4B.

Fig. 5

Nitrogenase activity of intact microbial mats and dispersed microbial mats Acetylene-reducing activity was measured under argon gas phase using intact mats and dispersed microbial mats. Error bars represent the standard deviation of three replicates.

Fig. 6

Effects of chemical conditions on nitrogenase activity of microbial mats Acetylene-reducing activity was measured using dispersed microbial mats in artificial hot spring water without Na₂S under the following conditions: “under argon gas”, “under H₂:CO₂ (4:1, v/v) gas”, “under H₂ gas”, and “under H₂:CO₂ (4:1, v/v) gas in artificial hot spring water containing no sodium sulfate”. Error bars represent the standard deviation of three replicates. Asterisks denote significant differences between complete (sample 2) and incomplete conditions (P values <0.05).