Abstract

In agricultural soils fertilized with a high amount of ammonium nitrogen, the pH decreases because of the oxidation of ammonia by nitrifiers. Molecular-based analyses have revealed that members of the genus Nitrospira dominate over other nitrifiers in some acidic soils. However, terrestrial Nitrospira are rarely cultivated and little is known about their ecophysiology. In addition, recent studies discovered a single microbe with the potential to oxidize both ammonia and nitrite (complete ammonia oxidizer; comammox) within Nitrospira, which had been previously recognized as a nitrite oxidizer. Despite their broad distribution, there are no enrichment samples of comammox from terrestrial or acidic environments. Here, we report the selective enrichment of both comammox and nitrite-oxidizing Nitrospira from the acidic soil of a heavily fertilized tea field. Long-term enrichment was performed with two individual continuous-feeding bioreactors capable of controlling ammonia or nitrite concentration and pH. We found that excessive ammonium supply was a key factor to enhance the growth of comammox Nitrospira under acidic conditions. Additionally, a low concentration of nitrite was fed to prevent the accumulation of free nitrous acid and inhibition of cell growth under low pH, resulting in the selective enrichment of nitrite-oxidizing Nitrospira. Based on 16S rRNA gene analysis, Nitrospira accounting for only 1.2% in an initial soil increased to approximately 80% of the total microorganisms in both ammonia- and nitrite-fed bioreactors. Furthermore, amoA amplicon sequencing revealed that two phylotypes belonging to comammox clade A were enriched in an ammonia-fed bioreactor. One group was closely related to previously cultivated strains, and the other was classified into a different cluster consisting of only uncultivated representatives. These two groups coexisted in the bioreactor controlled at pH 6.0, but the latter became dominant after the pH decreased to 5.5. Additionally, a physiological experiment revealed that the enrichment sample oxidizes ammonia at pH <4, which is in accordance with the strongly acidic tea field soil; this value is lower than the active pH range of isolated acid-adapted nitrifiers. In conclusion, we successfully enriched multiple phylotypes of comammox and nitrite-oxidizing Nitrospira and revealed that the pH and concentrations of protonated N-compounds were potential niche determinants.

Highlights

  • Nitrification is an important reaction composing the global nitrogen cycle

  • Considering the decrease in ammonia concentration caused by protonation, an excessive amount of ammonium was supplied into the bioreactor to enrich the comammox Nitrospira

  • On day 165–205, when the bioreactor was supplied with the medium containing 39 mM NH4Cl, the nitrate (NO3−) concentration in the bioreactor increased to 1.3 ± 0.8 mM on average, which shows an increase in nitrification activity (Figure 1A)

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Summary

Introduction

Nitrification is an important reaction composing the global nitrogen cycle. Nitrification acts as a production route of nitrate, a nitrogen source of soil plants. Despite the importance of nitrification, supplementation with excessive nitrogen fertilizers over-activates nitrification and induces environmental problems. Too much nitrate produced by nitrification leaches into groundwater, causing pollution and the economic loss of fertilizers (Schlesinger, 2009). Excessive nitrification causes the release of nitrous oxide, a greenhouse gas (Wrage et al, 2001) and soil acidification (Vitousek et al, 1997). Soils with lower pH were reported to generate more nitrous oxide (Mørkved et al, 2007; Liu et al, 2010; Bakken et al, 2012)

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