Investigating microbial and environmental drivers of nitrification in alkaline forest soil.

Ammonia oxidation is a key step in the biogeochemical cycling of nitrogen, and soils are important ecosystems for nitrogen flux globally. Approximately 25% of the world's soils are alkaline. While nitrification has been studied more extensively in agricultural alkaline soils, less is known about natural, unfertilized alkaline soils. In this study, microorganisms responsible for ammonia oxidation and several environmental factors (season, temperature, ammonia concentration, and moisture content) known to affect nitrification were studied in an alkaline forest soil with a pH ranging from 8.36 to 8.77. Ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea, and comammox were present, and AOB belonging to genera Nitrosospira and Nitrosomonas, originally comprising <0.01% of the total bacterial community, responded rapidly to ammonia addition to the soil. No significant difference was observed in nitrification rates between seasons, but there was a significant difference between in situ field nitrification rates and rates in laboratory microcosms. Surprisingly, nitrification took place under many of the tested conditions, but there was no detectable increase in the abundance of any recognizable group of ammonia oxidizers. This study raises questions about the role of low-abundance microorganisms in microbial processes and of situations where zero or very low microbial growth coincides with metabolic activity. In addition, this study provides insights into nitrification in unfertilized alkaline soil and supports previous studies, which found that AOB play an important role in alkaline soils supplemented with ammonia, including agricultural ecosystems.

Activity-based labelling of ammonia- and alkane-oxidizing microorganisms including ammonia-oxidizing archaea.

Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms.