Monitoring the Biochemical Activity of Single Anammox Granules with Microbarometers

Granule-based anaerobic ammonium oxidation (Anammox) is a promising biotechnology for wastewater treatments with extraordinary performance in nitrogen removal. However, traditional analytical methods often delivered an average activity of a bulk sample consisting of millions and even billions of Anammox granules with distinct sizes and components. Here, we developed a novel technique to monitor the biochemical activity of individual Anammox granules in real-time by recording the production rate of nitrogen gas with a microbarometer in a sealed chamber containing only one granule. It was found that the specific activity of a single Anammox granule not only varied by tens of folds among different individuals with similar sizes (activity heterogeneity) but also revealed significant breath-like dynamics over time (temporal fluctuation). Statistical analysis on tens of individuals further revealed two subpopulations with distinct color and specific activity, which were subsequently attributed to the different expression levels of heme c content and hydrazine dehydrogenase activity. This study not only provides a general methodology for various kinds of gas-producing microbial processes but also establishes a bottom-up strategy for exploring the structural-activity relationship at a single sludge granule level, with implications for developing a better Anammox process.