Context. Granular sludge exhibits unique features, thus being a favorable platform for the cultivation of slow-growing microorganisms, such as anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) microorganisms.
Gap.While granular anammox processes have been widely applied, little is known about how to speed up the granulation process of n-DAMO microorganisms, which grow even slower. In addition, rare information is available about how to distinguish the microbial reactions in different layers of stratified granules.
Aim.In this study, we aim to develop an operation strategy to obtain granules coupling anammox and n-DAMO microorganisms rapidly. Moreover, the microbial ecology of obtained granules will be investigated in-depth.
Methods. Anammox granules were used as biotic carriers to embed n-DAMO microorganisms. After peeling mature granules using different shear force, cryosection-16S rRNA gene amplicon sequencing and fluorescence in situhybridization (FISH) were applied to study the microbial stratification along granule radius. Mathematical modelling was applied to verified the interactions.
Findings.Combined anammox + n-DAMO granules were rapidly obtained within 6 months. To investigate microbial ecology of obtained granules, the microbial stratification along granule radius was elucidated by cryosection-16S rRNA gene amplicon sequencing, showing the dominance of n-DAMO archaea and anammox bacteria at inner and outer layers, respectively, which was validated by an established mathematical modelling. Moreover, the images of cryosection-FISH verified this stratification and also indicated a shift in microbial stratification. Specifically, n-DAMO bacteria and n-DAMO archaea attached to anammox granule surface initially, which moved to the inner layer after 4-months operation.
Utilization. (1) The approaches applied here could be used to study microbial ecology of other granular systems. (2) A practically useful nitrogen removal rate (1.0 kg N/m3/d) was obtained. (3) The obtained granules are ideal ecological platform to study the microbial interactions of methane and nitrogen cycles.
