抄録
In situ spatial organization of ammonia-oxidizing bacteria in wastewater biofilms was investigated using fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM) and digital image analysis. In situ hybridization of steady-state biofilm samples indicated more than 65% of ammonia-oxidizing bacteria present in the biofilm formed dense spherical clusters with the diameter of less than 10 μm and 10∼20% formed clusters with the diameter of more than 10 μm. Most of ammonia-oxidizing bacteria were present in states of small clusters (d<5 μm) in the surface biofilm, because the bigger clusters were imposed on the higher shear stress and consequently sloughed off.
On the other hand, about 4∼10 times higher bacterial population were found as the bigger clusters (d=5∼20 μm) in the bottom biofilm than in the surface, creating a spatial gradient of ammonia-oxidizing bacteria throughout the biofilm. Furthermore, evaluation of series of optical sections through the clusters taken by the CSLM showed that a cluster with the diameter of approximately 25 μm consisted of up to 9,000 rod-shaped cells. Areal cell density within the clusters was found to be in the range of 0.9∼1.1 cells·μm-2 with areal fraction of cells of 51 ± 6%, indicating that these clusters have relatively large openings. These experimental results suggested that the interior biofilm structure was porous and heterogeneous, the spatial distribution of ammonia-oxidizing bacteria was strongly influenced by the hydraulic shear stress and substrate concentration profiles within the biofilm.
