Supplementary Material
1. The granule morphology was periodically observed by scanning electron microscope
(SEM, Quanta 450, FEI, USA)
Figure S1, the SEM picture of the seed sludge (left) and the matured aerobic granular sludge
(right) on day 94.
2. Fluorescence in situ hybridization (FISH) was conducted according to the method described
by Neef et al., (1996). The seed sludge and100ml of sludge was periodically taken at the end of
the cycle from the reactor and stored in -20℃for further investigation. Probes used in this study
included EUB338 for detection of all Bacteria, and the combination of PAOmix (PAO464,
PAO651, PAO846) for PAOs. The GAOmix probe (GAOQ431 and GAOQ989) was used to
target GAOs. The samples were detected by a confocal laser scanning microscope (CLSM,
156 lympus FV1000, Japan).
FISH was employed to further determine community of the sludge. The results were listed in
Figure S2. PAOs and GAOs were barely observed in B and H, indicating that the amount of these
organisms was low in the seed sludge, while, PAOs and GAOs increased significantly along with
the running time as shown by D, F, J and K, which corresponding well with the previous
discussion.
Figure S2. Confocal laser scanning microscope images of FISH micrograph of sludge
(overlay of red PAOmix, bule GAO mix and green EUB338). A and G: FISH of all bacteria
of seed sludge combined with colored PAOs (A) and GAOs (G), B and H: FISH of PAOs (B)
and GAOs (H) of seed sludge, C and I, FISH of all bacteria of sludge combined with colored
PAOs (C) and GAOs (I) on day 50, D and J: FISH of PAOs (D) and GAOs (J) of sludge on day
50, E and K, FISH of all bacteria of sludge combined with colored PAOs (E) and GAOs (K) on
day 89, F and L: FISH of PAOs (F) and GAOs (L) of sludge on day 89. Bars represent 300um in
A to F and 298.83um in G to L
Neef A, Zaglauer A, Meier H, Amann R, Lemmer H, Schleifer KH. Population analysis in
a denitrifying sand filter: Conventional and in situ identification of Paracoccus spp. in
methanol-fed biofilms. Appl Environ Microbiol. 1996; 62: 4329-4339.