Supporting information

Microstructure of anammox granules and mechanisms endowing their intensity

revealed by microscopic inspection and rheometry

Ximao Lin, Yayi Wang*

*Corresponding author. Tel: +21 65984275; Fax: +21 65984275; E-mail:

wyywater@126.com; yayi.wang@tongji.edu.cn

State Key Laboratory of Pollution Control and Resources Reuse, College of

Environmental Science and Engineering, Tongji University, Siping Road, Shanghai

200092, P. R. China

Fluorescence in-situ hybridization (FISH)

FISH was conducted as described previously (Nielsen; et al. 2009). Prior to FISH

probing, the granular samples were fixed overnight in 4% (w/v) paraformaldehyde at

4 °C. Then, fixed granule samples were embedded in optimum cutting temperature

compound (TissueTek, Sakura Finetek, Torrance, CA, USA) and stored at −20 °C for

cryosectioning. Granules were then sectioned into 20 μm slices using a cryotome

(Leica). FISH was carried out on sliced granules with the following oligonucleotide

probes: Cy3-labeled EUBmix (EUB338, EUB338-II and EUB338-III (Daims et al.

1999)) for all bacteria, and Cy5-labeled AMX368 for all anammox bacteria (Schmid

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

et al. 2003). Micro-observations were realized with a CLSM (Leica TCS SP5 II

confocal spectral microscope imaging system, Germany), and analyzed with Leica

confocal software.

2

23

24

25

26

27

Figure S1. Confocal laser scanning microscopy image of fluorescence in-situ

hybridizationmicrographs of an entire granule section. (a) Anammox bacteria are

magenta; (b) overlay of red AMX368 and (c) blue EUBmix.

3

28

29

30

31

32

33

34

Figure S2 Size distribution of anammox granules after sieved at 1.0 mm

4

35

3637

38

39

40