Supplementary Information
Microbial community composition and ultrastructure of granules
from a full-scale anammox reactor
Gonzalez-Gil G. 1,2*, Sougrat R. 3, Behzad A. R. 3, Lens P.N.L. 2, Saikaly P.E. 1*
1
King Abdullah University of Science and Technology, Water Desalination and Reuse Center.
Division of Biological and Environmental Sciences and Engineering, Thuwal 13955-69000, Saudi
Arabia.
2
UNESCO-IHE, Westvest 7, 2611 AX Delft, The Netherlands.
3
King Abdullah University of Science and Technology, Advanced Nanofabrication Imaging and
Characterization, Thuwal 13955-69000, Saudi Arabia.
*Corresponding authors: g.gonzalezgil@unesco-ihe.org ; graciella.gil@gmail.com
pascal.saikaly@kaust.edu
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Details of DNA amplification and purification of amplicons
Extracted DNA was amplified in triplicates using platinum PCR SuperMix High Fidelity Taq
DNA Polymerase reagent, primer concentrations were 300 nM each and DNA template was
20 to 40 ng. The thermocycling consisted of an initial step at 94°C for 2 min, followed by 25
cycles at 94°C for 30 s, 56°C (53oC for archaea) for 40 s, and 72°C for 60 s, with a final
extension at 72°C for 7 min. Products were checked for correct length by gel electrophoresis.
For pyrosequencing, a second round of PCR (Behrendt et al 2012) was performed for
which the 454-adaptor sequence A (5’-ccatctcatccctgcgtgtctccgactcag- 3’) followed by a
sample specific 8 nt barcode sequence were attached to the 5’ end of the forward primers,
while the 454-adaptor sequence B (5’-cctatcccctgtgtgccttggcagtctcag-3’) was attached to the
5’ end of the reverse primers. The barcodes we used (Meyer et al 2008) met the following
criteria (Parameswaran et al 2007): (i) differed by at least 3 nt, (ii) did not contain
homopolymers, (iii) the end nt of the adapter differed from the start nt of the barcode, (iv) the
end nt of the barcode differed from the start nt of the primer and (v) barcodes had a similar
GC content. The 50 l PCR mixture contained 45 l of Platinum PCR SuperMix High
Fidelity Taq DNA Polymerase reagent (Invitrogen, Carlsbad, CA) and 500 nM of forward
primer, 300 nM of reverse primer and 2 l of template. The thermocycling consisted of an
initial step at 94°C for 2 min, followed by 12 cycles at 94°C for 30 s, 56°C for 40 s, and 72°C
for 60 s, with a final extension at 72°C for 7 min. For comparison of diversity indices,
pyrosequencing of three different anaerobic granules obtained from full-scale anaerobic
reactors treating different wastewaters was included in the analysis (Table 1). Products of
triplicate reactions were pooled for each granule sample (i.e., anammox and aerobic granules)
and then purified using the QIAquick kit PCR purification kit (Qiagen, Valencia, CA)
according to the manufacturer’s protocol and amplicon concentrations were quantified
(NanoDrop-1000 spectrophotometer, Thermo Scientific, Waltham, MA). Then, amplicons
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from different granules samples were mixed to obtain equal mass concentrations in the final
mixture. This mixture was further gel-purified using the QIAquick Gel extraction kit (Qiagen,
Valencia, CA) according to the manufacturer’s protocol.
Anammox activity of granules
The anammox activity of the granules was estimated by measuring the initial ammonium and
nitrite removal rate (measured value was 330 ± 16 mg N L-1 d-1) in a 4 L reactor operated at
about 20oC and inoculated with 12 gVSS L-1 of freshly collected anammox granules. The
reactor influent contained ammonium at ~100 mg L-1 and nitrite at ~131 mg L-1.
Table S1. Metagenomes used for comparisons.
Metagenome
Anammox granules
Anaerobic granules
EBPR west sludge
EBPR east sludge
Weebubbie cave slime
Hotspring slime
MG-RAST accession no.
Reference
This study
This sdudy
(Albertsen et al 2012)
(Albertsen et al 2012)
(Tetu et al 2013)
(Bhaya et al 2007)
4544122
4547780
4455295
4463936
4447951
4443746
Table S2. Brief metagenome summary. Full detailed metagenomes data and reads/sequences
statistics can be found in the MG-RAST data server.
Metagenome
MG-RAST
accession no.
Total
sequences
passing QC1
Sequences
containing
rRNA genes
Anammox granules
Anaerobic granules
4544122
4547780
147613
247735
2007
2256
1
Sequences
containing
predicted
proteins with
known function
93378
133984
passing QC, refers to reads that passed the quality control pipeline of the MG-RAST server.
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Figure S1. X-ray power diffraction (XRD) spectrum of the inorganic fraction of the
anammox granules. The mineral carbonate fluorapatite showed the best match for the peaks.
4
Figure S2. Phylogenetic diversity as a function of sequences showing that increased effort of
sequencing for anammox granules would hardly increase the phylogenetic diversity further.
For comparison, curves for anaerobic granules are presented. Anaerobic granules 1 and 2
refer to AnG 1 and AnG 2 from Table 1 from the main text.
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Figure S3. Archaeal community composition of anammox granules. A) Composition at the
order level. B) Composition at the genus level.
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Figure S4. The following figures show the functional comparison of anammox granules with
each of the other metagenomes. The figures were filtered so that only metabolisms where
significant differences were detected are displayed.
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Figure S5. Functional distribution of carbohydrates metabolism of various metagenomes.
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Figure S6. Comparison of enzymes related to the N2 cycle found in the anammox granules
with those found in other metagenomes.
Supporting references
Albertsen M, Hansen LBS, Saunders AM, Nielsen PH, Nielsen KL (2012). A metagenome of a full-scale
microbial community carrying out enhanced biological phosphorus removal. ISME J 6: 1094-1106.
Behrendt L, Larkum AWD, Trampe E, Norman A, Sorensen SJ, Kuhl M (2012). Microbial diversity of
biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella. ISME
J 6: 1222-1237.
Bhaya D, Grossman AR, Steunou A-S, Khuri N, Cohan FM, Hamamura N et al (2007). Population level
functional diversity in a microbial community revealed by comparative genomic and metagenomic
analyses. ISME J 1: 703-713.
Meyer M, Stenzel U, Hofreiter M (2008). Parallel tagged sequencing on the 454 platform. Nat
Protocols 3: 267-278.
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Parameswaran P, Jalili R, Tao L, Shokralla S, Gharizadeh B, Ronaghi M et al (2007). A pyrosequencingtailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic
Acids Res 35: e130.
Tetu SG, Breakwell K, Elbourne LDH, Holmes AJ, Gillings MR, Paulsen IT (2013). Life in the dark:
metagenomic evidence that a microbial slime community is driven by inorganic nitrogen
metabolism. ISME J 7: 1227-1236.
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