Supplementary Material

Materials and methods

High-throughput 16S rRNA gene amplicon sequencing

Each biofilm stored at -20°C was taken out from 50% ethyl alcohol solution and

cut into small pieces for DNA extraction. DNA was extracted using FastDNA™ Spin

Kit for soil (MP Biomedicals, Santa Ana, CA) according to the manufacturer’s manual.

The concentration and purity of DNA extracting solution were quantified by NanoDrop

spectrophotometer (ND-1000, Nanodrop Inc., DE, USA).

Each DNA template was diluted to 20 ng/μL to perform the qualitative PCR. PCR

amplification was performed in quadruplicate using a Applied Biosystems Veriti

Thermal Cycler (Life Technologies, USA) in a total volume of 50 μL containing 5 μL

10×PCR Buffer (TaKaRa, Japan), 4 μL MgCl2, 4 μL dNTP Mixture, 1 μL of each

forward and reverse primer, and 0.25 μL Ex Taq DNA polymerase, 2 μL DNA template.

The thermal cycles using the following conditions: an initial denaturation at 98°C for 5

min, and 20 cycles at 98°C for 30 s, 50°C for 30 s, and 72°C for 40 s, with a final

extension at 72°C for 10 min. Forward primer (5’-AGAGTTTGATYMTGGCTCAG-3’)

and reverse primer (5’-TGCTGCCTCCCGTAGGAGT-3’) and different 8-bases

barcodes and a Guanine were linked to the 5’ end of each primer were selected to target

V1V2 hypervariable regions of bacterial 16S rRNA gene. The barcodes added to the

forward and reverse primers for twelve samples were listed as follows: GAGAGAGAG

and GCAGAGATG, GAGAGCAGC and GCAGAGCTC, GAGATCATC and

GCAGATGAG, GAGATGAGC and GCAGCATGC, GAGATGCAG and

GCAGCTCTC, GAGCAGAGC and GCATCTGAG, GAGCAGCAG and

GCATGATGC, GAGCATCTG and GCATGCATG, GAGCTCAGC and GCTCAGATG,

GAGCTGATC and GCTCAGCTC, GATCAGATC and GCTCATGAG, as well as

GATCATCAG and GCTCTCATG.

Following amplification, 2 µL of PCR product was used for agarose gel (1%)

detection. The quadruplicate PCR reactions for each sample preparation were combined

and purified by E.Z.N.A.TM Cycle-Pure Kit (Omega Bio-tek Inc., USA). Purified

products were re-quantified by NanoDrop spectrophotometer (ND-1000, Nanodrop Inc.,

DE, USA).

High-throughput sequencing was conducted using Illumian sequencing platform

(Miseq, Illumina Inc., USA) at Jiangsu Zhongyijinda Analytical and testing limited

company (Jiangsu, China).

Data analysis

Sequencing data was estimated using Sickle to remove the bases of low quality

(Q<25) and any sequences with more than one N (https://github.com/najoshi/sickle).

Mothur program was used for sequence demultiplexing and filtration. General steps are

as follows: 1) Sequences were demultiplexed based on barcodes. 2) Read1 and Read2

were combined into contigs (parameter: minoverlap=10, maxhomop=8, maxambig=0).

3) Sequences which weren’t aligned to the V1V2 region were discarded. 4) Sequences

with no more than 3 different bases were pre-clustered into one sequence. 5) Chimera

was detected by comparing sequences with reference databases using the

chimera.uchime command packaged in Mothur. 6) The 16S rRNA gene sequences were

classified into operational taxonomic units (OTUs) at 3% cutoff (or 97% similarity).

The filtered sequences were classified by the stand-alone RDP classifier

(http://sourceforge.net/projects/rdp-classifier/).

Shannon diversity index (H) was used to study the microbial species diversity of

biofilms, it was calculated by the following equation (Shannon, 1948): H= - ∑Pi lnPi

(i=1, 2, 3…, S). Pi represents the relative abundance of the species and S represents the

number of microbial species.

Heat map was conducted using R program to analyze microbial community

structure in genus level. Genera with relative abundance > 0.10% (70 genera) in each

sample were selected and compared with relative abundances in other samples.

Fig. S1. Organic matter removal (A) and nitrification performance (B) in MBBRs

Fig. S2. Morphology comparison of biofilms formed over time during biofilm formation

and development in MBBR. Topographic image is 5×5 μm2 (A-C), the others (D-L) of

different days is 10×10 μm2. Figures A-L represent of the morphologies of 0 h, 1 hour, 3

hour, 1 day, 2 day, 4 day, 6 day, 10 day, 14 day, 20 day, 28 day and 45 day of biofilms.