Supporting Information Effect of Disinfectant, Water Age, and Pipe Materials on Bacterial and Eukaryotic Community Structure in Drinking Water Biofilm

AUTHOR NAMES. Hong Wang1, Sheldon Masters1, Marc A. Edwards1, Joseph O. Falkinham, III2, and Amy Pruden1*

RECEIVED DATE

AUTHOR ADDRESS. 1Via Department of Civil and Environmental Engineering, Virginia Tech,

Blacksburg, VA 24061; 2Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061

*CORRESPONDING AUTHOR. Via Department of Civil and Environmental Engineering,

Virginia Tech, Blacksburg, VA 24061. Email: [email protected]; Phone: (540) 231-3980; Fax: (540)

231-7916

KEYWORDS. Drinking water distribution systems, chlorine, chloramine, bacteria, eukaryote

Tables

Table S1 Physiochemical parameters in SDSs (± standard error) [1]

Disinfectant TOC DO concentration pH (mg/L) (mg/L) (mg/L) Chloraminated SDSs Iron 2.3d 0.15±0.03 7.24±0.06 1.72±0.14 4.08±0.27 Iron 5.7d 0.02±0.01 7.19±0.04 1.93±0.13 1.68±0.32 Cement 2.3d 0.36±0.11 7.45±0.06 2.28±0.13 5.89±0.25 Cement 5.7d 0.03±0.01 8.37±0.13 3.30±0.24 3.21±0.23 PVC 2.3d 0.17±0.05 7.24±0.05 2.10±0.14 5.24±0.36 PVC 5.7d 0.02±0.01 7.28±0.09 2.98±0.23 2.94±0.30 Chlorinated SDSs Iron 2.3d 0.75±0.13 7.44±0.05 1.64±0.14 5.58±0.16 Iron 5.7d 0.08±0.01 7.38±0.11 1.99±0.23 0.92±0.10 Cement 2.3d 1.99±0.37 7.66±0.03 2.69±0.44 7.21±0.11 Cement 5.7d 0.06±0.01 8.96±0.13 3.36±0.54 2.97±0.32 PVC 2.3d 2.57±0.43 7.55±0.04 1.91±0.16 7.25±0.50 PVC 5.7d 0.40±0.13 7.68±0.14 2.73±0.36 5.07±0.32

Table S2 Number of OTUs and Simpson index of SDS biofilm bacterial communities by pyrosequencing

Chlorinated SDSs Chloraminated SDSs

Number of OTUs Inverse of the Simpson Number of OTUs Inverse of the Simpson

index (1/λ) index (1/λ)

2.3d 5.7d 2.3d 5.7d 2.3d 5.7d 2.3d 5.7d

Iron 88±37 98±30 3.2±1.9 11.6±1.8 98±6 111±6 7.0±2.5 6.7±3.9

PVC NA 101±14 NA 9.2±0.7 81±14 92±27 6.3±2.6 8.4±1.1

Cement 98±3 102±2 4.8±3.2 5.8±1.0 90±12 82±20 5.2±2.0 4.3±1.2

Table S3 Number of T-RFs and Simpson index of SDS biofilm bacterial communities by T-RFLP

Chlorinated SDSs Chloraminated SDSs

Number of T-RFs Inverse of the Simpson Number of T-RFs Inverse of the Simpson

index (1/λ) index (1/λ)

2.3d 5.7d 2.3d 5.7d 2.3d 5.7d 2.3d 5.7d

Iron 8±1 21±5 2.6±0.9 4.3±0.6 20±3 22±6 5.1±0.3 5.9±2.6

PVC 13±2 17±8 3.8±0.5 5.8±3.6 20±0 17±4 4.2±0.5 5.1+4.8

Cement 14±3 15±2 3.1±0.8 4.6±1.9 14±3 15±1 3.3±0.4 4.5±0.4

Table S4 Comparison of dominant OTUs with dominant T-RFs at genus level taxonomic information of sequences

Genus-level identity OTUs from pyrosequencing T-RFs from T-RFLP2 Novosphingobium OTU2, OTU27 78.8 Hyphomicrobium OTU3 337.1 Acidovorax OTU4, OTU9 202.2 Azospirillum OTU5 78.8 Methylobacterium OTU6, OTU12, OTU25 342.7 Sphingomonas OTU7, OTU17, 78.8 Sphingopyxis OTU8 78.8 Methyloversatilis OTU10 202.2, 195.3 Brevundimonas OTU14 331.2 Pseudoxanthomonas OTU15 369.4 Pelomonas OTU16 197.2 Chryseobacterium OTU18 222.4 Mycobacterium OTU20 ND1 Bosea OTU22 339.9 Mesorhizobium OTU23 ND Mucilaginibacter OTU28 91.2 Xanthobacter OTU29 ND Afipia >>30 341.1 Alicycliphilus ND 203.6 Dechloromonas ND 205.3 Planctomyces ND 360.2 Microbacterium ND 362.7 Blastomonas ND 370.4 Vampirovibrio >>30 406 Note: 1) ND: not detected 2) Length of T-RFs for each clone were rounded up to the T-RFs found in biofilm sample T-RFLP profiles 3) “>>30” indicates that related genus were found in pyrosequencing OTUs in low abundance (i.e., outside the scope of the first 30 OTUs) 4) OTUs 1, 11, 13, 19, 21, 24, 26, 30 could not be classified at the genus level.

Table S5 Number of T-RFs and Simpson index of SDS biofilm eukaryotic communities by T-RFLP

Chlorinated SDSs Chloraminated SDSs

Number of T-RFs Inverse of the Simpson Number of T-RFs Inverse of the Simpson

index index

2.3d 5.7d 2.3d 5.7d 2.3d 5.7d 2.3d 5.7d

Iron 8±1 9±1 1.8±0.4 4.0±1.4 13±1 10±3 2.6±0.3 4.0±0.9

PVC 6±1 13±1 2.1±0.6 3.3±1.4 13±4 12±2 3.2±0.9 4.3±1.9

Cement 11±6 11±3 2.7±1.5 2.5±0.7 11±6 13±2 3.3±1.9 3.0±0.8

Table S6 Blast results for Legionella, mycobacteria, and Pseudomonas sequences

Representative Identity Gaps Bit E- Accession Closest Match The 2nd Closest Match OTU Score value No. OTU284 368/374 6/374 350 0 NR_074231. Legionella pneumophila subsp. Legionella pneumophila subsp. (98%) (1%) 1 pneumophila str. Philadelphia 1 pascullei strain U8W; ATCC strain Philadelphia 1 16S 33737 16S ribosomal RNA, partial ribosomal RNA, complete sequence (97%) sequence OTU903 364/368 0/368 356 0 NR_024969. Legionella waltersii strain 2074- Legionella pneumophila subsp. (99%) (0%) 1 AUS-E 16S ribosomal RNA, pneumophila str. Philadelphia 1 partial sequence strain Philadelphia 1 16S ribosomal RNA, complete sequence (97%) OTU1036 350/369 2/369 311 2e- NR_041790. Legionella maceachernii strain Tatlockia micdadei strain (95%) (0%) 164 1 ATCC35300 16S ribosomal ATCC33218 16S ribosomal RNA, RNA, partial sequence partial sequence (i.e., Legionella micdadei, 95%) OTU20 348/348 0/348 348 0 NR_042919. Mycobacterium mucogenicum Mycobacterium fortuitum strain (100%) (0%) 1 strain ATCC 49650 16S CIP 104534 16S ribosomal RNA, ribosomal RNA, partial sequence partial sequence (99%) OTU382 359/360 0/360 357 0 NR_102855. Mycobacterium avium 104 strain Mycobacterium colombiense (99%) (0%) 1 104 16S ribosomal RNA, CECT 3035 strain (99%) complete sequence OTU472 342/348 0/348 610 6e- NR_044840. Mycobacterium intermedium Mycobacterium saskatchewanense (98%) (0%) 175 1 16S ribosomal RNA, partial strain NRCM 00-250; ATCC sequence BAA-544 16S ribosomal RNA, partial sequence (98%) OTU1155 338/349 2/349 315 1e- NR_042919. Mycobacterium mucogenicum Mycobacterium pallens strain czh- (97%) (0%) 166 1 strain ATCC 49650 16S 8 16S ribosomal RNA, partial ribosomal RNA, partial sequence sequence (97%) OTU1318 351/363 3/363 325 4e- NR_041897. Mycobacterium branderi strain Mycobacterium kyorinense strain

(97%) (0%) 172 1 ATCC 51789 16S ribosomal KUM 060204 16S ribosomal RNA, partial sequence RNA, partial sequence (96%)

OTU1489 340/348 0/348 324 1e- NR_074427. Mycobacterium abscessus 16S Mycobacterium massiliense str. (98%) (0%) 171 1 ribosomal RNA, complete GO 06 strain GO 06 16S sequence ribosomal RNA, complete sequence (98%) OTU52 368/368 0/368 368 0 NR_074828. Pseudomonas aeruginosa PAO1 Pseudomonas resinovorans NBRC (100%) (0%) 1 strain PAO1 16S ribosomal 106553 strain NBRC 106553 16S RNA, complete sequence ribosomal RNA, complete sequence (99%)

Figures

Formatted: Font: (Default) Times New Roman, 14 pt, Bold

Figure S1 Simulated distribution systems

Figure S2 Relative abundance of bacterial phyla/classes (i.e., Proteobacteria) in simulated distribution systems (SDSs). Error bars represent the standard deviation of major bacterial phyla/classes among triplicate biofilm samples (only two samples included chlorinated iron 2.3d and cement 2.3d SDSs).

(a) Chlorinated SDSs (b) Chloraminated SDSs

Figure S3 Multidimensional scaling analysis (MDS) of SDS biofilm bacterial community composition derived from T-RFLP

(a) water age =2.3d (b) water age= 5.7d

Figure S4 Multidimensional scaling analysis (MDS) of SDS biofilm bacterial community composition derived from pyrosequencing

References 1. Wang, H.; Masters, S.; Hong, Y.; Stallings, J.; Falkinham, J., III; Edwards, M.; A, P. Effect of disinfectant, water age, and pipe material on occurrence and persistence of Legionella, mycobacteria, Pseudomonas aeruginosa, and Two Amoebas. Environ. Sci. Technol. 2012, 46, (21), 11566-11574.