PCE Dechlorination by Non-Dehalococcoides in Microbial Electrochemical System

PCE dechlorination by non-Dehalococcoides in microbial electrochemical system

Jaecheul Yu, Younghyun Park, Van Khanh Nguyen, Taeho Lee †

Department of Environmental Engineering, Pusan National University, Busan, Korea

†Corresponding author

Address: Department of Environmental Engineering, Pusan National University, Busan 609-735.

Tel: +82-51-510-2465.

Fax: +82-51-514-9574.

Email address:

Material and Methods

PCR-DGGE

Bacterial 16S rRNA genes were amplified from the extracted microbial community DNA by PCR with primers Eub 8F (5-AGA GTT TGA TCM TGG CTC AG -3) and Eub 1389R (5- ACG GGC GGT GTG TAC AAG -3). The PCR products were then used as templates for a nested PCR using primers Eub 340F-GC (5- CGC CCG CGC GGC GGC CGG GGC GGG GGC ACG GGG - GGA GRA AAG CAG GGG ATC G -3) and Eub 518R (5- WTT ACC GCG GCT GCT GG -3) to amplify the variable (V3) region of 16S rRNA. PCR amplifications were performed in a thermal cycler (MyCycler; BioRad Lab. Inc.; CA, USA). Each PCR mixture contained 2 μL of PCR products, 0.25 μL (10 pmol) of each primer, 10 μL (10 mmol) of each dNTP, 2.5 μL of 10× PCR buffer, and 0.125 μL of Taq DNA polymerase (Solgent Co. Ltd.; Deajeon, Korea), in a final volume of 50 μL. The tubes were first denatured for 9 min at 95°C, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 2 min, and primer extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min. All of the PCR products were also purified using a PCR purification kit (Solgent Co. Ltd.; Daejeon, Korea) according to the manufacturer’s instructions. DGGE was first performed at 20 V for 20 min and then at 200 V for 6 h on an 8% polyacrylamide gel that contained a linear gradient ranging from 30% to 60% of denaturant (100% denaturant constituted 7 mol/L urea and 40% formamide) using a Decode Universal Mutation Detection System (Bio-Rad; Hercules, CA, USA). After electrophoresis, the gel was stained in ethidium bromide for 30 min. The band profile was visualized using an ultraviolet transilluminator (Uvitec; Cambridge, UK) and photographed with a digital camera (Olympus 720 UZ; Olympus Optical Co. Ltd.; Japan).

Pyrosequencing

The bacterial 16S rRNA genes were amplified by PCR with primers Eub 27F (5- GAG TTT GAT CMT GGC TCA G -3) and Eub 518R (5- WTT ACC GCG GCT GCT GG -3). The PCR products were pyrosequenced using the Genome Sequencer FLX system (Roche Diagnostics Co.; Branford, CT, USA) by Macrogen Inc. (Deajeon, Korea). The obtained 16S rRNA sequences were processed by the Ribosomal Database Project’s pyrosequencing pipeline. Phylotypic identification was based on 16S rRNA sequence homology by performing a nucleotide BLAST search on the website of the National Center for Biotechnology Information.

Table S1. Phylotypes of the extracted DGGE bands of the 16s rRNA gene fragments

Band name / MV* / Biofilm/suspension / Taxonomic group / Closet 16S rRNA gene sequence / Accession
No. / Similarity
(%) / Assumed putative role / Ref
#1 / - / Suspension / Gammaproteobacteria / uncultured Acinetobacter sp. / EF593051 / 94 / EET/Dechlorination / [12, 23]
#2 / - / Biofilm / Bacteroidetes / uncultured Bacteroidetes bacterium / EU887995 / 97 / H2 production / [20]
#3 / - / Suspension / Deltaproteobacteria / uncultured delta proteobacterium / FM206230 / 98 / Dechlorination/ H2 production / unpublished
#4 / - / Biofilm / Firmicutes / Clostridium-like species / U27711 / 98 / Dechlorination/ H2 production / [11]
#5 / - / Biofilm / Betaproteobacteria / Thiobacillus sp. E4IPC-4826 / DQ133429 / 98 / EET / [4, 23]
#6 / - / Biofilm / Environmental samples / uncultured bacterium / HM149204 / 98 / EET / unpublished
#7 / - / Biofilm / Firmicutes / Clostridium bifermentans / HQ013322 / 99 / Dechlorination/ H2 production / [5]
#8 / - / Suspension / Environmental samples / bacterium DCE29 / AJ249260 / 99 / Dechlorination/ H2 production / [8, 9]
#9 / + / Biofilm / Gammaproteobacteria / Pseudomonas sp. PCP2.1 / AY131332 / 99 / EET / [13]
#10 / + / Suspension / Bacteroidetes / uncultured Dysgonomonas sp. / FJ393099 / 99 / EET / [2]
#11 / + / Suspension / Environmental samples / uncultured bacterium / DQ471352 / 98 / Dechlorination / [1]
#12 / + / Biofilm / Gammaproteobacteria / Citrobacter freundii / EU046372 / 98 / EET/ H2 production / [17, 22]
#13 / + / Suspension / Gammaproteobacteria / Pantoea agglomerans / AY335552 / 96 / Dechlorination/ H2 production / [10, 16]
#14 / + / Suspension / Bacteroidetes / uncultured bacterium / FJ393131 / 99 / EET / [2]
#15 / + / Biofilm / Firmicutes / Clostridium bifermemtans / HQ013322 / 99 / Dechlorination/ H2 production / [5]

* +: with MV and -: without MV** EET: Extracellular electron transfer

Table S2. Five most dominant bacterial 16S rRNA gene sequences in the cathode compartment of an MES

Conditions / Name / Distribution
(%) / Taxonomic group / The Closest 16S rRNA gene sequence / Accession
No. / Similarity
(%) / Assumed putative roles / Ref
M1 / EC1 / 18.5 / Gammaproteobacteria / Enterobacter sp. 2019 / JX566554 / 99 / Dechlorination/ H2 production / [10, 17]
EC2 / 13.5 / Gammaproteobacteria / Enterobacter sp. AJ2 / KJ913658 / 97 / Dechlorination/ H2 production / unpublished
EC3 / 7.9 / Gammaproteobacteria / Enterobacter cloacae strain AJ1 / KJ872526 / 99 / Dechlorination/ H2 production / unpublished
EC4 / 3.2 / Gammaproteobacteria / Enterobacter sp. NJ-64 / AM421983 / 98 / Dechlorination/ H2 production / unpublished
EC5 / 3.2 / Environmental sample / Uncultured bacterium DCE29 / AF349765 / 97 / Dechlorination/ H2 production / [8, 9]
M 3 / Suspension / AS1 / 25.7 / Gammaproteobacteria / Acinetobacter haemolyticus / AM184255 / 96 / EET/Dechlorination / [12, 23]
AS2 / 3.7 / Gammaproteobacteria / Uncultured Aeromonas sp. clone O-1 / AB745447 / 99 / EET / [6]
AS3 / 3.6 / Environmental sample / Uncultured bacterium clone / FJ393105 / 97 / EET / [2]
AS4 / 3.0 / Gammaproteobacteria / Uncultured Aeromonas sp. OTU-9-AB / JQ624317 / 99 / EET / [6]
AS5 / 2.9 / Environmental sample / Uncultured bacterium 9E / AF427913 / 100 / Dechlorination / [14]
Biofilm / AE1 / 10.5 / Alphaproteobacteria / Rhodopseudomonas palustris / AB127985 / 100 / EET/H2 Production/Dechlorination / [3, 7, 21]
AE2 / 10.1 / Alphaproteobacteria / Rhodopseudomonas palustris CS-1 / JQ863308 / 99 / EET/H2 Production/Dechlorination / unpublished
AE3 / 7.1 / Betaproteobacteria / Uncultured Azonexus sp. clone SD2 / JN860153 / 99 / Dechlorination/H2 production / [18]
AE4 / 4.9 / Betaproteobacteria / Uncultured bacterium QEDN6BE11 / CU925083 / 100 / Unknown / [15]
AE5 / 4.6 / Environmental sample / Uncultured bacterium clone / FJ393105 / 99 / EET / [2]
M4 / Suspension / BS1 / 22.4 / Gammaproteobacteria / Pseudomonas aeruginosa / KJ865913 / 98 / EET / [13]
BS2 / 20.6 / Firmicutes / Clostridium sp. 6-62 / AB596881 / 99 / H2 production/Dechlorination / [11]
BS3 / 8.7 / Gammaproteobacteria / Enterobacter sp. Ls104 / KJ730211 / 99 / Dechlorination/ H2 production / [10, 17]
BS4 / 7.0 / Firmicutes / Clostridium bifermentans strain DPH-1 / Y18787 / 100 / Dechlorination/H2 production / [5]
BS5 / 5.1 / Firmicutes / Clostridium sp. 6-62 / AB596881 / 99 / H2 production/Dechlorination / [11]
Biofilm / BE1 / 21.7 / Gammaproteobacteria / Enterobacter cloacae strain AJ1 / KJ872526 / 99 / Dechlorination/ H2 production / [10, 17]
BE2 / 11.5 / Gammaproteobacteria / Pseudomonas aeruginosa strain VSS6 / KJ528948 / 99 / EET / [13]
BE3 / 7.0 / Synergistetes / Uncultured Cloacibacillus sp. / FR693794 / 97 / EET / [19]
BE4 / 5.0 / Betaproteobacteria / Uncultured Comamonadaceae bacterium / FJ393075 / 99 / EET / [2]
BE5 / 4.5 / Gammaproteobacteria / Citrobacter freundii strain Y9 / KF781347 / 99 / EET/ H2 production / [17, 22]

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