1
2
Supplementary material to
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Geobacter, Anaeromyxobacter and Anaerolineae populations are
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enriched on anodes of root exudate driven microbial fuel cells in
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rice field soil
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7
8
MATERIALS AND METHODS
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Microbial fuel cells
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Two series (A and B) of planted MFCs (PMFCs) were constructed and operated including
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open circuit and unplanted controls. All PMFCs were constructed using rice field soil as
12
support for rice plants. Soil was sampled in 2006 from a drained rice field of the Italian Rice
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Research Institute “Instituto Sperimentale per la Cerealicoltura” near Vercelli (Po River
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valley, Italy). Soil parameters were as described previously (Holzapfel-Pschorn& Seiler,
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1986). The soil was air dried, sieved (mesh size 5 mm) and stored at room temperature as
16
described previously (Chin & Conrad, 1995).
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PMFCs series A and B were operated for 104 and 90 days in 2007 and 2008, respectively, in a
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greenhouse at the Max Planck Institute for Terrestrial Microbiology, Marburg. All series
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included duplicate PMFCs (PMFC-A1, PMFC-A2; PMFC-B1, PMFC-B2), one open circuit
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control for each series (OC-A and OC-B) and for series A, also an unplanted control (NP-A).
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Plastic containers were filled with 3 kg of rice field soil and flooded with water leaving a 5
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cm layer of overlying water. In each PMFC and controls, two anodes of 10 cm by 10 cm were
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placed vertically in the soil matrix giving a total anode surface (TAS) of 425 cm2 and a
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cathode of 10 cm by 10 cm was placed in the overlying water layer. Carbon felt anodes and
1
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cathodes (Alfa Aesar, Ward Hill, USA; 3.18 mm thick) were attached to an insulated cable
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with a graphite rod (5 mm diameter, Thielmann Graphite, Grolsheim, Germany). Closed
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electrical circuits had an external resistance of 470 Ω for series A, and 470 Ω and 100 Ω (470
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Ω until day 45 and 100 Ω from day 45 until the end of the experiment) for series B. Three two
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week old plants (Oryza sativa cultivar Koral) were planted in each pot (except for the
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unplanted control). Fertilizer (5 mL / kg soil; urea [45g/L], Na2HPO4 x 2H2O [17g/L] and KCl
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[50g/L]) was added twice during the first weeks. Series A and B were operated in a
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greenhouse with light: dark cycles of 12h:12h at an average temperature of 25 °C. The electric
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potential (mV) was recorded every 15 minutes with a data logger (Agilent 34970A, Agilent
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Technologies, Böblingen) and current densities were calculated as reported previously (Logan
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et al., 2006). At the end of the incubation, anodes and bulk soil were sampled and stored at -
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80°C for further molecular analysis.
37
38
Nucleic acid extraction, PCR and TRFLP
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RNA extractions (n=4) with 0.5g of anode material or bulk soil each were performed using a
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bead-beating protocol as described previously (Lueders et al., 2004). TRFLP analysis was
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performed according to Egert et al. (2003). Briefly, 16S rRNA was reversely transcribed and
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PCR amplified using a single step RT-PCR system (Access Quick, Promega, Mannheim,
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Germany). 5’ 6-carboxyfluorescein labelled (FAM) primers were used to specifically amplify
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bacteria (FAM-Ba27f and Ba907r) and archaea (Ar109f and FAM-Ar912rt). PCR products
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(~100 ng) were digested with restriction enzymes MspI or TaqI (Promega) for bacteria and
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archaea, respectively, and size separated on an ABI PRISM 3130 Genetic Analyzer (Applied
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Biosystems, Weiterstadt, Germany). TRFLP electropherograms were analyzed with
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GeneMapper Software 4.0 (Applied Biosystems). Tables were generated for each sample with
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peak size vs. fluorescence intensity and terminal restriction fragments (TRFs) that differed by
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±0.5bp in different profiles were considered as identical in order to compare TRFLP profiles
2
51
between different samples. The peak heights were standardized to the minimum sample
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according to Dunbar et al.(2000) using TRFs between 50 and 900 bp. Discrepancies between
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in silico and observed TRF sizes might occur (Schutte et al., 2008) and therefore, TRFLP
54
analysis was performed for relevant clones and in vitro TRFs were determined (Table S1).
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Cluster analysis using Unweighted Pair Group Method with Arithmetic Mean (UPGMA)
56
algorithm, and Bray-Curtis similarity index was performed using PAST software (Hammer et
57
al, 2001).
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Clone libraries and phylogenetic analysis
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16S rRNA reverse transcripts from PMFC-A2 were amplified using primers specific for
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Bacteria (Ba27f, 5`-AGA_GTT_TGA_TCC_TGG_CTC_AG-3´, Edwards et al., 1989 and
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Ba907r,5´-CCA_TCA_ATT_CCT_TTR_AGT_TT-3´ modified after Muyzer et al., 1995) and
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Archaea (Ar109f, 5’- ACKGCT CAG TAA CAC GT -3´ Großkopf et al, 1998, and
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Ar912rt: 5’- GTG CTC CCC CGC CAA TTC CTT TA -3´ Lueders & Friedrich, 2002).
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Amplicons were cloned into Escherichia coli JM109 competent cells (Promega) using
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pGEM®-T Easy Vector System II (Promega). Clones were selected randomly and checked for
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correct insert size via agarose gel electrophoresis.
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In addition, bacterial clone libraries were also constructed for control anode samples (OC-A
68
and NP-A). Samples OC-A and NP-A were sequenced by Qiagen (Hilden, Germany) and
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sample PMFC-A2 by ADIS (Max Planck Institute for plant breeding research, Cologne). Raw
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sequence data were processed using SeqMan software (DNAStar, Madison, WI). Clone
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libraries were screened for chimera by using Bellerophon (Huber et al., 2004) and Mallard
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software (Ashelford et al., 2006). Putative chimera were verified by fractional treeing
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(Ludwig et al., 1997) and excluded from further analysis. Bacterial phylogenetic analysis was
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conducted using ARB 5.1 software package (Ludwig et al., 2004; http://www.arb-home.de).
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16S rRNA sequences from all three samples were added to the database and aligned with the
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Fast Aligner tool of the ARB software. Reference sequences were downloaded from the ARB
3
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Silva database or GenBank (National Center for Biotechnology Information-NCBI,
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http://www.ncbi.nlm.nih.gov/), added to the ARB database, and phylogenetic trees were
79
constructed by the neighbor joining method. Archaeal phylogenetic analysis was conducted
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using MEGA version 5 software package (Tamura et al. 2011). Reference sequences were
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downloaded from GenBank database (National Center for Biotechnology Information-NCBI,
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http://www.ncbi.nlm.nih.gov/), and phylogenetic trees were constructed by the neighbor
83
joining method. In silico terminal restriction fragment sizes (in silico TRFs) were obtained by
84
searching restriction sites of the restriction enzymes MspI (C|CGG) and TaqI (T|CGA).
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Nucleotide sequences were deposited into GenBank under accession numbers JF325892-
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JF326184.
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FIGURES AND TABLES
Figure S1. Current density (mAm-2 TAS) profiles of planted (filled square: PMFC-A, filled
-2
m2)
Current density (mA /m
TAS)
triangles: PMFC-B) and unplanted control (filled circle: NP-A) during operation time.
35
30
25
20
15
10
5
0
0
10
20
30
40
50
60
70
80
90
100
110
Time (days)
89
90
Figure S2.
Phylogenetic tree showing the relationships of 16S rRNA clone sequences related to Archaea.
Clones obtained in this study from planted sediment microbial fuel cell (MFC) were included
in the phylogenetic trees. The in silico TRF sizes are as indicated in brackets in base pairs.
4
Bootstrap values were obtained from 1000 replications. The scale bar represents 2 %
sequence divergence. GenBank accession numbers of reference sequences as indicated.
MFC-A33
59 MFC-A76
MFC-A70
MFC-A13
MFC-A34
MFC-A29
MFC-A31
MFC-A8
MFC-A53
67
MFC-A54
MFC-A52
99 MFC-A1
Methanosarcina (184bp)
NR 074110 Methanosarcina acetivorans strain C2A
MFC-A71
63 MFC-A28
MFC-A43
NR 074253 Methanosarcina barkeri str. Fusaro
99
MFC-A62
MFC-A22
MFC-A7
91 MFC-A25
63 MFC-A60
99
MFC-A26
88 KF760660 Uncultured archaeon clone C6P100 (rice field soil)
99
MFC-A6
KC784742 Uncultured archaeon clone UP1cl46 (rice field soil)
72
MFC-A16
52
MFC-A2
99
MFC-A10
94
MFC-A74
NR 102903 Methanosaeta concilii strain GP6
59
MFC-A56
MFC-A14
52 85
Methanosaeta (282bp)
69 MFC-A59
MFC-A20
70
MFC-A30
81
AM778305 Uncultured Methanosaetaceae archaeon clone D64AR30R6 (rice field soil)
99
99 MFC-A36
MFC-A67
MFC-A24
MFC-A40
99 MFC-A17
MFC-A51
99 MFC-A68
MFC-A69
72 AM495388 Uncultured archaeon clone LL1Soil 17 (rice root)
MFC-A42
97
MFC-A41
99 FM957907 Uncultured archaeon partial 16S rRNA gene clone CKAR19
78
99
NR 074232 Methanocella arvoryzae strain MRE50
JN048683 Methanocella conradii HZ254
99
Methanocella (391bp)
71 MFC-A37
KF269297
Uncultured archaeon clone A3-28 16S (paddy soil)
84
MFC-A49
65 58 MFC-A65
89 NR 074192 Methanocella paludicola strain SANAE
MFC-A19
98
MFC-A45
98 MFC-A63
NR 074177 Methanospirillum hungatei strain JF-1
NR 041669 Methanofollis ethanolicus strain HASU
87
MFC-A3
89
99
MFC-A5
99 AY607227 Uncultured Methanomicrobiaceae archaeon clone X4Ar01 (flooded soil)
Unc. Methanomicrobiales (82bp)
95
MFC-A32
MFC-A46
74 MFC-A50
75
99 MFC-A77 (391bp)
KF303784.1| Uncultured archaeon clone Dp-12C-141 (rice rizosphere)
MFC-A55
AB479390 Methanoregula formicicum SMSP
99 MFC-A58
Methanoregula (391bp)
92 AJ879005 Uncultured Methanomicrobiaceae archaeon clone LrhA13 (rice rizosphere)
95 MFC-A11
88
NR118098 Methanomassiliicoccus luminyensis strain B10
MFC-A18 (379bp)
99
99 KJ604059 Uncultured archaeon clone J1105242 C-41 M13F-47 (paddy field soil MFC)
99
MFC-A64 (390bp)
FR865353 Uncultured archaeon clone t30d34La7 (rice field soil)
99 MFC-A72 (201bp)
99
KC437233
Uncultured archaeon clone CRYS SPR A04H
88
MFC-A61 (738bp)
NR
102913
Nitrosopumilus maritimus SCM1 strain SCM1
98
NR 102916 Candidatus Nitrososphaera gargensis Ga9.2
94
AB182772 Uncultured archaeon clone: pTN-23 (rice paddy soil)
99
99 MFC-A75 (184bp)
NR 102775 Thermotoga maritima strain MSB8
91
92
0.02
93
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Table S1. Clone sequence analysis and TRF assignment. The table shows phylogenetic affiliations, in silico and in vitro TRF length, and relative
abundance of 16S rRNA clones retrieved from plant microbial fuel cells (PMFC), open circuit control (OC) and unplanted control (NP).
Plant microbial fuel cell (PMFC-A2)
Phylogenetic group
Deltaproteobacteria
Geobacter (Cluster 1, 2, 3)
Other Geobacteraceae
Myxococcales
Anaeromyxobacter (Cluster 4)
Clone
abundance
(%) n=88
In silico TRFs
(bp)
In vitro
TRFs (bp)(a)
49
Clone
In silico TRFs
abundance
(bp)
(%) n= 77
In vitro
TRFs (bp)(a)
33
19
161/163, 130
2
161/163
159, 127
5
3
20
15
Open circuit control (OC-A)
Unc. Myxococcales (Cluster 5)
1
444
Other Myxococcales
4
141,128, 78
Unc. Deltaproteobacteria (Cluster 6)
8
483, 490, 469,
211, 485
Other
0
Chloroflexi
21
125, 155
441
10
8
2
481, 489, 467,
208, 482
161/163
In silico TRFs
(bp)
In vitro
TRFs (bp)(a)
16
161/163, 66
59
3
161/163
10
129, 160,
444, 158, 130,
164
478, 509
0
5
Clone
abundance
(%) n=62
31
161/163
20
129, 160
Unplanted control (NP-A)
155
5
129
441, 155,
129.5, 160
2
444
3
141, 510
0
187, 164
12
0
24
Anaerolineae (Cluster 7)
17
64, 117, 121,
128, 155, 163,
167, 466, 511,
512, 516, 520,
576
Dehalococcoides (Cluster 8)
5
131, 164
1
Alphaproteobacteria
7
150/152,128,
446,132
10
Betaproteobacteria
5
490,456,492,496
20
Acidobacteria
3
3
201, 97
2
Actinobacteria
6
204, 201
140/142, 139,
163, 272, 124
8
140/142,159,163
6
Bacteroidetes/Chlorobi
1
93
4
89,93,491
2
93
Chlamydiae/Verrucomicrobia
1
493
0
3
421, 497
2
272
10
86, 157, 527,
453, 466, 511,
574
18
511
7
Firmicutes
3
518/520, 153
10
Gemmatimonadales
2
82, 79
0
150/152,128,
463, 439
483, 490, 457,
141, 418, 432,
157, 454, 488,
492, 139, 228
518/520, 153,
272
11
11
64, 155, 201,
452, 509, 511,
515, 516, 522,
527
164, 189, 216,
458
150/152,128,171,
68
139, 141, 488,
492, 430, 125,
496
292
140/142, 162,
132
0
6
Gammaproteobacteria
Planctomycetes
0
0
OP10
1
1
0
133
0
493
0
2
206
6
265,133,304
Characteristic TRFs length (bp) for different phylogenetic groups as indicated. TRF detected for more than one clone within the group is indicated as boldface.
a
Discrepancies between in silico and in vitro TRF sizes might occur (Schutte et al., 2008).
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Table S2. Sequence identity in percentage obtained from the similarity matrix of clone
clusters 1 to 8 depicted in the phylogenetic trees (Figure 2).
Cluster
1
2
3
4A
4B
4C
5
6
7
8
Sequence/species
Sequence identity (%)
Rice field soil clone sequences/Geobacter
chapellei (U41561)
98-100/92-95
Geobacter chapellei (U41561)
95-98
Geobacter bremensis (U96917)
95-99
Anaeromyxobacter dehalogenans (AF382400)
97
Anaeromyxobacter dehalogenans (AF382400)
96-97
Anaeromyxobacter dehalogenans (AF382400)
94-95
Myxococcus fulvus (AJ233918)
<87
Rice field soil clone sequences
92-97
Anaerolineae thermolimosa (AB109437)
77-88
Dehalococcoides ethenogenes (AF004928)
80-88
8
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