Supporting Information
Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional
metaproteomics (protein-SIP)
Florian-Alexander Herbst1,2, Arne Bahr3, Marcia Duarte4, Dietmar H. Pieper4, Hans-Hermann
Richnow3, Martin von Bergen1,4,7, Jana Seifert1,8* and Petra Bombach3,6**
1
UFZ - Helmholtz Centre for Environmental Research, Department of Proteomics,
Permoserstrasse 15, D-04318 Leipzig, Germany
2
University of Freiburg, Faculty of Biology, Schaenzlestrasse 1, D–79104 Freiburg, Germany
3
UFZ
-
Helmholtz
Centre
for
Environmental
Research,
Department
of
Isotope
Biogeochemistry, Permoserstrasse 15, D-04318 Leipzig, Germany
4
Helmholtz Center for Infection Research-HZI, Microbial Interactions and Processes
Research Group, Inhoffenstraße 7, 38124 Braunschweig, Germany
5
UFZ - Helmholtz Centre for Environmental Research, Department of Metabolomics,
Permoserstrasse 15, D-04318 Leipzig, Germany
6
Isodetect GmbH - branch Leipzig, Permoserstrasse 15, D-04318 Leipzig, Germany
7
University of Aalborg, Department of Biotechnology, Chemistry and Environmental
Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark
8
Institute of Animal Nutrition, University of Hohenheim, Emil-Wolff-Str. 10, 70599 Stuttgart
1
Material and Methods
In situ microcosms The in situ microcosms consisted of perforated Teflon tubes of 5 cm
length and 1 cm diameter with a perforation of 1 mm which were filled with 1 g of preheated
(4 h at 300°C) activated carbon pellets (Biocoal, Silcarbon Aktivkohle GmbH, Kirchhundem,
Germany) (Fig. 1). Following sterilization and hydration of the BACTRAPs by autoclaving at
121°C, the BACTRAPs were each loaded with 25 mg [13C6]-naphthalene, a 1:1 mixture of
[13C5]- and [13C6]-fluorene or their unlabeled counterparts via dropping the compounds diluted
in 1 mL n-hexane on the activated carbon pellets. For each groundwater well, one BACTRAP
with labeled and one BACTRAP with unlabeled naphthalene or fluorene was prepared.
Additionally, one BACTRAP loaded with unlabeled fluorene was prepared to collect
indigenous groundwater microorganisms at the plume fringe as inoculum for laboratory
microcosms (see chapter Laboratory microcosms). After an incubation period of two days in
a desiccator under reduced pressure (60 mbar), BACTRAPs were transported in sterile water
to the field site. All BACTRAPs were fastened to a tether with 15 cm of space between the
C and 12C-PAH BACTRAPs and deployed at 0.8 – 1.4 m below groundwater level directly in
13
the Quaternary aquifer. The BACTRAP for the laboratory microcosm was deployed 30 cm
below the 12C-fluorene BACTRAP. BACTRAPs loaded with naphthalene were installed in the
wells UA 92005 (contaminant source) and RKB 127 (plume fringe). The fluorene-BACTRAPs
were installed in the wells RKB 139 (contaminant source) and RKB 222 (plume fringe). After
100 days (naphthalene) and 68 days (fluorene) of incubation, the BACTRAPs were
recovered and immediately transferred to sterile falcons on dry ice. BACTRAPs were stored
at -80°C until further analysis.
Laboratory microcosm Laboratory microcosm was set up using one
12
C-fluorene BACTRAP
as inoculum. The BACTRAP was incubated in well RKB222 (plume fringe) also used for the
in situ fluorene-amended BACTRAPs (see above). After 100 days of incubation, the
BACTRAP was recovered and immediately transferred to a flask completely filled with oxic
2
mineral media [1] (Fig. 1). Additionally, a groundwater sample from the well was collected
serving as media and additional inoculum for the laboratory microcosm. Half of the activated
carbon pellets (0.5 g) was transferred to a sterile 240 mL serum flask filled with 100 mL
groundwater of the respective well. The microcosm was spiked with 3 mg of
12
C-fluorene and
was closed gastight with a Teflon-coated crimp cap. The microcosm was incubated for 126
days on an orbital shaker (90 rpm) at 14°C in the dark in order to mimic the field conditions.
At regular intervals, the dissolved oxygen concentration was measured using oxygen sensor
spots (SP-PSt3-NAU-D5-YOP, PreSens - Precision Sensing GmbH, Regensburg, Germany).
A detailed description of the oxygen measurements are given in Rosell et al. (2010) [2]. If the
dissolved oxygen concentration decreased below 1 mg/L, the microcosm was spiked with
oxygen according the starting concentrations. After 126 days of incubation the activated
carbon pellets were removed under sterile conditions and were immediately frozen at -80°C.
The medium was filtrated through a 0.2 µm filter (Supor 200; Pall GmbH, Dreieich, Germany)
under sterile conditions and also stored at -80°C.
DNA extraction
Total nucleic acids were extracted each from 0.5 g activated carbon pellets of the
12
C-
naphthalene and 12C-fluorene BACTRAP using a bead beating protocol described elsewhere
[3]. Nucleic acid extracts were suspended in 30 µl molecular-grade nuclease-free water and
stored frozen at -20°C.
Illumina sequencing
For an overview of the microbial community, total DNA was used as template to amplify the
V1 region of the 16S rRNA gene using primers based on the previously described 27F and
338R primers [4, 5]. Barcoded amplicon mixtures were sequenced on a MiSeq Sequencer
(Illumina) (Camarinha-Silva, unpublished data) and forward end sequence reads of a length
of 120 nucleotides were used for manual annotation, using the Ribosomal Database Project
(RDP) database [6]. Between 142 and 263 unique sequences were analyzed per sample.
3
Results
Fig. S1 Relative abundances of phylogenetic groups based on the v1 hypervariable region of
the 16S rRNA gene .
References
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sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer
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[6] Cole, J. R., Wang, Q., Cardenas, E., Fish, J., et al., The Ribosomal Database Project:
improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009, 37, D141145.
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