Supplementary Information (doc 39K)

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Methane oxidation coupled to oxygenic photosynthesis in anoxic waters
Jana Milucka, Mathias Kirf, Lu Lu, Andreas Krupke, Phyllis Lam, Sten Littmann,
Marcel M.M. Kuypers, Carsten J. Schubert
Supplementary Material and Methods
Study site
Lago di Cadagno is a meromictic lake located in the Piora valley in the
southern Alps of Switzerland (46°33’N, 8°43’E; 1923 m altitude). It covers an area of
26 ha with a volume of 2.4x106 m3 and has a maximum water depth of 21 m in
summer (Del Don et al., 2001). The water column is characterized by a permanent
chemocline separating the oxic epilimnion from the anoxic, sulfidic hypolimnion. The
chemocline moves between 9 and 14 m throughout the year (Del Don et al., 2001),
but is very stable during the summer (C. J. Schubert, unpubl. data). Overall, the
position of the chemocline has been stable between 1985 and today (Del Don et al.,
2001). The bottom waters below the chemocline are permanently anoxic (Wagener et
al., 1990). The supply of sub-aquatic spring water flowing through gypsum-rich
dolomites has resulted in relatively high concentrations of sulphate (1.5 mmol l-1),
hydrogen carbonate (between 2-3 mmol l-1 during sampling), calcium and magnesium.
High concentrations of sulphide (1 mmol l-1) in the water column are a result of
ongoing sulphate reduction in the hypolimnion and the sediment (Hanselmann and
Hutter, 1998).
CARD-FISH and cell-marking with laser micro-dissection system
20 ml of water from
13
CH4 incubation was fixed in 2% formaldehyde (final
concentration) for 45 minutes and filtered onto 0.2 μm pore-sized Au/Pd–coated
polycarbonate filters (Merck Millipore, Billerica, MA, USA). Dried filters were kept
1
frozen until further processing. Catalysed reporter deposition–fluorescence in situ
hybridization (CARD–FISH) procedure was performed as described previously
(Musat et al., 2008). For hybridization with the Anme I–350 probe (Boetius et al.,
2000), cells were first permeabilized with proteinase K (15 μg/ml; 3 min at RT). For
hybridization with probes ANME II–538 (Treude et al., 2005), Mgamma705 and
Mgamma84 (Eller et al., 2001), and Alfa968 and Malfa450 (Eller et al., 2001) the
permeabilisation was done with lysozyme (10 mg/ml; for 40 min at 37 °C). Following
formamide concentrations were used 20% for Mgamma705+Mgamma84, and
Malfa450; 35% for Alfa968 and 40% for AnmeI–350 and AnmeII–238. All filters
were counterstained with DAPI. After drying, small circles (5 mm diameter) were cut
out of the filter and individual fields of view containing labeled cells were marked
with a laser in a Laser Micro-dissection microscope (DM 6000 B, Leica
Microsystems, Wetzlar, Germany). Subsequently, the filter was embedded in
Citifluor/Vectashield® and fluorescent images of the marked fields of view were
obtained with an epifluorescence microscope (AxioPlan Microscope, Carl Zeiss, Jena,
Germany). The filters were ultimately washed in 96% ethanol and, after drying,
mounted onto a nanoSIMS holder.
Functional gene analyses
DNA extraction was performed using the UltraCleanTM Microbial DNA
Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA). All PCR reactions were
performed with the PrimeSTAR® HS DNA Polymerase (Takara Bio/Clontech, SaintGermain-en-Laye, France). PCR products were checked with 2% agarose gel
electrophoresis, visualized with SYBR Green staining and subsequently gel-purified
with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). For samples
with positive PCR results clone libraries were constructed using the TOPO TA
2
Cloning Kit for Sequencing (pCR4 vector, Invitrogen, San Diego, CA, USA). Ninetysix colonies were randomly picked from each clone library and PCR-screened with
the primers M13F-M13R to check for inserts. Before sequencing, PCR products were
purified with Sephadex®G-50 superfine columns (Sigma-Aldrich, St. Louis, MO,
USA). DNA sequencing was done with the BigDye Terminator cycle sequencing kit
version 3.1 (Applied Biosystems, Carlsbad, CA, USA) and analyzed on an ABI
PRISM® 3130xL genetic analyzer (Applied Biosystems, Carlsbad, CA, USA).
The acquired DNA sequences were aligned with the CLC Sequence Viewer software
package (CLC Bio/Qiagen, Aarhus, Denmark). Detailed sequence alignment and
phylogenetic analyses were conducted based on the translated amino acids sequences
via the ARB software package (Ludwig et al., 2004). PmoA sequences from this
study were aligned with an existing pmoA database with approximately 7000
sequences (Ruff et al., 2013). Phylogenetic trees were calculated with the maximum
likelihood algorithm PHYML (100 bootstraps) and amino acids were weighted
according to the JTT model.
Nanometer–scale secondary ion mass spectrometry (nanoSIMS)
Image acquisition: All analyzed filters were pre–sputtered with a Cs+ beam of
~500 pA to remove surface contaminations and to implant Cs+ ions. For analyses, the
samples were sputtered with a 0.8–1.3 pA Cs+ primary ion beam focused into a spot
of ~100 nm diameter that was scanned over the sample with an image size of 256x256
pixel and a counting time of 1 ms per pixel. Different amounts of planes were
recorded for individual fields of view (between 22x22 μm or 30x30 μm). The
instrument was tuned for high mass resolution (5,000–7,000 Cameca) of individual
masses (12C–,
13 – 12 14
C,
C N–,
32 –
S ). All respective mass peaks were tuned directly on
3
the sample. For measurement of isotopes, t0 samples from the respective experiments
were analysed for natural abundance.
Data processing: Ion–count images were processed using [email protected]
(Polerecky et al., 2012). For each field of view, the scanned planes were aligned and
accumulated. Subsequently, regions of interest (corresponding to individual cells)
were defined by interactive thresholding (Polerecky et al., 2012), using images of
12 14
C N or
32
S as masks. For each region of interest,
13
C/12C,
12 14
C N/12C and
32
S/12C
ratios were calculated.
Scanning Electron Mircoscopy (SEM)
Fixed samples on gold/palladium-sputtered 0.2 µm polycarbonate filters
(Merck Millipore, Billerica, MA, USA) were examined in an ESEM Quanta 250 FEG
(FEI, Eindhoven, Netherlands) operating at 10 kV and equipped with a double energy
dispersive X-ray spectrometer system (Bruker Nano GmbH, Berlin, Germany) using
Flash 6/30 detectors with an energy resolution < 123 eV Mnkα..
Chlorophyll a measurements
Water (100 ml) from the chemocline and the turbidity maximum
(corresponding to sulfide oxidation zone) was filtered onto pre-combusted GF/F glass
fiber filters (Merck Millipore, Billerica, MA, USA). Chlorophyll a concentrations
were
measured
after
extraction
in
ethanol
by
high-performance
liquid
chromatography (Jasco MC2010 PLUS detector, Jasco, Tokyo, Japan) using a
LiChroCart 250-4 column (Merck Millipore, Billerica, MA, USA).
4
Supplementary references
Boetius A, Ravenschlag K, Schubert CJ, Rickert D, Widdel F, Gieseke A et al. (2000)
A marine microbial consortium apparently mediating anaerobic oxidation of methane.
Nature 407: 623-626.
Del Don C, Hanselmann KW, Peduzzi R, Bachofen R (2001) The meromictic alpine
Lake Cadagno: Orographical and biogeochemical description. Aquatic Sciences 63:
70-90.
Eller G, Stubner S, Frenzel P (2001) Group-specific 16S rRNA targeted probes for the
detection of type I and type II methanotrophs by fluorescence in situ hybridisation.
FEMS Microbiology Letters 198: 91-97.
Hanselmann K, Hutter R (1998). Geomicrobiological coupling of sulfur and iron
cycling in anoxic sediments of a meromictic lake: sulfate reduction and sulfide
sources and sinks in Lake Cadagno. . In: Peduzzi R, Bachofen R, Tonolla M (eds).
Lake Cadagno: a meromictic Alpine lake Documenta dell'Istituto Italiano di
Idrobiologia. pp 85-98.
Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar et al. (2004)
ARB: a software environment for sequence data. Nucleic Acids Research 32: 13631371.
Musat N, Halm H, Winterholler B, Hoppe P, Peduzzi S, Hillion F et al. (2008) A
single-cell view on the ecophysiology of anaerobic phototrophic bacteria.
5
Proceedings of the National Academy of Sciences of the United States of America 105:
17861-17866.
Polerecky L, Adam B, Milucka J, Musat N, Vagner T, Kuypers MMM (2012)
[email protected] - a tool for the analysis of nanoSIMS data in environmental
microbiology. Environmental Microbiology 14: 1009-1023.
Ruff SE, Arnds J, Knittel K, Amann R, Wegener G, Ramette A, Boetius A (2013)
Microbial communities of deep-sea methane seeps at Hikurangi continental margin
(New Zealand). PLoS ONE 8(9): e72627. doi:10.1371/journal.pone.0072627
Treude T, Knittel K, Blumenberg M, Seifert R, Boetius A (2005) Subsurface
microbial methanotrophic mats in the Black Sea. Applied and Environmental
Microbiology 71: 6375-6378.
Wagener S, Schulz S, Hanselmann K (1990) Abundance and distribution of anaerobic
protozoa and their contribution to methane production in Lake Cadagno (Switzerland).
FEMS Microbiology Ecology 74: 39-48.
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