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Supplementary Materials and Methods
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Origin and enrichment of samples
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Microbial biofilms were scraped off moist limestone near the entrance of Jenolan caves (Blue Mountains, NSW,
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Australia) and packed into shaded zip-lock bags for transportation back to the laboratory. In the lab, biofilms
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were aliquoted into Erlenmeyer flasks containing sterile freshwater based f/2 medium and maintained at 28°C.
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The cultures were shaken at 100 rpm under a 12/12 h light-dark cycle and near infrared radiation (NIR) was
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provided by narrow band LEDs (centered at 720 nm, Epitex Inc., Japan) at an irradiance of 20-40 µmol photons
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m-2 s-1. Irradiance was measured and adjusted by use of a calibrated spectroradiometer (JAZ-ULM, Ocean
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Optics). After several months of incubation, photopigments in the enrichments were screened via HPLC (see
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below) and promising candidates (=containing Chl f) were transferred repeatedly and maintained in fresh f/2
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medium.
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High-performance liquid chromatography (HPLC)
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For HPLC analysis, samples of enrichment culture were centrifuged at 13,000 x g, the supernatant was removed
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and the cell pellet was resuspended in cold acetone:methanol (7:2 by vol) and sonicated for 20s using a
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Misonix sonicator 4000 (Qsonica LLC., Newtown, CT, USA) according to (Frigaard et al. 1996). The cells were
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incubated for 2 min on ice in complete darkness, centrifuged again and the supernatant transferred into a fresh
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Eppendorf tube. Ammonium acetate (15 µl; 1.0 M) was added to the extracts (150 µl) to further improve
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pigment resolution before subsequent injection and pigment separation on a HPLC (Agilent 1260 infinity HPLC,
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Agilent Technologies, Santa Clara, CA, USA) equipped with a Nova-pak C18 column (dimensions: 3.9x 300 mm).
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Culture extracts were run with solvent A (methanol:acetonitrile:water, 42:33:25 by vol) and solvent B
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(methanol:acetonitrile:ethyl acetate, 39:31:30 by vol) in a gradient comprising 40% solvent B at time of
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injection, a linear increase to 100% B at 60 min and back to 40% B in 3 minutes. The flow rate was kept
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constant at 1 ml min-1 and the column was kept at a temperature of 30°C. A spectral detector (1260 Infinity
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Multiple Wavelength Detector, Agilent Technologies, Santa Clara, CA, USA) was used for the detection of
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compound-specific absorption characteristics at wavelengths between 190-950 nm. Photopigments were
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identified manually by carefully detecting absorption from the resulting HPLC chromatograms as stated in
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(Frigaard et al. 1996).
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Hyperspectral imaging
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Enrichment culture samples were mounted on microscopy slides under a coverslip and were screened for the
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occurrence of Chl f with a hyperspectral image scan unit (100T-VNIR, Themis Vision, Bay Saint Louis, MS, USA)
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mounted on a compound microscope (Axioscope FS, Zeiss, Germany) using the built-in halogen lamp for
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illumination. Hyperspectral image stacks were corrected (in Hypervisual 3.0, Themis Vision) for background
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noise under darkness and normalized to percent (%) transmission using hyperspectral image stacks recorded in
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regions without cells as 100% transmission (Kühl and Polerecky 2008). Reflectance spectra averaged over
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particular areas of interest (AOI) were subsequently calculated and extracted from the hyperspectral image
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stack by the system software.
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Microsensor measurements
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Clusters of cells were removed from Chl f enrichment cultures and placed onto solidified 1% low-melting point
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agarose in a deep-well microscopy slide. Cells were then overlaid with a thin layer of agarose and placed into
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darkness for solidification. A petri dish filled with pre-warmed f/2 medium was aerated using an aquarium
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pump and the cell-containing microscopy slide placed within. A Clark-type O2 microsensor (tip size ~20 µm;
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Revsbech 1989) was mounted on a motorized micromanipulator (Pyro-Science GmbH, Aachen, Germany) and
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connected to a picoammeter (Unisense PA2000, Unisense A/S, Aarhus, Denmark). The sensor was linearly
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calibrated from measurements in O2-free medium (by the addition of sodium dithionite) and fully aerated
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medium at experimental temperature and salinity. The position where the microsensor tip touched the cell
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cluster was determined by visual inspection using a stereo microscope mounted on a stand. All microsensor
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signals were recorded on a strip-chart recorder (Kipp and Zonen, B.V., Delft, Netherlands) and via an A/D
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converter (AD-216, Unisense A/S, Aarhus, Denmark) and gross-photosynthesis (in units of nmol O2 cm-3 s-1) was
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quantified using the O2 microsensor light-dark shift method (Kühl et al. 1996; Revsbech et al. 1983).
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Transmission electron microscopy
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Samples were fixed with 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.4) for 24 h. Samples
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were then rinsed three times in 0.15 M sodium cacodylate buffer (pH 7.4) and subsequently postfixed in 1%
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w/v OsO4 in 0.12 M sodium cacodylate buffer (pH 7.4) for 2 h. The specimens were dehydrated in graded series
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of ethanol, transferred to propylene oxide and embedded in Epon according to standard procedures. Sections,
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approximately 80 nm thick, were cut with a Reichert-Jung Ultracut E microtome and collected on copper grids
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with Formvar supporting membranes. Sections were stained with uranyl acetate and lead citrate and were
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examined with a Philips CM 100 TEM (Philips, Eindhoven, The Netherlands), operated at an accelerating
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voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k x 2k CCD camera and
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the ITEM software package.
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Molecular analysis
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DNA from an enrichment culture was extracted with the FastDNA for soil kit (MP Biomedicals, France) using the
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manufacturers standard protocol. DNA concentration was quantified using a Qubit system (Invitrogen, Life
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Technologies Europe, USA), adjusted to 5 ng µl-1 using water and stored at -20°C until further use. From the
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DNA, the V4 region of the 16S rDNA gene was amplified using the modified primers 515F
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(GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACNNGGGTATCTAAT). The PCR mix (20 µl) contained: 2.0 µl
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of 10X AccuPrime PCR Buffer II containing 15 mM MgCl2, 0.12 µl AccuPrime Taq DNA Polymerase (2 U µl-1, Life
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Technologies, Carlsbad, CA, USA), 1.0 µl of each primer (10 µM), 2 µl DNA template and water to a total of 20
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µl. The first PCR mix was incubated according to following conditions: initial activation of the “hot start”
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polymerase at 94°C for 2 min, followed by 30 cycles of 94°C for 20 s, 56°C for 20s and 68°C for 30s, and final
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extension at 68°C for 5 min. The amplified PCR product was incubated at 70°C for 3 min then placed on ice to
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minimize hybridization between specific PCR products and short non-specific amplicons. The concentration of
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amplified DNA was measured by Pico Green (Life Technologies) and LightCycler 96 (Roche, Basel, Switzerland).
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Addition of adapters and Index to DNA fragments was done in a second PCR containing: 2.0 µl 10X AccuPrime
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PCR Buffer containing 15 mM MgCl2, 0.12 µl AccuPrime Taq DNA Polymerase (2 U µl-1, Life Technologies), 1.0 µl
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of each barcoded fusion primers (10 µM), 2 µl of 10X diluted PCR product from first PCR and water to a total of
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20 µl. The PCR incubation conditions for second PCR were: an initial activation of the “hot start” polymerase at
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94°C for 2 min, followed by 15 cycles of 94°C for 20 s, 56°C for 20s and 68°C for 30s, and final extension at 68°C
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for 5 min. The amplified PCR product was incubated at 70°C for 3 minutes and then directly placed on ice. The
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amplified PCR product was purified by Agencourt AMPure XP (Beckman Coulter, Pasadena, CA, USA). The
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concentration of amplified DNA was measured by Pico Green (Life Technologies) on a LightCycler 96 (Roche)
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and hereafter concentrated with the DNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA, USA).
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Sequencing of the 16S rDNA gene was done using MiSeq reagent kit v2 (500cycles) and a MiSeq sequencer
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(Illumina inc., San Diego, CA, USA). All unprocessed sequences were deposited ar EMBL under the accession
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number PRJEB7980. Miseq reads were classified using the Illumina 16S Metagenomics Basespace app. and
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reads classified down to the order Synechococcales (=the only oxygenic phototrophs present) were extracted
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from the fastq files (=6.210 reads of a total of 57.627 reads). The UPARSE pipeline (Edgar 2013) was used to
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cluster the reads into a single OTU at 97%. Briefly, paired end reads were merged with usearch using the
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options fastq_truncqual 3 and fastq_maxdiffs 2, for quality control. Merged reads were dereplicated and
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sorted with usearch. Reads were clustered with usearch -cluster_otu, discarding singletons as recommended.
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The resulting representative sequence (GTGCCAGCAGCCGCGGTAAGACGGAGGAGGCAAGCGTTATCCGGAATTATT
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GGGCGTAAAGCGTCCGCAGGTGGCTTCTCAAGTCTGCTGTCAAAGCCCAGGGCTCAACCCTGGATAGGCAGTGGAAACTGGGTCGCTA
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GAGTACGGTAGGGGTAGAGGGAATTCCCGGTGTAGCGGTGAAATGCGTAGATATCGGGAAGAACACCAGTGGCGAAGGCGCTCTAC
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TGGACCGTAACTGACACTCATGGACGAAAGCTAGGGGAGCGAAAGGGATTAGAAACCCCAGTAGTCC)
was used for tree
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building with reference sequences downloaded from SILVA (http://www.arb-silva.de/). Reference sequences
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and the representative sequence were aligned using MUSCLE (Edgar 2004) as implemented in SeaView (Gouy
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et al. 2010). Maximum-likelihood phylogenetic tree reconstruction was done in MEGA6 (Tamura et al. 2013)
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using the implemented GTR (I + G) model with five discrete gamma categories. Branch support was estimated
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by performing 100 bootstrap replications (Felsenstein 1985).
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Supplementary references
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Edgar RC. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids
Res. 32:1792–7.
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Edgar RC. (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods
10:996–8.
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Felsenstein J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution (N. Y).
39:783–791.
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Frigaard N, Larsen KL, Cox RP. (1996). Spectrochromatography of photosynthetic pigments as a fingerprinting
technique for microbial phototrophs. FEMS Micriobiology Ecol. 20:69–77.
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Gouy M, Guindon S, Gascuel O. (2010). SeaView version 4: A multiplatform graphical user interface for
sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27:221–4.
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Kühl M, Glud RN, Ploug H, Ramsing NB. (1996). Microenvironmental Control of Photosynthesis and
Photosynthesis -coupled respiration in an epilithic cyanobacterial biofilm. J. Phycol. 32:799–812.
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Revsbech N. (1989). An oxygen microsensor with a guard cathode. Limnol. Oceanogr. 34:474–478.
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Revsbech N, Jorgensen B, Blackburn T, Cohen Y. (1983). Microelectrode studies of the photosynthesis and 0 2,
H2S, and pH profiles of a microbial mat. Limnol. Ocean. 28:1062–1074.
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Tamura K, Stecher G, Peterson D. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol.
Biol. Evol. 1–12.
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Supplementary Figure legends:
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Figure S1: HPLC chromatograms and spectral characteristics of eluents from cells enriched under NIR. (A)
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Samples contained detectable amounts of Chl f (at ~44.5 min) and Chl a (~49 min) absorbing maximally at 704-
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708 and 665 nm, respectively. (B) Absorption spectrum of HPLC-extracted Chl f (in acetone:MeOH) with
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maximum absorption occurring at 404 and 704 nm.
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Figure S2: Order level taxonomy of enrichment cultures containing Chl f. Results were generated using Illumina
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16S rDNA gene amplicon sequencing on the MiSeq system. Data were analyzed as stated in the materials and
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methods section and here displayed are the 57,627 reads classified to the order level. Reads belonging to the
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Synechococcales, the only order in the sample known to contain oxygenic phototrophs, were extracted (=6,210
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reads) and clustered, resulting in a single OTU. The latter OTU was hereafter used to construct a Neighbor-
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joining tree.
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