Dec 9 2009 (2) Common. Mostly variable length filaments of. Oscillatoria spp. (35-80 µm). One. Nostocaceae filament observed (39. µm). Rare. Not observed.
SUPPLEMENTARY INFORMATION DOI: 10.1038/NGEO2925
In the format provided by the authors and unedited.
Microbial formation of labile organic carbon in Antarctic glacial environments Glacial organic carbon, not just a legacy of the past H. J. Smith1,2, R. Foster4,5, D. M. McKnight6, J. T. Lisle7, S. Littmann5, M. M. M. Kuypers5, and C. M. Foreman1,3** 1
Center for Biofilm Engineering, 2Land Resources and Environmental Sciences, 3Chemical and Biological Engineering, Montana State University, Bozeman, Montana 59717, USA,
4
Department of Ecology, Environment, and Plant Sciences, Stockholm University, Stockholm 10691, Sweden
5
Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany
6
INSTAAR, University of Colorado, Boulder, Colorado 80309, USA
7
US Geological Survey, St. Petersburg Costal & Marine Science Center, St. Petersburg, FL 33701, USA
Supplementary Information: Supplementary Information Content: Supplementary Figures (S1-S4) Supplementary Tables (S1-S5)
NATURE GEOSCIENCE | www.nature.com/naturegeoscience
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
1
Supplementary Figures:
Supplementary Figure 1: Excitation emission matrices (EEMs) of CG stream water, successive incubations, and organic matter transformations. EEMs from: (a.) Cotton Glacier stream water, (b.) exuded 13C-labeled exudates after 72 hrs incubation and removal of unincorporated 13CDIC, (c.) organic material after a 24 hr feedback of exuded OC to the in situ Cotton Glacier stream microbial community,
Supplementary Figure 2. PICRUST generated predicted genetic function profile of the Cotton Glacier Stream bacterial community, providing functional evidence for the degradation of in situ OM dominated by amino acid-like fluorescence.
Supplementary Figure 3. Example of analyzed Alphaproteobacteria sp. cells enriched in a 13Clabeled algal amino acid (13C-AA) mixture (a.) Epifluorescence overlay used to confirm cell identification of Alphaproteobacteria sp. cells hybridized with ALF1B and ALF968 oligonucleotide probes (used in equal amounts). (b.) nanoSIMS isotope ratio image for the 13 12 C/ C ratio for cells enriched in 13C labeled AA mixture, white lines indicate ROIs used for enrichment calculations of analyzed bacterial cells. (c.) Summary of nanoSIMS analyses of 13CAA enriched cells reported in atom % and (d.) reported in fmol C cell-1 d-1 for Alphaproteobacteria sp., Bacteroidetes sp., Betaproteobacteria sp., and Polaromonas sp. The whiskers represent the 25th and 75th percentile (lower and upper quartiles, respectively) the mean is shown as a solid line, and outliers are represented by (•).
0.4
Cyanobacteria
0.3
Frequency5
Diatoms
0.2
0.1
0
5
15
25
35
45
55
65
75
85
95 105 115 125
>
Filament5(cyanobactiera)5or5cell5(diatom)5length5(μm) Supplementary Figure 4. Size histogram of cyanobacteria filaments and diatom cell lengths from 10 Cotton Glacier samples imaged by FlowCAM. In total 219 cyanobacteria filaments and 25 diatom cells were observed.
Supplementary Tables:! Supplementary Table 1. Summary of FlowCAM images, including organism abundance, description, and size. Occurrence designations: Abundant: > 10 filaments or cells, Common: 310 filaments or cells, Rare: < 3 filaments or cells. Cyanobacteria of the family Oscillatoriaceae appeared to be either of the genera Oscillatoria or Phormidium, while Nostocaceae had wider filaments with beaded cells. Rotifers or protists were not observed in the planktonic community Sample date (#)
Filamentous cyanobacteria
Pennate diatoms
Dec 9 2009 (1)
Occurrence Common
Occurrence Rare
Description/comments Not observed
Dec 9 2009 (2)
Common
Rare
Not observed
Dec 13 2010 (1)
Abundant
Description/comments Mostly variable length filaments of Oscillatoria spp. (18-48 µm). One Nostocaceae filament observed (52 µm). Mostly variable length filaments of Oscillatoria spp. (35-80 µm). One Nostocaceae filament observed (39 µm). Mostly variable length filaments of Oscillatoria spp. (20-178 µm). Few Nostocaceae filaments observed (14-76 µm).
Rare
1 Luticola spp. (24 µm) 1 Muelleria spp. (28 µm)
Dec 13 2010 (2)
Abundant
Rare
1 Luticola spp. (22 µm) 1 Muelleria spp. (35 µm)
Dec 15 (1)
Common
Rare
1 Muelleria spp. (40 µm) 1 Navicula spp. (23 µm)
Dec 23 2009 (1)
Abundant
Rare
1 Luticola spp. (39 µm) 1 Stauroneis spp. (45 µm)
Dec 23 2009 (2)
Common
Mostly variable length filaments of Oscillatoria spp. (14-113 µm). Few Nostocaceae filaments (27-45 µm). Only Oscillatoria spp. filaments with variable lengths observed (1863 µm) Mostly variable length filaments of Oscillatoria spp. (15-100 µm), Nostocaceae, common, (15-65 µm). Few shorter filaments of Phormindium spp.also present but rare (7-23 µm). Mostly variable length filaments of Oscillatoria spp. (24-95 µm) and Nostocaceae (28-46 µm)
Common
1 Humidophila spp. (17 µm) 3 Muelleria spp. (35-65 µm) 1 Navicula spp. (25 µm)
Dec 29 2010 (1)
Abundant
Mostly variable length filaments of Oscillatoria spp. (18-120 µm), while shorter (10-50 µm) Nostocaceae also common. Few short Phormidium spp. filaments (10-22 µm) present, but rare.
Common
4 Humidophila spp. (15-20 µm); 3 Muelleria spp. (45-55 µm); 1 Navicula spp. (28 µm).
Dec 30 2009 (1)
Abundant
Mostly Oscillatoria spp. of variable length (18-184 µm), some short Nostocaceae filaments (15-24 µm).
Common
2 Luticola spp. (18-28 µm) 2 Stauroneis spp. (38-44 µm)
Dec 30 2009 (2)
Common
Mostly Oscillatoria spp. filaments of variable length (20-70 µm), few Nostocaceae filaments (20-30 µm)
Rare
1 Luticola spp. (22 µm)
Supplementary Table 2. Summary of cell diameter measurements and nanoSIMS analysis for the various target groups identified by CARD-FISH. SD is the standard deviation, and N is the number of cells analyzed for a particular isotopic tracer (13C-labeled exudates or 13C- labeled algal amino acids). Average DOC uptake rates of 13C-labeled exudates and 13C-labeled amino acids ±1SD. Enrichment that was not above natural abundances is considered not enriched and below detection (ND).
Target Bacterial Group & Carbon Substrate
Cell diameter (µm) (mean ± SD)
Biovolume (µm3) (mean ± SD)
N
Time (h)
DOC uptake rate (fmol C cell-1 h-1) (mean ± SD)
Alphaproteobacteria 13 C Exudates 13 C Algal Amino Acids
16 16
24 72
0.871 ± 0.229 1.012 ± 0.241
0.418 ± 0.104 0.631 ± 0.127
ND 2.88 ± 2.02
Bacteroidetes 13 C Exudates 13 C Algal Amino Acids
15 15
24 72
0.861 ± 0.106 0.566 ± 0.170
0.346 ± 0.103 0.114 ± 0.0926
.298 ± .118 .468 ± .370
Betaproteobacteria 13 C Exudates 13 C Algal Amino Acids
16 19
24 72
0.764 ± 0.224 0.809 ± 0.282
0.290 ± 0.220 0.384 ± 0.139
.239 ± .183 2.39 ± 1.83
Polaromonas 13 C Exudates 13 C Algal Amino Acids
23 25
24 72
0.786 ± 0.109 0.755 ± 0.212
0.269 ± 0.131 0.275 ± 0.123
.245 ± .111 2.88 ± 1.94
Supplementary Table 3: Summary of 16S rRNA targeted oligonucleotide probes, target organisms, competitor sequences and formamide (FA) concentrations, and references for the probes selected in this study. All FA concentrations used were the optimal concentrations recommended by each respective publication. Probe and Target Organism
Target Position
Target
% FA
Reference
338-355
16S
35
Amann et al. (1990)
338-355
16S
35
Damis et al. (1999)
338-355
16S
35
Damis et al. (1999)
338-355
16S
35
Wallner et al. (1993)
319-336
16S
35
Manz et al. (1996)
319-336
16S
35
Manz et al. (1996)
CGT TCG YTC TGA GCC AG GGT AAG GTT CTG CGC GTT
19-35
16S
20
Manz et al. (1992)
968-985
16S
20
Neef (1997)
GCC TTC CCA CAT CGT TT GCC TTC CCA CTT CGT TT
1027-1043
23S 23S
35
Manz et al. (1992)
GCC TTC CCA CTT CGT TT GCC TTC CCA CTT CGT TT
1027-1043
23S 23S
35
Manz et al. (1992)
828-845
16S
20
Loy et al. (2005)
Probe Sequence (5'-3')
For Bacteria (used in equal amounts) EUB338-I EUB338-II EUB338-III Antisense of EUB338 NON338 Most Flavobacteria, some Bacteroidetes and some Sphingobacteria CF319a CF319b Alphaproteobacteria, some Deltaproteobacteria, Spirochaetes (used in equal amounts) ALF1B ALF968 Gammaproteobacteria GAM42A Competitor Betaproteobacteria BET42a Competitor Polaromonas Pomo828
GCT GCC TCC CGT AGG AGT GCA GCC ACC CGT AGG TGT GCT GCC ACC CGT AGG AGT ACT CCT ACG GGA GGC AGC
TGG TCC GTG TCT CAG TAC TGG TCC GTA TCT CAG TAC
CTA ATG CAC CCA ACA ACC
Supplementary Table 4: Summary of previously identified fluorophore designations (Coble 1996, Marhaba et al. 2000) adapted from D’Andrilli et al. 2013, of the Ex/Em maxima for fluorescing regions present in this study. Fluorophore Designation
Ex/Em Max (nm)
Compositional Description
CG Stream Water Ex/Em Max (nm)
Exudate Ex/Em Max (nm)
Exudate Feedback Ex/Em Max (nm)
B
225-237/309-321 275/310
Protein-like (Tryrosine)
270/300
280/304
240/326
T
225-237/340-381 275/340
Protein-like (Tryptophan)
240/326
280/326
240/328
A
237-260/400-500
Humic-like
240/400
240/452
240/400
Supplementary Table 5. Parameters and equations used in estimating the cell specific C and N assimilation rates. Sources for the parameters and references for the equations are given where appropriate. Parameter t d BV Ccell Ccon 13 C/12Ci
Definition Time Cell diameter Biovolume of a sphere Initial carbon content Initial carbon content Natural abundance of cells
Units
Equation
µm µm3 pgC pmolsC 13 C/12C
BV = π/6 x d Log(C)=-3.63 + 0.863 (Log BV)* Ccell/12.01
13
C/12Cf Cex CSR
Enrichment of cells at time final 13 C excess Labeling percent
13
C/12C Atom% %
(13C/12Cf – 13C/12Cti)/100
FC FR
Cell specific C assimilation Cell specific C fixation rate
pmols C pmols C cell-1 h-1
FC = (13Cex x Ccon)/CSR Fc/t
13
* LOG: logarithms base 1
Source or Reference Measured Measured from ROI diameter Sun & Liu 2003 Verity et al. 1992 Time 0 filter from bulk analyses or ROIs of time 0 cells ROIs at time final Calculated from the measured natural 13C abundance. Foster et al. 2011
