Carbon Flow from Volcanic CO2 Into Soil Microbial Communities of a Wetland Mofette

Supplementary Information

Carbon flow from volcanic CO2 into soil microbial communities of a wetland mofette

Felix Beulig1, Verena B. Heuer2, Denise M. Akob1,3, Bernhard Viehweger2, Marcus Elvert2, Martina Herrmann1,4, Kai-Uwe Hinrichs2 and Kirsten Küsel1,4

1 Aquatic Geomicrobiology, Institute of Ecology, Friedrich Schiller University Jena, Dornburger Str. 159, D-07743 Jena, Germany

2 Organic Geochemistry Group, Dept. of Geosciences and MARUM Center for Marine Environmental Sciences, University of Bremen, P.O. Box 330 440, D-28334 Bremen, Germany

3U.S. Geological Survey, 12201 Sunrise Valley Drive, MS 430, Reston, Virginia 20192 USA

4 German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, D-04103 Leipzig, Germany

Corresponding author:

Kirsten Küsel

Friedrich Schiller University Jena

Institute of Ecology

Aquatic Geomicrobiology Group

Dornburger Str. 159,

07743 Jena

Germany

Subject category: Microbial ecology and functional diversity of natural habitats

Keywords: Mofette soils; methanogens; acetogens; Acidobacteria; CCS

Supplementary Methods

Sulfate, nitrate and Fe(II) analysis in pore-waters

Pore-water was filtered (0.45 μm, PVDF) and then either stored at −20 °C for nitrate analysis or acidified and stored at 4 °C for sulfate analysis. Fe(II) was determined with the phenanthroline method (Tamura et al.1974) after extraction in 0.5 N HCl for 1 h at 22 °C. Sulfate and nitrate were analyzed using the barium chloride method (Tabatabai, 1974) and the salicylate method (Vijayasarathy, 2008), respectively.

Lipid analysis

One to 2 g (dry weight) soil from the unlabeled and 13CO2 incubations were lyophilized and extracted using a modified Bligh–Dyer procedure (Sturt et al., 2004) with addition of 10 μg of 2-methyl-octadecanoic acid and 1-Nonadecanol as internal standards for final fatty acid and ether lipid quantification, respectively. Polar lipid derived fatty acids (PLFAs) were released from the total lipid extracts by mild alkaline hydrolysis using 6% KOH in methanol according to Elvert et al. (2003). PLFAs were converted to fatty acid methyl esters (FAME) using boron trifluoride in methanol. Neutral lipids, which have been recovered after the saponification reaction, were separated by liquid chromatography using a NH2-solid phase extraction (cf. Hinrichs et al., 2000). The obtained alcohol fraction (eluted with DCM:Acetone = 9:1) was subjected to an ether cleavage reaction using boron tribromide followed by subsequent reduction with lithium triethylboronhydride (Lin et al., 2010), thus producing hydrocarbons from core ether lipids (e.g., phytane and biphytane). Both FAMEs and ether-cleaved hydrocarbons were analyzed by GC/MS for identification (Agilent 6890 coupled to Agilent 5973 GC-MSD), GC/FID (Thermo Finnigan Trace GC) for quantification and irm-GC/MS (Thermo Electron Trace GC ultra coupled to Thermo Finnigan Delta plus XP) for stable carbon isotopic composition using conditions reported previously (Elvert et al., 2003; Lin et al., 2010). δ13C values of FAMEs were corrected by mass balance with regard to added methyl groups (Rieley, 1994). Fatty acids are presented as the total number of carbon atoms, followed by a colon and the number of double bonds. The prefixes ai and i denote methylation in anteiso- and iso-position, respectively. 10Me signifies a methyl group in C-10 position. Analysed fatty acids included (C14:0, iC15:0, aiC15:0, iC16:0, C16:1ω7c, C16:0, 10MeC16:0, iC17:0, aiC17:0, C17:0, C18:1ω9, C18:1ω7c, C18:0, C20:0).

Thermodynamic Calculations

In situ standard free energies (ΔG0in situ) for acetoclastic and hydrogenotrophic methanogenesis (Eq. (1) and (2)), as well as acetogenesis (Eq. (3)) were calculated using the SUPCRT92 software package (Johnson et al. 1992) with the slop07 database for a soil temperature of 285 K and 1 atm pressure.

(1)

(2)

(3)

Non-standard free energies (ΔG'in situ) were then calculated based on in situ pore water data according to ΔG'in situ = ΔG0in situ + R  T  ln Q, where R is the ideal gas constant, T is the in situ temperature, and Q denotes the activity quotient of reactants and products. Bicarbonate concentrations were calculated based on the Henderson–Hasselbalch equation with the assumption of an effective pKa of 6.1 according to pH = pKa + lg ([HCO3-] / [CO2,aq])

References

Lin YS, Lipp JS, Yoshinaga M, Lin S-H, Elvert M, Hinrichs KU. (2010). Intramolecular stable carbon isotopic analysis of archaeal glycosyl tetraether lipids. Rapid Commun Mass Spectrom, 24 (19), 2817-2826.

Elvert M, Boetius A, Knittel K, Jørgensen BB. (2003). Characterization of specific membrane fatty acids as chemotaxonomic markers for sulfate-reducing bacteria involved in anaerobic oxidation of methane. Geomicrobiol J20 (4): 403-419.

Hinrichs KU, Summons RE, Orphan V, Sylva SP, Hayes JM. (2000). Molecular and isotopic analysis of anaerobic methane-oxidizing communities in marine sediments. Org Geochem 31, 1685–1701.

Johnson JW, Oelkers EH, Helgeson HC. (1992). SUPCRT92: A software package for calculating the standard molal thermodynamic properties of minerals, gases, aqueous species, and reactions from 1 to 5000 bar and 0 to 1000°C. Comput Geosci 18: 899–947.

Rieley G. (1994). Derivatization of organic compounds prior to gas chromatographic–combustion–isotope ratio mass spectrometric analysis: identification of isotope fractionation processes. Analyst119: 915–919.

Sturt HF, Summons RE, Smith K, Elvert M, Hinrichs KU. (2004). Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry--new biomarkers for biogeochemistry and microbial ecology. Rapid Commun Mass Spectrom18: 617–628.

Tabatabai MA (1974). A Rapid Method for Determination of Sulfate in Water Samples. Environ Letters7: 237–243.

Tamura H, Goto K, Yotsuyanagi T, Nagayama M (1974). Spectrophotometric determination of iron(II) with 1,10-phenanthroline in the presence of large amounts of iron(III). Talanta21: 314–318.

Vijayasarathy PR (2008). Determination of nitrate in water. In: Engineering Chemistry. Prentice-Hall India: New Delhi, p 228.

Supplementary Tables

Table S1. Soil solid phase geochemical characteristics from different depths of the wetland mofette soil and reference soil obtained in April 2011. All values are means±standard deviation (N = 3).

Sample / pH
(10mM CaCl) / TOC
[w.-%] / N
[w.-%] / C/N / DAPI cell counts
[x108 cells g(dry weight)-1]
Wetland mofette soil
0–10 cm / 4.49±0.04 / 17.6±3.9 / 1.5 ±0.5 / 12.1 ±1.2 / 1.39 ±0.42
10–25 cm / 4.61±0.07 / 31.7 ±7.2 / 1.3 ±0.3 / 24.4 ±2.4 / 0.86±0.34
25–40 cm / 4.62±0.02 / 25.2 ±9.1 / 1.1 ±0.3 / 22.6 ±2.4 / 0.71±0.17
Wetland reference soil
0–10 cm / 5.43±0.04 / 6.3 ±1.2 / 0.55 ±0.12 / 11.6 ±0.5 / 1.68 ±0.27
10–25 cm / 5.41±0.02 / 3.4 ±0.9 / 0.31 ±0.07 / 10.9 ±0.6 / 2.11±0.66
25–40 cm / 5.33±0.06 / 2.6 ±2.3 / 0.24 ±0.19 / 10.2 ±1.1 / 2.11±0.61

Table S2. TOC, total PLFA and ether lipid concentrations and TOC, fatty acid and ether-lipid hydrocarbon 13C-label incorporations during the mofette 13CO2-labeling experiment. All values are means ± standard deviation (N = 2).

Compound / Concentration
[μg C g soil-1 (dry weight)] / initial δ13C
[‰ vs VPDB] / δ13C after 28 days
[‰ vs VPDB] / 13CO2 incorporation after 28 d[ng C g soil-1 (dry weight) d-1]
0–10 cm / 25–40 cm / 0–10 cm / 25–40 cm / 0–10 cm / 25–40 cm / 0–10 cm / 25–40 cm
TOC / 220,000 ±20,000 / 380,000 ±30,000 / -26.4±0.2 / -26.8±0.1 / -19.9 ±0.2 / -23.3 ±0.1 / 494 ±14 / 512 ±10
Individual PLFAs
C14:0 / 20.4 ±3.0 / 25.2 ±2.4 / -30.7 ±0.4 / -32.2 ±0.6 / 3.5 ±0.1 / -22.4 ±1.9 / 0.27 ±0.00 / 0.10±0.02
iC15:0 / 30.4 ±2.1 / 6.8 ±1.1 / -25.9 ±1.7 / -24.2 ±2.6 / 130.7 ±1.8 / 73.2 ±1.1 / 1.86 ±0.04 / 0.26 ±0.04
aiC15:0 / 20.0 ±1.2 / 5.5 ±1.1 / -27.1 ±1 / -27.7 ±1.4 / 195.7 ±13.1 / 143.9 ±5.8 / 1.75 ±0.11 / 0.37 ±0.06
iC16:0 / 10.4 ±0.7 / 2.9 ±0.9 / -28.6 ±0.4 / -27.5 ±1.9 / 94.1 ±0.7 / 44.7 ±6.2 / 0.50±0.00 / 0.08 ±0.03
C16:1ω7c / 14.3 ±1.2 / 3.0 ±0.5 / -24.8 ±0.2 / -24.6 ±0.9 / 114.4 ±6.4 / 56.6 ±4.1 ¥ / 0.78 ±0.04 / 0.19 ±0.06 ¥
C16:0 / 130.5 ±5.4 / 80.8 ±9.0 / -29.8 ±0.1 / -32.3 ±0.5 / 9.3 ±0.6 / 39.9 ±0.6 / 2.00±0.03 / 2.29 ±0.02
10MeC16:0 / 13.7 ±1.2 / 2.3 ±0.3 / -25.9 ±0.7 / -26.7 ±0.1 / 34.5 ±2.3 / 30.8 ±0.9 ¥ / 0.32 ±0.02 / 0.11 ±0.00¥
iC17:0 / 14.6 ±0.8 / 3.3 ±0.7 / -24.7 ±2 / -26.0±1 / 22.5 ±1.4 / -3.5 ±0.8 / 0.27 ±0.02 / 0.03 ±0.01
aiC17:0 / 7.0 ±0.5 / 2.7 ±0.8 / -27.8 ±1.5 / -26.6 ±1.9 / 53.1 ±3.8 / -5.3 ±1.5¥ / 0.22 ±0.01 / 0.05 ±0.02 ¥
C17:0 / 18.6 ±0.6 / 5.2 ±1.1 / -27.4 ±0 / -27.9 ±1.5 / -10.6 ±0.2 / -32.0 ±1.9 / 0.12 ±0.00 / B.D.
C18:1ω9 / 57.2 ±6.0 / 18.5 ±3.9 / -31.2 ±0.5 / -30.5 ±0.4 / 34.0 ±0.8 / 11.1 ±0.5 / 1.46 ±0.03 / 0.3 ±0.02
C18:1ω7 / 21.5 ±1.7 / 5.5 ±0.9 / -27.8 ±1.7 / -32.5 ±1.5 / 58.0 ±3.6 / -13.2 ±0.8¥ / 0.72 ±0.04 / 0.08 ±0.01 ¥
C18:0 / 52.0 ±6.3 / 37.3 ±4.5 / -29.2 ±0.1 / -29.1 ±0.1 / -8.6 ±1.4 / -32.2 ±0.5 / 0.42 ±0.03 / B.D.
C20:0 / 43.1 ±9.7 / 40.5 ±8.5 / -30.9 ±0.4 / -30.0±0.4 / -29.7 ±0.0 / -30.5 ±0.2 / 0.02 ±0.01 / B.D.
Sum of individual PLFAs
453.6 ±40.6 / 239.8 ±35.8 / 10.7 ±0.4 / 3.9 ±0.3
Individual ether lipids
Phytane / 10.2 ±2.4 / 40.9 ±12.3 / -30.1 ±0.4 / -29.1 ±0.6 / 70.0 ±0.8 / -25.5 ±0.7 / 0.40 ±0.01 / 0.01 ±0.01
Biphytane / 9.3 ±2.7 / 40.0 ±16.3 / -27.2 ±0.3 / -26.6 ±1.5 / 12.3 ±0.3 / -18.8 ±0.1 / 0.14 ±0.0 / 0.03 ±0.01
Sum ofindividual ether lipids
19.5 ±5.1 / 80.9 ±28.6 / 0.57 ±0.01 / 0.04 ±0.02

¥based on the incorporation after 14 d, as irm-GC-MS signals were too low for quantification

B.D. below detection

Table S3. Phylogenetic affiliation of bacterial 16S rRNA reads with ≥5% sequence read abundance in different SIP-fractions of the unlabeled control and 13CO2 treatment.

Taxon / Relative sequence abundance (%) by DNA-SIP Fractions¥
13CO2 treatment / Unlabeled control treatment
Heavy
(ρ > 1.736
g ml-1) / Medium
(ρ = 1.736 – 1.719 g ml-1) / Light
(ρ < 1.719
g ml-1) / Heavy
(ρ > 1.736
g ml-1) / Medium
(ρ = 1.736 – 1.718 g ml-1) / Light
(ρ < 1.718
g ml-1)
Acidobacteria / 36.6 / 55.0 / 11.5 / 25.1 / 51.8 / 16.1
Acidobacteriaceae / 35.2 / 54.9 / 11.5 / 23.8 / 51.4 / 15.9
Chloroflexi / 11.8 / 3.0 / 2.8 / 3.3 / 3.8 / 4.7
Uncl.* KD4-96 Chloroflexi / 10.9 / 1.4 / 0.1 / 2.0 / 2.5 / 0.3
Cyanobacteria / 5.8 / 2.5 / 0.4 / 7.1 / 3.5 / 0.5
Uncl. Cyanobacteria / 5.0 / 2.5 / 0.3 / 7.1 / 3.5 / 0.5
Bacteroidetes / 1.8 / 2.5 / 26.1 / 2.0 / 0.5 / 19.8
Porphyromonadaceae / 0.4 / 0.5 / 6.4 / 0.4 / 0.1 / 5.3
Uncl. Sphingobacteriales / 0.9 / 1.5 / 13.5 / 0.7 / 0.4 / 9.8
Uncl. Bacteroidetes / 0.5 / 0.5 / 3.5 / 0.6 / 0.1 / 5.6
Alphaproteobacteria / 13.5 / 8.5 / 1.8 / 16.5 / 9.7 / 2.3
Uncl. Rhizobiales / 7.4 / 6.4 / 1.4 / 10.2 / 6.8 / 1.9
Uncl. Bacteria / 14.4 / 12.1 / 37.0 / 30.9 / 13.2 / 35.3
Others¢ / 6.1 / 4.5 / 12.9 / 6.1 / 8.0 / 10.0

*Uncl., unclassified

¥Ctrl, unlabeled control treatment; 13CO2, 13CO2 labeled treatment; The number of sequences in each dataset was normalized by randomized subsampling in MOTHUR v1.7 to the sample with the lowest amount of sequence reads (2063) after cleaning and denoising (sequences <250 bp, primer mismatches, quality scores <30); coverage was >91% for all samples at a cutoff of 3% sequence dissimilarity; Only major bacterial families with ≥5% sequence read abundance are presented and therefore the sum of their sequence abundance does not necessarily equal that of their class or phylum

¢Actinobacteria, Caldiserica, Candidate Divisions OD1, OP10, OP11, SR1, TG-1, TM6, TM7, Chlorobi, Fibrobacteres, Firmicutes, Nitrospirae, Planctomycetes, Beta-, Delta-, Gammaproteobacteria, Verrucomicrobia

Table S4. Phylogenetic affiliation of archaeal 16S rRNA reads with ≥5% sequence read abundance in different SIP-fractions of the unlabeled control and 13CO2 treatment.

*Uncl., unclassified; MCG, Miscellaneous Crenarchaeotic Group

¥Ctrl, unlabeled control treatment; 13CO2, 13CO2 labeled treatment; The number of sequences in each dataset was normalized by randomized subsampling in MOTHUR v1.7 to the sample with the lowest amount of sequence reads (1351) after cleaning and denoising (sequences <250 bp, primer mismatches, quality scores <25); coverage was >96% for all samples at a cutoff of 3% sequence dissimilarity; Only major archaeal families with ≥5% sequence read abundance are presented and therefore the sum of their sequence abundance does not necessarily equal that of their phylum

Supplemental Figure Legends

Figure S1 Relative nucleic acid distribution in CsCl density gradients of unlabeled control treatment (circles) and labeled 13CO2 treatment (triangles) SIP incubation of 0–10 cm depth mofette soil. Also shown are the relative bacterial (grey bars) and archaeal (black bars) 16S rRNA gene copies (mean±SD, N = 3 replicates) of the combined subfractions "Light", "Medium" and "Heavy" from the unlabeled control treatment (filled bars) and 13CO2 treatment (dashed bars).

Figure S2 Gel electrophoresis results offhs gene PCR amplification (ca. 1.1 kbp) from fractions 1–12 of the unlabeled control and 13CO2 treatment SIP incubation of 0–10 cm depth mofette soil, analyzed on a 1% agarose gel. Fractions 5–6 of the 13CO2 treatment were considered the 'labeled' fractions, as fhsamplicons could not be detected in fractions 5–6 of the unlabeled control treatment. Other fractions 7–10 of the 13CO2treatment and fractions 5–10 of the unlabeled control treatment were considered as 'unlabeled' and 'combined control', respectively.

Figure S3Gel electrophoresis results of mcrA gene PCR amplification(ca. 0.5 kbp) from fractions 1–12 of the unlabeled control and 13CO2 treatment SIP incubation of 0–10 cm depth mofette soil, analyzed on a 1% agarose gel. Fractions 5–6 of the 13CO2 treatment were considered 'labeled' fractions, as fhsamplicons could not be detected in fractions 5–6 of the unlabeled control treatment. Other fractions 7–10 of the 13CO2 treatment and fractions 5–10 of the unlabeled control treatment were considered as 'unlabeled' and 'combined control', respectively.

FigureS4Phylogenetic tree of (a)fhsdeduced amino acid sequences (226 positions) and (b) mcrA deduced amino acid sequences (161 positions) constructed as a consensus tree of neighbor joining (1000 bootstraps) and parsimony (1000 bootstraps) analyses. The dashed box highlights sequences branching within the 'acetogen Cluster A'. Information in brackets: relative number of sequences from the 'labeled' fractions of the 13CO2 treatment / 'combined control' fractions of the unlabeled control treatment; acetogen similarity score (HSc). Marked in grey are clusters with sequences from the 13C-labeled fractions. Black rectangles depict nodes with >75% bootstrap support based on parsimony analyses. Bar indicates an estimated 0.1 changes per amino acid.

Figure S5Pore-water profiles of Eh, Fe(II), nitrate and sulfate concentrations in mofette (circles) and reference (triangles) soils in June 2011 (open symbols) and August 2012 (filled symbols).

Figure S6Pore-water profiles of Eh, Fe(II), nitrate and sulfate concentrations in mofette soil from November 2011 parallel to the 13C-CO2 labeling experiment.

Figure S7Potential effect of elevated CO2 concentrations on standard free energies (ΔG0; pH = 7) of hydrogenotrophic methanogenesis and acetogenesis at different levels of H2. Shaded areas demonstrate levels of mofette CO2 concentration. Dashed line represents average CO2 concentrations in mofette pore waters from November 2011.

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

Figure S6

Figure S7