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Supplemental Material and Methods
Site description
The sampling area is located in the Russian discontinuous permafrost zone
(62°57'E, 67°03'N). The sampling area has a mean annual temperature of -5.8°C and
a mean annual precipitation of 505 mm. Cryoturbated peat circles (4 to 25 m in
diameter) which develop through soil mixing as a result of frost action, are located on
a peat plateau containing thick, unturbated peat soil. Peat circles lack vascular plant
cover and show only sporadic growth of mosses and lichens (Repo et al., 2009). The
vegetation of the peat plateau consists mainly of Ledum decumbens, Rubus
chamaemorus, different mosses (e.g. Dicranum sp.) and lichens (e.g. Cladina sp.); in
situ pH approximates 3 to 4 (Repo et al., 2009). Total carbon is 54% and 47%, total
nitrogen is 2.3% and 0.8%, and nitrate content is 30 to 55 (app. 1 to 2 mM in pore
water) and < 0.1 µg NO3—N gDW-1 (< 3 µM in pore water) in cryoturbated and
unturbated soil, respectively. Cumulative N2O emissions in the field from the
cryoturbated soil during the snow-free season (138 days) are 1.2 ± 0.3 g N2O m-2,
while N2O emissions from the unturbated soil are negligible (Repo et al., 2009). The
topsoil has been identified as the most important source of N2O emitted from peat
circles (data not shown), thus our study focuses on the upper soil layer. In situ soil
temperature in 2-5 cm of depth is up to 12 - 14 ° C during the vegetation period, and
air temperatures of 20-30 °C occur (Marushchak et al. 2011; Repo et al. 2009; Atlas
of the Komi Republic on Climate an Hydrology, Moscow, 117 pp.). Soil of the upper 5
cm was sampled from three cryoturbated peat circles and three adjacent, unturbated
control areas in September 2010 for microcosm experiments and DNA extraction.
Roots were removed by hand-picking from unturbated soil, and soil for microcosms
studies was stored at 4°C until further processed. Soil for DNA extraction was
suspended in RNAlater (Qiagen, Hilden, Germany) immediately after sampling to
avoid decomposition of nucleic acids, and stored at -20 °C upon arrival at the
laboratory. Experiments were conducted within 2 month after sampling. Moisture
content of soil samples was 71% and 81% in cryoturbated and unturbated peat soil,
respectively.
Apparent Michaelis-Menten kinetics
Calculation of apparent Michaelis-Menten constants (Km) and maximum
reaction velocities (vmax) were based on the production of N2O in microcosms
supplemented with either nitrate or nitrite and acetylene. Headspace concentrations
of N2O were determined via gas chromatography (as described in Palmer et al.,
2010) within 8 h of incubation, and N2O production rates were calculated in the linear
phase by linear regression of three or four data points (R2 values were always
greater than 0.92).
Apparent Michaelis-Menten kinetics were fitted to the data using the program
GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA) for calculation
of Km and vmax according to the following equation (Segel, 1993):
,
where [S] is the concentration of nitrate or nitrite.
Analytical methods
pH was analyzed in water-soil solutions of cryoturbated and unturbated peat
soil with a pH electrode (InLab 422; Mettler Toledo GmbH, Gießen, Germany) (1:10
dilution of soil with double-distilled water). Moisture content was determined by
weighing the soil before and after drying at 60°C for 3 days. Nitrate was analyzed
with a Dionex DX-100 ion chromatograph equipped with an IonPac AS4A-SC ionexchange column and an ED40 electrochemical detector (Sunnyvale, CA, USA) after
water extraction.
N2O was quantified with a Hewlett-Packard 5980 series II gas chromatograph
(Hewlett-Packard Paolo Alto, CA, USA) equipped with an electron capture detector
(Palmer et al., 2010).
Organic acids and alcohols were determined by ion exclusion chromatography
with a Hewlett-Packard 1090 high-performance liquid chromatograph equipped with
security guard cartridges (50 x 7.8 mm; Phenomenex, Torrance, CA, USA) and a
Rezex ROA-Organic Acid H+ (8 %) column (300 x 7.8 mm; Phenomenex) at 60 °C,
as well as a variable wavelength detector at =210 nm and refractive index detector
(both series 1200, Agilent Technologies, Böblingen, Germany). The mobile phase
was 4 mM H3PO4 at a flow rate of 0.8 ml min-1 (modified after Wüst et al., 2009). The
lower limits of quantification for organic acids and alcohols were 10 and 100 µM,
respectively, when 50 µl of filtered (0.2 µM pore diameter) aqueous sample was
injected.
DNA extraction
Nucleic acids were extracted from triplicate cryoturbated and unturbated peat
soil samples to account for lateral heterogeneity in microbial communities. A beadbeating protocol tailored for the efficient removal of PCR-inhibiting humic acids by
aluminum sulfate precipitation prior to cell lysis was applied (Peršoh et al., 2008). In
brief, the minimal amount of aluminum sulfate for efficient humic acid removal
depends on the humic acid content of a soil sample, and was experimentally
determined to be 800 and 600 µl of a 0.2 M aluminum sulfate solution per 0.5 g of
soil samples from cryoturbated and unturbated peat soil, respectively. Following bead
beating, nucleic acids were purified by two organic solvent extractions and
isopropanol precipitation to obtain high quality DNA. DNA yields were 0.1 to 1.9 µg
DNA per gram freshweight of soil. A260/A230 values approximating 1 to 2 indicated
DNA with moderate to low humic acid content. DNA extracts from triplicate
extractions per soil were pooled for subsequent analyses.
Amplifikation of narG, nirK, nirS, and nosZ
PCR reaction mixtures contained 10 µl of 2.5 x Mastermix (5 Prime GmbH,
Hamburg, Germany), 5 µl of PCR Enhancer (5 Prime GmbH), 1 µl of 3% BSA, 0.5 µl
of MgCl2 (25 mM), and 0.1-1 µl of each primer (100 pmol/µl; Biomers, Ulm,
Germany). Touchdown PCR was performed for all target genes with a PeqStar 96
Universal Gradient Cycler (Peqlab, Erlangen, Gemany) and was preceeded by an
initial denaturation (95°C for 5 min). Denaturation and elongation per cycle were at
95°C for 0.5 min and 72°C for 1 min, respectively. The annealing temperature was
lowered by 0.5 °C per cycle from 64 to 59 in 10 precycles, followed by 35 cycles with
annealing at 54.7, 56.3, 57.7, 59.2, 60.8, 62.1, 63.0, and 63.6 °C for 1 min.
Quantitative PCR
Quantitative kinetic real-time PCRs (qPCRs) were performed with an iQTM5
Real-Time qPCR cycler (Bio-Rad, Munich, Germany). All qPCR reactions were run
with DNA extracts from 3 replicate cryoturbated and 3 unturbated sites in 6 technical
replicates per DNA extract. Negative controls with sterilized water instead of DNA
template were included in every PCR setup. Standard curves were set up by serially
diluting M13uni/rev PCR products of a pGEM-T vector with the appropriate insert
from 109 to 101 target gene copies µl-1 for every primer set. DNA concentrations were
determined fluorimetrically as described above. The standard curve was obtained by
the Bio-Rad iQ5 optical system software version 2.0 using linear regression of
threshold cycle numbers (cT) versus log copy numbers of targets. qPCR reactions
were performed in 20 µl reaction mixtures that were composed of SensiMix Plus
SYBR Green & Fluorescein (Bioline GmbH, Luckenwalde, Germany), 1.2 µl 50 mM
MgCl2 (Bioline GmbH), 150 ng/µl bovine serum albumin, 0.2 - 1.6 pM of each primers
(Biomers, Ulm, Germany), 5 µl of template DNA and sterilized deionized water.
Thermal protocols and primers were as described (see above; Table 1). Melting
Curve analyses were performed from 65 to 95 oC with increments of 0.2 oC per cycle.
Agarose gel electrophoresis, melting curve analysis, and sequencing of amplicons
generated with the same primers indicated that the amplification was specific. The
lower limits of quantification were 101 gene copy numbers µl-1 of DNA extract. 16S
rRNA gene copy numbers were determined concomitantly for all environmental
samples in order to quantify microorganisms that harbor genes associated with
denitrification in soil relative to the total bacterial population (Muyzer et al., 1993;
Zaprasis et al., 2010).
Inhibition of qPCR was assessed according to Zaprasis et al. (2010). All
environmental DNA samples were diluted 100-fold to reduce inhibition by coextracted PCR-inhibiting substances (e.g., humic acid) from soil.
Gene copy
numbers in environmental samples were calculated according to the standard curve
and were corrected for inhibition. Soil DNA was spiked with pure standard DNA to
increase target gene copy numbers per reaction by more than 10-fold. Gene copy
numbers obtained by qPCR of spiked and non-spiked DNA extracts were termed S
and ns, respectively. C was the gene copy number measured for the standard DNA.
The inhibition factor (IF) was calculated as IF = (S - ns) / C and ranges from 0
(complete inhibition) to 1 (no inhibition). ns was divided by IF to approximate real
gene copy numbers in DNA extracts. IF depends on the abundance of the gene
analyzed as well as the quality of the individual DNA extracts (Zaprasis et al., 2010),
and varied from 0.2-0.8, 0.5-0.8, 0.4-1, 0.5-0.8, and 0.5-0.6 for qPCR analyses of
narG, nirK, nirS, nosZ, and 16S rRNA genes respectively. Unless otherwise
indicated, inhibition corrected structural gene copy numbers were normalized per ng
DNA and 16S rRNA gene copy numbers, since such normalized gene copy numbers
are less sensitive to varying DNA extraction efficiencies (that is, DNA extraction bias)
than copy numbers per gram dry weight soil.
Supplemental References
Wüst PK, Horn MA, Drake HL. (2009). Trophic links between fermenters and methanogens
in a moderately acidic fen soil. Environ Microbiol 11:1395-1409.