Supplementary methods
Analysis of H2 and organic acid production
To measure H2 production, 25 L of headspace gas was withdrawn by volumetric syringe
from each of six replicate vials at each time point for each control or manipulation
experiment and injected directly onto a gas chromatograph with quantification of H2 by a
mercuric oxide detector. To analyze organic acids, the entire liquid phase (4 mL) of each
of three replicates for each time point for each control or manipulation experiment was
sampled (with the associated incubation sacrificed). Liquid was filtered through 0.2 m
syringe-driven filters for storage in glass vials at -20OC. Organic acids (C1-5) were
quantified via high-pressure liquid chromatography (Albert and Martens, 1997).
Manipulation experiments
To identify the location of H2 production in microbial mats, incubations were carried out
using sectioned mat cores.
Mat cores were sectioned using a sterile scalpel blade
yielding the following mat layers; 0-2 mm, 2-4 mm and 4-15 mm. Mat cores were
sectioned and incubated under normal light conditions only and not for any of the
following manipulation experiments described below.
Incubations that employed
addition of ammonium (NH4Cl, 8.8 mM final concentration) to field site water to
suppress N2-fixation (diazotrophy) were performed to assess co-metabolic production of
H2 via nitrogenases. To assess the importance of phototrophs (oxygenic and anoxygenic)
in H2 production, microbial mat cores were deprived of light by wrapping aluminium foil
around serum vials prior to sunrise.
Assessment of the importance of oxygenic
phototrophy in H2 production was carried out by inhibition of photosystem II (PSII) by
addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 20 M final concentration)
to field site water (Bebout et al., 1993; Oettmeier, 1992). Mat cores were incubated in
DCMU amended field site water for 1 hour prior to the beginning of the incubation. To
confirm that PSII was effectively inhibited using DCMU, a Walz diving-pulse amplitude
modulation fluorometer (Diving PAM; Heinz Walz, Germany) was used to measure
changes in PSII chlorophyll fluorescence by determination of the quantum PSII yield
(PSII). Changes in PSII were analyzed using the saturation pulse method (Fleming
et al., 2007; Genty et al., 1989). PSII values are relative and range from 0 or no
electron transfer (inferring 0% PSII activity) to 1 or 100% electron transfer (inferring
100% PSII activity). Campbell et al. (1998) provide further information and critical
reviews of this technique’s application in PSII chlorophyll fluorescence analysis of
cyanobacteria.
Nucleic acid isolation from microbial mats
All standard precautions to mitigate against RNase contamination were employed. These
precautions included: working on ice, use of and frequent changing of gloves,
decontamination of surfaces and non-disposable equipment using RNase™ Zap (Ambion,
TX, USA), use of disposable RNase/DNase free- plasticware and use of prepackaged
RNase/DNase free filter tips. Frozen biomass cores were sectioned using a sterile scalpel
blade to obtain the upper 2 mm phototrophic layer. This biomass was then transferred
into a 2 ml tube containing 0.5 ml of RLT™ buffer (Qiagen, Netherlands) with 5 μl
Nucleoguard™ (AmpTec, Germany). Cells within the biomass were disrupted using a
rotor-stator homogenizer (Omni, GA, USA). The suspension was transferred into a new
sterile 2 mL screw cap tube containing zirconium beads, and agitated for 40 sec in a
FastPrep™ instrument (MP Biomedicals, OH, USA) to further disrupt cells.
This
mixture was centrifuged at 8,000g for 1 min to remove debris. Supernatant from at least
three cores was mixed and split into separate aliquots for RNA and DNA isolation.
Aliquots of supernatant for RNA isolation were processed first by adding 1 ml of acidphenol mixture. This was vortexed for 10 sec, incubated at room temperature for 5 min
followed by centrifugation at 8,000g for 5 min. The aqueous phase was transferred to a
new tube and passed through a 20-gauge needle 5x to shear high MW DNA. Sheared
supernatant was then transferred to gDNA eliminator columns (Qiagen) and centrifuged
at 8,000g for 30 sec. One volume of 80% ethanol was added to the eluate and vortexed
for 10 sec. The Qiagen RNeasy Mini Kit protocol was then carried out to isolate RNA
(Qiagen).
Aliquots of supernatant for DNA isolation were processed by adding 1 ml of an acidphenol mixture. This was vortexed for 10 sec, incubated at room temperature for 5 min
followed by centrifugation at 8,000g for 5 min. The aqueous phase was transferred to a
new tube, 1 volume of 100% ethanol was added and the solution was vortexed for 10 sec.
DNA was then isolated using the QIAamp DNA Mini Kit as per manufacturers protocol
(Qiagen).
Nucleic acids were quantified by fluorometric quantitation using the Qubit instrument
(Invitrogen, CA, USA).
RNA purification and cDNA synthesis
Prior to cDNA synthesis, traces of contaminating DNA were removed from isolated RNA
using the Turbo DNA-free kit (Ambion) to as per manufacturers protocol using two
rounds of enzymatic digestion. DNA-free RNA was then further purified using the
RNeasy MinElute protocol (Qiagen). RNA (1 ug) was reverse-transcribed to cDNA
using random hexamers (100 ng) and the superscript III reverse transcriptase (RT) as per
manufacturers protocol (Invitrogen). DNA contamination in RNA extracts was
determined by performing cDNA synthesis reactions without RT. Aliquots of (-)RT
reactions were then subjected to PCR in the same way as aliquots of (+)RT reactions.
References
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