SULFUR AND METHANE CYCLING DURING THE HOLOCENE IN ACE LAKE (ANTARCTICA) REVEALED BY LIPID AND DNA STRATIGRAPHY - Past and Present Water Column Anoxia - page 46

in fluorescence due to the binding of the fluorescent dye SYBR Green. Reaction
mixtures (25 µl) contained 12.5 µlofiQ TM SYBR Green Supermix (Biorad),
10 µg of BSA, 0.5 µM of primers, and ultra pure sterile water (Sigma). 15 ng
of template DNA from each sample was added to each real time PCR reaction.
Duplicate reactions of samples were run. For the calibration of samples, differ-
ent amounts of genomic DNA of the GSB Chlorobium phaeovibrioides DSMZ
269 T (ranging between 0.1 and 1590 picogram) was subjected to real time PCR.
Control reactions included: one reaction without template DNA as a control for
contamination during pipetting, one control for contamination during the DNA
extraction procedure, and three control reactions were performed with 15 ng of
DNA from Escherichia coli , Flavobacterium sp., and Mycobacterium interjec-
tum to monitor the specificity of the reactions. After the total amount of DNA of
GSB per sample was determined by quantitative PCR, the amount of DNA from
the phylotypes AL-GSB 1 and 2 was integrated from the intensities of the DGGE
bands.
3. RESULTS
Carotenoids. The carotenoids chlorobactene, isorenieratene and β-isoreni-
eratene of the obligate anoxygenic photolithotrophic GSB (members of the
family Chlorobiaceae) were first detected just beneath the oxic-anoxic inter-
face (OAI) located at a depth of 11.7 m in the water column of Ace Lake (Fig.
3a). Chlorobactene (structure I; Fig. 4) was the most predominant carotenoid
of GSB with a concentration of up to 4.3 mg.L − 1 in the euxinic chemocline.
In water layers below the photic zone its concentration was a factor of two
lower. Isorenieratene and β-isorenieratene (structures II, III; Fig. 4) concentra-
tions were much lower throughout the anoxic water column with maximum
concentrations of 0.3 mg.L − 1 in the chemocline (Fig. 3a).
Within the Holocene sediment record of Ace Lake, carotenoids of GSB were
absent in the ancient freshwater sediments of Unit III (Fig. 5d). Chlorobacte-
ne, isorenieratene and β-isorenieratene were first detected from the sediments
which were deposited after seawater entered the lake (9400 years BP, Unit
II). Chlorobactene concentrations were between 3 and 17 times higher than
isorenieratene plus β-isorenieratene concentrations within the sediment layers
of units I and II (Fig. 5d). The average concentration of chlorobactene in the
older layers of Unit II (between 130 and 78 cm) was 6 mg.g − 1 TOC. The chlo-
robactene concentration increased significantly in sediment layers shallower
than 79 cm reaching a maximum concentration of 258 mg.g − 1 TOC between
37 and 39 cm, but declined again at shallower depths (Unit I; saline, lacustrine,
sulfate-depleted). These carotenoids were almost absent between 17 and 19 cm
(Fig. 5d) with a chlorobactene content of only 0.2 mg.g − 1 TOC.