Environmental Engineering Reference
In-Depth Information
horizontal fragments and 41 out of 74 slices with 2 cm space intervals were
used for lipid and 16S rDNA analysis.
2.3 Calibration of Sediment Ages
Accelerator mass spectrometry (AMS) radiocarbon (
14
C) dating of selected
bulk sediment from the sediment core (sections 1
−
3 cm, 17
−
19 cm, 33
−
35
cm, 63
149 cm) was
carried out at the R.J. Van der Graaff laboratory, University of Utrecht, The
Netherlands. Standard techniques of calibration [49] were used to express the
sediment ages in calendar years before the present (BP), assuming reservoir
ages of 115 years (Unit I; closed saline lacustrine lake system), and 500 years
for Unit II (marine inlet period) [11], and 0 years for the freshwater lacustrine
period (Unit III) [53].
−
65 cm, 89
−
91 cm, 117
−
119 cm, 133
−
135 cm, and 147
−
2.4 Carotenoid Analysis
Freeze-dried and ground sediment samples of 0.1 to 0.7 g were ultrasonically
extracted with acetone (3
, 3 min). Samples were centrifuged at 3000 rpm for 5
min. The supernatants were decanted, combined and concentrated using rotary
vacuum evaporation. The extracts were subsequently dried under a gentle flow
of nitrogen. The residue was redissolved in dichloromethane and applied to a
silica column and the apolar carotenoids eluted with dichloromethane, dried
under nitrogen and the residue dissolved in acetone. Care was taken during
the entire sample preparation procedure to avoid exposure of the samples to
light and heat. This carotenoid fraction was then immediately analyzed on a
HP 1100 series HPLC equipped with an auto-injector and photodiode array
detector. Separation was achieved on a ZORBAX Eclipse XDB-C
18
column
(2.1
×
150 mm, 3.5µm; Agilent Technologies, USA), maintained at 25
◦
C, with
a linear gradient from 65% solvent B to 80% solvent B in 45 min, with solvent A
being methanol/water (4:1, v/v) and solvent B acetone/methanol/water (19:1:1,
v/v/v). The flow rate was 0.3 ml/min. Detection was achieved by in-line UV-
detection (250
×
700 nm). Chlorobactene and isorenieratene were quantified by
comparing their UV responses at 462 nm (λ
max
of chlorobactene in the mobile
phase) and 454 nm (the λ
max
of isorenieratene in the mobile phase) of known
amounts of an authentic β-carotene standard (Aldrich) and correcting for the
difference in extinction coefficients [5].
In order to examine whether chlorobactene was accompanied by its diage-
netic products (e.g. partly hydrogenated chlorobactane), ca.3goftwosediment
sections (37
−
−
−
87 cm) were ultrasonically extracted using mix-
tures of dichloromethane and methanol. An aliquot of the total lipid extract,
with an internal standard (6,6-d
2
-2-methylheneicosane) was hydrogenated for
1 h with PtO
2
and a few drops of acetic acid. The resulting fraction was chro-
39 cm and 85
