Environmental Engineering Reference
In-Depth Information
2.3 Bulk Organic Carbon
Particulate organic carbon and nitrogen (POC, PON) concentrations were
routinely measured on materials retained on GF/F filters from samples collected
at 15 depths over the entire water column (see [32]). Aliquots of the GF/F-filtrate
were acidified and stored at 5
◦
C in precombusted 40 ml EPA bottles with acid-
washed Teflon-lined caps until analysis for DOC. DOC was measured using a
Shimadzu TOC-5000 Total Organic Carbon Analyzer [47].
2.4 Microbial Dynamics
Prokaryotes and flagellated protozoa were enumerated in DAPI-stained sam-
ples from 15-18 depths as described in [56]. Viral-like particles (VLP) were
enumerated in the same preserved samples by epifluorescence microscopy at
1000x magnification according to [36]. VLP from 0.3 to 2-ml subsamples re-
tained on 0.02 µmAl
2
O
3
Anodisk 25-mm membranes (Whatman International
Ltd.) were stained for 15 min with SYBR Green I fluorochrome (Molecular
Probes, Inc.) [see ref. 57 for details]. Ciliated protozoa were enumerated by a
similar technique differing only in that cells from 150-ml samples were cap-
tured on 2.0µm Nuclepore membranes and stained with acridine orange. Based
on size and gross morphological features, ciliates were enumerated separately
to the class or sub-order taxonomic levels.
Prokaryotic biomass was estimated from 200 randomly selected cells in
each sample by visually sorting into eight size classes based on their linear
dimensions, approximated with an ocular micrometer at 1000x magnification.
Cellular carbon biomass (C) was estimated from biovolume (V) using an al-
lometric carbon to volume extrapolation function, C = 0.12 V
0.72
[37]. Cell
carbon content estimates varied from 25 to 79 fg C cell
−
1
(
x
+
1SD=47
+
10, n = 37) within oxic layer samples. Heterotrophic bacterial net production
(BNP), acetate turnover and chemoautotrophic production were determined
throughout the water column by
3
H-leucine,
14
C-acetate and
14
C-bicarbonate
assimilation, respectively, as described in [14] and [56]. All rate measurements
were obtained from samples incubated under in situ redox and temperature
conditions by immersing vials sealed without a headspace (described above) in
water baths. Inhibition and stimulation experiments were performed by adding
inhibitor (N-Serve) or putative stimulants (S
2
O
3
,S
0
,NO
3
−
,MnO
2,
Fe
2
O
3
)
to replicate
14
C-bicarbonate assimilation sample bottles, then incubating and
processing in parallel with the chemoautotrophy assay [56].
