Journal of Plankton Research Vol.18 no.6 pp.969-986.19%
Abundance, size distribution and bacterial colonization of
transparent exopolymeric particles (TEP) during spring in the
Kattegat
Xavier Mari and Thomas Ki0rboe
Danish Institute for Fisheries Research, Department of Marine and Coastal
Ecology, Charlottenlund Castle, DK-2920 Charlottenlund, Denmark
Abstract. The abundance, size distribution and bacterial colonization of transparent exopolymeric particles (TEP) were monitored in the Kattegat (Denmark) at weekly intervals throughout the spring
(February-May) encompassing the spring diatom bloom. These recently discovered particles are
believed to be formed from colloidal organic material exuded by phytoplankton and bacteria, and may
have significant implications for pelagic flux processes. During this study, the number concentration of
TEP (>1 u,m) ranged from 3 x 103 to 6 x 10* mH and the volume concentration between 0.3 and 9.0
p.p.m.; they were most abundant in the surface waters subsequent to the spring phytoplankton bloom.
The range of TEP (encased) volume concentration was similar to that of the phytoplankton, although
at times TEP volume concentration exceeded that of the phytoplankton by two orders of magnitude.
The TEP size distribution followed a power law, with the abundance of particles scaling with particle
diameter" * ". The seasonal average estimate of (3 (2.3) was not significantly different from three, consistent with TEP being formed by shear coagulation from smaller particles. However, date-specific
estimates of f3 differed significantly from three, probably because TEP are fractal. All TEP were colonized by bacteria, and bacteria were both attached to the surface of and embedded in TEP. Yet the
number of attached bacteria per TEP was related neither to the surface area nor the volume, but rather
scaled with TEP size raised to an exponent of —1.5. We argue that this is consistent with TEP being
fractal. Between 0.5 and 20% of the total bacterial population were attached to TEP. Crude estimates
of TEP carbon concentrations combined with considerations of turnover times suggest that TEP and
their colloidal precursors may represent a hitherto understudied but potentially significant pathway for
dissolved organic carbon in the ocean.
Introduction
Transparent exopolymeric particles (TEP) are a group of recently discovered particles in the ocean (Alldredge etal,, 1993). TEP are presumably formed from dissolved or colloidal exopolymers exuded by phytoplankton or bacteria (e.g.
Alldredge et ai, 1993; Ki0rboe and Hansen, 1993) and their physical structure
resembles a highly dispersed matrix of fibrils with —99% water by weight (Sutherland, 1972). TEP range in size from <2 to >100 ^m in diameter (Alldredge et ai,
1993; Ki0rboe and Hansen, 1993; Passow et ai, 1994), and their abundance and size
distribution in the ocean appears to be related to the concentration and composition of the phytoplankton (Passow and Alldredge, 1994). According to the scarce
information available so far, concentrations of TEP vary by four orders of magnitude with a maximum of up to ~5000 ml 1 (Passow et al., 1994).
Owing to the physicochemical properties of TEP and because of the high concentrations at which they may occur, these particles may have important implications for food web structure and flux processes in the ocean: they may act as
sorption sites for solutes (Morel and Gschwend, 1987; Wells and Goldberg, 1991)
and attachment sites for bacteria (Alldredge etal., 1993), as food for particle grazers (Carman, 1990; Decho and Moriarty, 1990; Shimeta, 1993), and they may
© Oxford University Press
969
X-Mari and T.Kierboe
coagulate with other particles, including phytoplankton (Ki0rboe and Hansen,
1993; Passow et ai, 1994), into marine snow aggregates and thus enhance the vertical transport of substances by sedimentation. The significance of sinking aggregates, whether composed of phytoplankters or other particles, for vertical material
fluxes in the ocean has now been generally acknowledged (Fowler and Knauer,
1986; Alldredge and Silver, 1988). Information on the formation and occurrence of
TEP in various regions of the oceans is necessary to evaluate the potential significance of TEP to processes in the pelagic zone.
The scope of this study was, thus: (i) to describe the occurrence, abundance and
size distribution of TEP during one spring season, including the spring bloom, in
coastal water; (ii) to examine what fraction of the bacterial population is attached
to TEP; (iii) to discuss the formation and fate of TEP, and the implications of TEP
for material fluxes.
Method
Sample collection
Seawater samples were collected at about weekly intervals from February to May
1994 at a 30 m deep coastal station in the southern Kattegat (56°15'N, 12°00'E)
with 301 Niskin bottles. The Kattegat is characterized by a northward surface flow
of brackish water originating from the Baltic and a southwardflowof more saline
water near the bottom; the two layers are separated by a strong pycnocline (10-20
m depth) during most of the year. The vertical stratification was determined by
CTD casts on each sampling occasion. Samples were collected in the middle of the
surface mixed layer, at the pycnocline and in the middle of the bottom layer.
Nutrients and phytoplankton pigments
The concentrations of inorganic silica (SiO2) and nitrate (NO3) were measured by
an autoanalyser according to the methods of Grasshof (1976). Chlorophyll a and
phaeopigments were determined spectrophotometrically in 90% acetone extracts
of 3 1 seawater samplesfilteredonto 47 mm Whatman GF/Cfilters(Strickland and
Parsons, 1972).
Staining and enumeration of TEP
Semi-permanent slides of TEP were prepared largely following Passow et al.
(1994). Several 1-20 ml aliquots of each sample were filtered through 0.2 urn
Nuclepore filters at a low and constant vacuum pressure (200 mbar). TEP retained
on the filter were stained with 500 |il of an aqueous solution of 0.02% alcian blue
and 0.06% acetic acid. After staining, the filter was transferred to a microscope
slide and prepared according ta the Filter-Transfer-Freeze (FTF) technique
(Hewes and Holm-Hansen, 1983) as follows. The slide was quickly frozen (by
freeze spray) and the filter was then carefully peeled off, leaving the stained particles on the slide. Two drops of a gelatin solution (2.5% gelatin and 20% glycerol
in 0.22 \y.mfilteredseawater), maintained at 3O-35°C, were then applied to the
sample and allowed to harden in a horizontal position for 25 min. Coverslips were
970
Transparent exopolymeric partides in the Kattegat
finally placed on the slides using two drops of a glycerine solution (20% glycerol in
0.22 |un filtered seawater). Because the alcian blue staining depends on the pH
(Parker and Diboll, 1966), all samples were prepared fresh to avoid a modification
of the pH. Blanks were prepared with a 10 ml sample filtered twice through 0.2 p.m
Nuclepore filters, in order to remove the TEP and other suspending particles. The
blanks were then filtered, stained and prepared as above. Blanks were always
insignificant.
About 100 TEP were counted and sized on each slide at 250x magnification in a
compound light microscope connected to a computer through a colour video camera. TEP contour lines were traced manually, so that individual TEP and other
particles (i.e. algae) were distinguishable. By a semi-automatic image-analysis system, the cross-sectional area of each TEP was measured, and its equivalent spherical volume (ESV) and diameter (ESD) were calculated. Our estimates of TEP
volumes are encased volumes in that porosity is ignored. For each sample, the
particle size distribution, the number concentration and the total volume concentration of TEP were recorded. Since TEP may flatten somewhat on the filter and
are not necessarily spherical in seawater, the calculation of TEP volume potentially overestimates the real volume and, therefore, volume concentration of TEP.
However, previous comparisons of TEP volume determined microscopically and
by electronic particle counter showed very good correspondence (Ki0rboe and
Hansen, 1993).
The abundance of TEP on the filter had to be kept within limits for the precise
determination of abundance and size distribution. If too dense, TEP coagulate in
the filter funnel and TEP abundance decreases and average size increases. If too
sparse, counting statistics are bad. We therefore filtered a series of aliquots, typically 4-6, of increasing volume (1-20 ml) for each sample and plotted the abundance of TEP versus filtered volume. In calculating size and abundance, we
excluded those subsamples that exceeded the linear range. As a result, a total of
between 200 and 600 TEP were sized and counted in each sample (average 370).
TEP size distributions
Particle size distributions are often described by power relations of the type N =
k'dp-*, or dAVd(dp) = kdp^+ ", where dN is the number of particles per unit volume
in the size range dp to [dp + d(dp)] (e.g. McCave, 1984). The constant k depends on
the concentration of particles and p describes the size distribution; the smaller (J is,
the smaller the fraction of small particles. We estimated k and p from regressions
of log[dA7d(dp)] versus log[dp]. The magnitude of (3 and comparisons of the theoretical power law and observed TEP size distributions may provide information on
the formation and fate of TEP.
Bacterial abundance and fraction of bacteria attached to TEP
Total bacterial abundances were determined in 5 ml samples filtered onto 0.2 (xm
Nuclepore filters after staining with 0.1 jig ml 1 4',6'-diamidino-2-phenylindole
(DAPI) (Porter and Feig, 1980; King and Parker, 1988). Bacteria were counted in
10fieldson each slide at lOOOx magnification in an epifluorescence microscope.
971
X.Mari and T.Kierboe
The number of bacteria attached to TEP were counted after double staining
with DAPI and alcian blue (Passow and Alldredge, 1994). After staining, filters
were transferred to slides and prepared according to the FTF technique. All samples were prepared fresh. Bacteria associated with 20 TEP on each slide were enumerated by switching between UV and visible light. The individual TEP were
sized. Because TEP are three dimensional, the entire volume of each TEP was
examined by changing the microscope focusing to count all the associated bacteria.
A potential source of error is suspended bacteria retained by the filter beneath a
TEP. However, according to filter area, filtered volume and observed concentrations of bacteria, this would add only ~4% of attached bacteria to the smallest
TEP (3 u.m) and ~13% to the largest (30 urn), and was thus ignored.
A relationship between TEP size and number of attached bacteria was calculated for each sample in order to estimate the fraction of total bacteria that was
attached to TEP. The number of attached bacteria can be fitted to a power law
relationship, n = adpb, where n is the number of bacteria per TEP, dp is the equivalent spherical TEP diameter, and a and b are constants for a given sample. Numbers of associated bacteria and TEP diameter were plotted in log-log coordinates
to obtain a and b. The fraction of attached bacteria was calculated by combining (i)
the relationship described, (ii) the size distribution of TEP and (iii) the total concentration of bacteria in seawater.
TEP production by bubbling
It has been suggested that TEP are formed by coagulation from small organic particles or colloidal organic material. To examine the potential source and mode of
formation of TEP, we attempted to produce TEP by bubbling solutions of exudates from the diatom Thalassiosira excentrica in a small pilot laboratory experiment. The bubbling method has been used in previous studies to produce
aggregates from dissolved organic material derived from macrophytes (Kepkay
and Johnson, 1988; Alber and Valieda, 1994) or phytoplankton (Mopper et ai,
1995). Thalassiosira excentrica were grown in batch cultures on f/2 media with silica. A dense, 10-day-old culture wasfiltered(200 mbar) through a 2 (xm Nuclepore
filter to remove particles larger than 2 u.m, and the filtrate was diluted five times
with 0.22 p.m filtered seawater. This solution was added to a 42 cm high 2 1 plastic
cylinder, covered with foil, and bubbled with atmospheric air in the dark for 24 h.
Bubbles were produced by an ordinary aquarium frit fitted on line with a 0.22 u.m
airfilter.Samples of 2 ml were collected in the middle of the column every 30 min
during the first 2 h. After 24 h, samples were collected at the surface, middle and
bottom of the column. All samples were immediately filtered and stained for TEP
as above, and the concentration and size distribution of TEP recorded.
Results
Pigments and nutrients
The concentration of chlorophyll a was low (<0.6 \ig H) until the beginning of
March, increased thereafter topeak in the middle of March (14 u.g chlorophyll I"1 in
the surface mixed layer) and subsequently declined to pre-bloom concentrations
972
Transparent exopolymeric partides in the Kattegat
(Figures la, 2a and 3a). This pattern was most pronounced in the surface mixed
layer and at the pycnocline, whereas the signal was very weak in the bottom layer.
The temporal variation in phaeopigments followed that of chlorophylls. The concentrations of nitrate and silica in the surface layer declined almost continuously
between the beginning of March and the beginning of April, from, respectively, 6
to 15 u.M to practically zero (Figure lb and c). We took no samples between March
17 and April 6, but the continuous decline in nutrient concentrations suggests that
the phytoplankton bloom peaked close to April 1, which is typical for the region
(e.g. Ki0rboe and Nielsen, 1994).
Occurrence and size distribution of TEP in the ocean
TEP occurred in significant concentrations (i.e. >103 ml"1) on all sampling
occasions and at all sampling depths.
The TEP concentration in the surface mixed layer was relatively low (<7 x 103
TEP ml"1) until the middle of March, increased thereafter by about one order of
magnitude at the beginning of April (6.4 x 104 TEP ml'), and subsequently
declined to —104 TEP ml"1 (Figure 2b). The highest TEP concentration occurred
liooo u>
I"
|80O
g
a.
3
70
80
90
100
110
120
130
140
Julian day number
Fig. 1. Seasonal variation in the water column of (a) concentration of chlorophyll a, (b) inorganic silica
and (c) inorganic nitrate.
973
X.Mari and T.Kiarboe
Chlorophyll
a
—•— Phacopigmenti
«J S —
E c oo
s
10/02 16/02
24/02 03/03
10/03 17/03
06/0411/04
21/0426/04
03/05
10/02 16/02
24/02 03/03
10/03 17/03
06/0411/04
21AM26/O4
O3«5
I S eu
c
JL
-
n r
l(V02 16«2
24/02 03/W
10/03 17/03
06/0411/04
21/0426/04
03/05
10/02 16/02
24/02 O3A13 10/03 17/03
06/0411/04
21/0426/04
03/05
Date
Fig. 2. Mixed surface layer seasonal variation of (a) concentration of pigments, (b) number concentration of TEP (± SD), (c) volume concentration of TEP, (d) concentration of bacteria and fraction of
total bacteria attached to TEP.
subsequent to the phytoplankton peak. The concentration increased again to 3 x
104 TT£P ml-1 at the beginning of May. As in the surface mixed layer, TEP concen974
Transparent exopotymeric particles in the Kattegat
14
a
—Cr-
2
He
IP
.,
CUoroftylia
—•— Phaeopigments
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8
6
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/
\ ^
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i
i
i
i
i
i
i
i
10/02 16/02
24/02 03/03 10/03 17/03
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03/05
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06/0411/04
21/0426/04
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:
nfi
10/02 16/02
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ii
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\
v7
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-•
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10/02 16/02
i
t
24/02 03/03 10/03 17/03
1
06/0411/04
1
21/042404
1
_
03/05
Date
Fig. 3. Pycnocline layer seasonal variation of (a) concentration of pigments, (b) number concentration
of TEP (± SD), (c) volume concentration of TEP, (d) concentration of bacteria and fraction of total
bacteria attached to TEP.
tration in the pycnocline was relatively low until the middle of March, increased
thereafter by a factor of about three and remained constant during the rest of the
study period (Figure 3b). In the bottom layer, the concentrations were always high
975
JCMari and T.Kierboe
(3-5 x 104 TEP ml1) and seemingly independent of the variations occurring in the
surface mixed layer (data not shown).
The volume concentration of TEP varied between 0.3 and 9 p.p.m. (Figures 2c
and 3c), and was on average higher in the bottom layer (x = 3.9 ± 2.2) than at the
pycnocline (x = 2.2 ± 2.4) and in the surface mixed layer (x = 2.3 ± 2.4). The temporal variation in volume concentration largely followed the number concentrations at all three depth strata, i.e. with a significant peak at the beginning of
April at the two shallow depths, and no pronounced pattern near the bottom.
The power relation fitted the TEP size distributions relatively well (Figure 4),
except that the smallest size classes appeared to be under-represented. This may
be a sampling artefact, since TEP may pass through pores that are considerably
smaller than their nominal ESD due to their flexible character (Alldredge et al,
1993) and/or an artefact due to the enumeration technique, since the observation
of TEP < 3 n.m is very difficult at a magnification of 250x. It may also be the case
that 3-4 u,m ESD TEP slough off diatoms and are born into the size distribution. In
calculating the regressions, we considered only particles larger than 3-4 u.m ESD.
In the surface mixed layer, estimates of 3, the exponent of the power relation,
varied between about one and about three (Figure 4). It tended to increase during
the course of the study period, i.e. small particles become relatively more abundant
with time. A similar pattern was observed at the pycnocline, while there was no
obvious pattern in the bottom layer (data not shown).
Bacterial colonization of TEP
Double staining with DAPI and alcian blue demonstrated that all TEP were colonized by bacteria. Bacteria were both attached to the surface of and present inside
TEP.
The number of bacteria per TEP increased with TEP size and bacterial numbers
scaled with TEP diameter raised to an exponent of ~1.5 (range 0.9-1.9) (Figure 5).
Thus, bacteria per TEP volume decrease with size. The relationship between the
number of attached bacteria and TEP size appears to vary somewhat during the
study period, in particular in the surface layer and at the pycnocline. The
regression lines for the surface and pycnocline samples cluster in two groups: one
group consisting of samples taken during the period subsequent to the phytoplankton bloom (April 6-21 in the surface layer and April 6-26 at the pycnocline) and
one consisting of all other observations. Within these groups, the regression lines
do not differ statistically, whereas the difference between groups is statistically
significant both in the surface layer and at the pycnocline (P < 5%). These differences may reflect differences in the dynamics (formation and degradation) of TEP.
There were no consistent trends in the bottom layer.
Because of the different scaling in bacterial number with TEP size, it is not
straightforward to examine the temporal variation in bacterial density on TEP. To
approach this, we calculated from the above regressions and for each sampling
date and depth the expected number of bacteria attached to TEP with a size equal
to the overall average size (ESD = 5.3 jtm) in surface waters (Figure 6). It shows
that the number of bacteria per 'standard size' TEP varies from ~3 to 10, corresponding to 4 x 1010-13 x 1010 bacteria ml ' of TEP. In the surface mixed layer and at
976
Transparent exopolymeric partides in the Kattegat
100000
10/02
16/02
10000
1000
lo» k - 4.4
100
10
1
24/02
100000
10000
03/03
P-07
k)gk-4.0
p-2.1
k>gk-4.8
10/03
17/03
p-u
p-2.5
lot k " 5-5
1000
100
10
1
100000
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lot k - 4.4
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100
8
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11/04
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P-24
logk-5.8
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100000
10000
1000
100
10
26/04
21/04
1
p-3.2
logk-6.2
100000
$-4.4
kjgk-6.6
10000
1000
100
1
10
100 I
100
10
TEP equivalent spherical diameter, d Qim)
Fig. 4. Differential TEP size distribution in the surface mixed layer as recorded on individual sampling
days. Regression lines (dNld[dp] = kdf*' ") have been fitted to the data, but utilizing only observations
described by closed symbols.
the pycnocline, there was a consistent pattern in the bacterial density in TEP; it
increased continuously from the beginning of March, peaked immediately
subsequent to the phytoplankton bloom at the beginning of April, and then
declined again. In the bottom layer, bacterial density in TEP increased more or less
977
X.Mari and T.Kierboe
100
10/03
03/03
10
100
b-l^J
k>t • - O.OS
r-O.SO
100
21/04
b-1.00
lot •-0.12
r-0.90
03/05
10
100
I
10
100
TEP equivalent spherical diameter, dp (um)
Fig. 5. Surface mixed layer number of bacteria attached to a TEP (n) as a function of its size (dp, u.m) as
recorded on individual sampling days. Regressions were fitted to the observations (n = ad*).
continuously during the study period. The density of bacteria in TEP was
unrelated to ambient concentrations of both suspended bacteria and TEP (either
as volume or as number concentration).
978
Transparent exopolymeric partides in the Kattegat
1WD2 16A32
03*3
06*41 MM
10*3 17/03
21/006*4
03/05
Date
Fig. 6. Seasonal variation in the number of bacteria attached to TEP (as bacteria per TEP and bacteria
per TEP volume) for a standard size TEP particle (of ESD = 5.3 p.m) in the surface mixed layer ( -).
at the pycnocline (—) and in the bottom layer (—).
The fraction of seemingly free bacteria that were in fact associated with TEP
varied between 0.5 and 18.4% in the surface mixed layer, between 1.9 and 10.8% at
the pycnocline, and between 5.6 and 20.1% in the middle of the bottom layer.
Overall averages for the three layers were 5.0 ± 5.4% (surface), 5.5 ± 2.8% (pycnocline) and 13.3 ± 4.7% (bottom), and thus appeared to be highest in the bottom
layer (P < 0.01). The temporal patterns in the fraction of TEP-associated bacteria
(Figures 2d and 3d) were similar in the surface and pycnocline layers: an elevated
fraction on March 17 and April 6, when both phytoplankton and TEP were abundant, and relatively lower values during the rest of the study period. In the bottom
layer, the attached fraction increased throughout the study period (data not
shown).
Bacterial abundance was positively related to the volume concentration of TEP
at all three sampling depths, but the correlation was only statistically significant (P
< 5%) in the bottom layer (r = 0.60 in the mixed surface layer, r = 0.70 in the
pycnocline and r = 0.73 in the middle of the bottom layer).
Occurrence and size distribution of TEP produced by bubbling
TEP < 30 n,m occurred in the filtrate at a relatively high number concentration (1.5
x 104 ml-1), but relatively low volume concentration (6 p.p.m.), even before bubbling commenced (Figure 7). This may be due to flexible TEP > 2 \im passing the
filter, and/or because coagulation occurred during handling and set-up of the
experiment. TEP volume concentration increased dramatically in the course of the
first 90 min of the bubbling experiment, to 800 p.p.m., and particles larger than 30
H-m were found. After 24 h, the number concentration of small TEP was high (~ 105
ml-') in the surface of the column, but most larger TEP had settled, resulting in a
very high volume concentration of TEP near the bottom (2250 p.p.m.). Significant
numbers and volumes of TEP were, thus, formed by coagulation due to bubbling.
The particle size spectra of TEP were constructed as above, but we excluded
particles larger than 40 u,m from the regression analyses because of the poor counting statistics. The particle size spectra observed in the bubbling experiment were
similar to those recorded in the field, with estimates of p between two and three.
979
X.Maii and T.Kierfooe
100000
TOmin
T 24 houn (surface'
10000
P-2.K
P-3.02
log k - 6.72
6ppm
1000
lot k - 6.90
17 ppm
100
10
I
1
10000
T30min
r
1000 r
100
o
\
10 -
^
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°
1
1
1
p-2.16
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lot k - 6.03
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o
\
:
T 24 hours (middle)
0
10
10O0O0
z
IOOO
d
100
T 24 houn (bottom]
T60min
1,0000
100
3 itoooo
H
P - 1.96
talk-5.38
2250 ppm
p-2.62
tog k-6.42
315 ppm
1000
T90min
10000
P-2.98
log k - 6 . 7 1
SOOppm
IOOO
100
10
I0OOOO
T 120 min
10000
P-ZI6
tog k - 5.90
370 ppm
IOOO
100
10
1
10
100
1000
TEP equivalent ipherical diameter, dp (fun)
Fig. 7. Differential TEP size distribution in the bubbling column as recorded during 24 h. Regression
lines {dNld[dr] = kdp-<* * ") have been fitted to the data, but utilizing only observations described by
closed symbols.
Discussion
Abundance and size distribution of TEP
At this coastal study site, we found TEP in significant concentrations on all
sampling occasions and at all sampling depths. A similar study by Passow and
980
Transparent exopolymeric partides in the Kattegat
Alldredge (1994) found concentrations of TEP in the coastal waters off California
that were somewhat lower than reported here, up to 5000 ml"1, which is an order of
magnitude smaller than our peak value. However, they considered only TEP
larger than 3 (xm; the majority of TEP recorded in the present study were <3 p.m,
which may account for the difference. Passow and Alldredge (1994) also reported
TEP concentrations as total cross-sectional area per volume of water; their values
ranged between 0.2 and 2000 mm211. If we make a similar calculation for our data,
wefinda very similar range, between 100 and 1650 mm21"1. Since the small particles
contribute only little to total cross-sectional area, this is a more reasonable comparison. In northern Adriatic waters, Schuster and Herndl (1995) found low concentrations of TEP, 0-600 ml"1, which are two orders of magnitude lower than
found here. This difference may be due to a different enumeration technique since
Schuster and Herndl (1995) counted TEP directly on thefilters.Thus, the limited
data so far available suggest that the values reported here are typical for coastal
areas.
The volume concentration of TEP in the size window examined here (~ 1-100
(xm) is similar to or exceeds that of the phytoplankton. For example, by assuming a
carbon to chlorophyll ratio of 30 (\ig p.g-') and a specific carbon content of phytoplankton of 10~7 p.g C n.nr3 (Strathmann, 1967), then 1 \ig chlorophyll H corresponds to a phytoplankton volume concentration of 0.3 p.p.m. Thus, the observed
range of chlorophyll concentration at the surface (0.2-14 \ig 1-') corresponds to
phytoplankton volume concentrations between 0.1 and 4 p.p.m., while TEP
ranged between 0.3 and 9 p.p.m. Subsequent to the phytoplankton bloom, when
the concentration of TEP was high and that of phytoplankton was low, TEP volume concentration exceeded phytoplankton concentration by about two orders of
magnitude. TEP are therefore potentially important for all processes that depend
on particle concentration (e.g. coagulation) and need be taken into account.
Sources and formation of TEP
TEP are believed to be formed from colloidal organic material that, by
coagulation, forms subsequently larger and larger fractal particle aggregates
(Wells and Goldberg, 1993) that eventually appear as TEP (Alldredge etai, 1993;
Kepkay, 1994). Reported concentrations of colloidal organic particles in the ocean
are on the order of 109 mh' (Wells and Goldberg, 1992), and concentrations of
small 1-5 u,m colloidal aggregates are on the order of 105 ml 1 (Wells and Goldberg,
1993). Thus, concentrations expressed as particle size spectra are ~1010 and 2 x 104
particles ml 1 unr1, respectively, in these size intervals. Interestingly, extrapolations of the present size distributions of TEP > 3 u.m (Figure 4) to those particle
sizes are similar to the concentrations actually reported. We consider this to support the suggested source and mode of formation of TEP in the ocean.
The result of our bubbling experiment is also consistent with TEP being formed
by coagulation, although the source of TEP in these experiments was not necessarily only colloidal material; particles up to 30 p.m occurred from the very beginning, but particles of all sizes were formed during the experiment, and total particle
volume increased by several orders of magnitude. The similarity between the TEP
size spectra observed in these experiments and those recorded at our study site
981
X.Mari and T.Kierboe
may suggest that the main mode of formation was similar, i.e. that coagulation was
important in the sea.
There are other potential ways that TEP can be formed (Jackson, 1995). For
example, Ki0rboe and Hansen (1993) observed sheets of mucus (TEP) apparently
being secreted directly from diatoms in culture, and Alldredge el al. (1993) found
TEP in the ocean of a shape (e.g.films,strings) that is not immediately consistent
with formation by coagulation.
Despite several possible modes of formation, the primary ultimate source of
organic material (and TEP) in the ocean is phytoplankton production, even in
coastal regions (Smith and Hollibaugh, 1993). Accordingly, the abundance of TEP
in the euphotic zone appears to be related to the occurrence of phytoplankton
(Passow and Alldredge, 1994), albeit not in a simple way (Figures 2 and 3). We
found that TEP was positively related to bacterial concentration, possible because
both TEP and bacteria depend on dissolved (including colloidal) organic material.
However, the abundance of TEP peaked 1-2 weeks after the height of the phytoplankton bloom. Kepkay etal. (1993) described the variation in concentration of
low molecular (LOC) and colloidal dissolved organic carbon (COC) during a
spring bloom in a temperate coastal bay. Consistent with our observation, they
found maximum concentrations of LOC and COC to lag behind the phytoplankton maximum by ~1 and 2 weeks, respectively. Whether this time lag is due to the
time required for LOC to coagulate into COC and further into TEP, or simply
because concentrations do not necessarily reflect production (due to sinks), is
unclear.
If the size distribution of TEP is caused by coagulation processes, and assuming
that coagulation is driven primarily by turbulent shear, then the steady-state size
distribution would have an exponent, fi, equal to three (McCave, 1984).
Coagulation due to Brownian motion is insignificant for particles larger than ~1
u.m (McCave, 1984), and coagulation due to differential settling can probably be
ignored since TEP consist mainly of water (Sutherland, 1972) and, thus, sink very
slowly. Different values of [3 may result if TEP are fractal (3 < 3; Jiang and Logan,
1991), the size distribution is not in steady state and/or if other processes (e.g.
grazing) are involved. The overall average 3 in our surface samples was 2.3 ± 0.76
(95% confidence limits) and, thus, barely different from the expected value of
three. However, several of the individual p estimates deviate significantly from
three. If we assume that the TEP size distributions are in steady state, then the
fractal dimension, D, of TEP can be estimated (shear coagulation) as D = 2(3 - 3
(Jiang and Logan, 1991). The overall average 3 for the surface samples (2.3) would,
thus, imply a fractal dimension of 1.6. The relationship between the number of
attached bacteria and the size of individual TEP is, in fact, consistent with TEP
being fractal and having a fractal dimension of this magnitude (see below).
Passow and Alldredge (1994) also considered the size distribution of TEP and
observed that when diatoms were abundant, and during what they considered the
'early fioccing state of a diatom bloom', the size distribution of TEP either exhibited very low 3 values or did not follow a power law. They considered this a result
of TEP coagulating with phytoplankton cells to form phytoplankton-TEP marine
982
Transparent exopolymeric particles in the Kattegat
snow particles. We did not observe any such relationship between TEP size distribution and abundance of phytoplankton; at the peak of the diatom bloom, p = 2.5.
TEP as attachment sites for bacteria
Since Alldredge etal. (1993) originally suggested that a significant fraction of the
apparently freely suspended bacteria is in fact associated with TEP, estimates of
this fraction have declined from high (28-68%) (Alldredge etal., 1993) to lower
values: 2-25% (Passow and Alldredge, 1994), 0-20% (average < 5%) (Schuster
and Herndl, 1995) and 0.5-20% (average of the order of 5%) (present study). We
conclude that although all TEP are associated with bacteria, the significance of this
association for bacterial population dynamics appears to be considerably less than
envisaged by Alldredge etal. (1993).
In contrast to Passow and Alldredge (1994), we found very tight correlations
between the size of TEP and the number of attached bacteria (Figure 5). We found
that bacteria are present both on the surface of and inside TEP, but the abundance
of attached bacteria scales neither with the equivalent surface area (°° r2), nor with
the equivalent volume (°° r3) of TEP, but rather with the radius raised to an average
exponent of ~1.5. This may, of course, simply be because TEP are not spherical,
but it may also be related to the mode of TEP formation and to the fractal character
of TEP. Assume (for a moment) that the number of bacteria per TEP (n) is proportional to its solid volume (v,); i.e. v, oo n. The total encased volume of a TEP (v,)
is vt = 4/3irr\ and its porosity (p) is defined asp = 1 - vjv,. From fractal geometry, we
find that particle porosity scales with particle size (e.g. Logan and Wilkinson,
1990):
(l-p)oo/•*>-*
where D is the fractal dimension of the aggregate. Hence:
(1 -p) = vJv, = n/
Thus, the exponents of the relationships between the number of bacteria per TEP
and TEP size (Figure 5) are, under the above assumptions, estimates of the fractal
dimensions of TEP. Although our estimates of TEP fractal dimensions derived this
way (~15) are similar both to those estimated above from size distributions and to
those estimated for marine snow particles, and also consistent with shear
coagulation (Logan and Wilkinson, 1990; Logan and Kilps, 1995), they must not be
accepted uncritically because bacteria cannot be considered conservative tracers.
The attached bacteria may both multiply and degrade TEP material, thereby
changing the n versus dp relationship. The seasonal variation in bacterial density in
TEP (Figure 6) may be the result of such complex interactions. However, we conclude that the observed relationship is consistent with the current comprehension
of the mode of TEP formation.
983
X.Mari and T.Kisrboe
Dynamics and implications of TEP
Dissolved organic carbon (DOC) constitutes by far the most significant pool of
organic carbon in the ocean. Evidence is now accumulating that while bulk DOC
(= truly dissolved + colloidal) has very long residence times, the colloidal fraction
has much shorter turnover times due to involvement in biological (grazing, bacterial uptake) and physical (coagulation) processes; thus, COC appears to be a
reactive component of DOC (Benner et al., 1992; Kepkay, 1994; Wells and Goldberg, 1994). Recent estimates suggest that COC constitutes >10% (Koike et al.,
1990), >30% (Benner et al., 1992) or -16% (Kepkay et al., 1993) of bulk DOC.
Depending on the (so far unknown) carbon content and turnover rate of TEP,
COC and TEP may represent a significant pathway for DOC in the ocean, which is
partly alternative to that traditionally considered the most important one (mineralization by bacteria).
We do not know much about the eventual fate of TEP. It has been demonstrated
that TEP may coagulate with phytoplankton or other particles and subsequently
sink out of the water column. TEP may also be digested by the attached bacteria
(Smith et al., 1992) and return to solute form without being respired. Finally, TEP
may be consumed by planktonic particle grazers. It has been demonstrated that
several microphageous protozoans (Shimeta, 1993; Tranvik etal, 1993), as well as
larvaceans, may graze on colloidal TEP (Flood et al., 1992) and grazing may likewise occur on TEP in the size range considered here (e.g. by copepods). However,
there is so far limited information on this available in the literature and the relative
significance of the potential sinks for TEP is unknown.
Acknowledgements
This research was supported by a fellowship to X.M. from the Danish Ministry of
Education (1994-9432-3) and by grants to T.K. from the Danish Natural Science
Research Council (11-0420-1) and the US Office of Naval Research (N00014-93-10226). Uta Passow, Bruce Logan, Ian Jenkinson, George Jackson and Morten S0ndergaard provided constructive criticism of an earlier draft of the manuscript.
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Received on September 12.1995; accepted on January 22. 1996
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