Journal of Experimental Marine Biology and Ecology
307 (2004) 17 – 34
www.elsevier.com/locate/jembe
Colloidal organic carbon and trace metal
(Cd, Fe, and Zn) releases by diatom exudation
and copepod grazing
Wuchang Zhang 1, Wen-Xiong Wang *
Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay,
Kowloon, Hong Kong, PR China
Received 25 June 2003; received in revised form 15 January 2004; accepted 21 January 2004
Abstract
Colloidal macromolecular organic compounds are important intermediaries between solution
and particle phases and play a critical role in the biogeochemistry of trace metals and organic
carbon. The releases of colloidal organic carbon and trace metals (Cd, Fe, and Zn) mediated by
copepod grazing and decomposition, and direct diatom exudation, were examined using a
radiotracer approach. The colloidal phase was operationally defined in this study as the size fraction
between 5 kDa and 0.2 Am and the dissolved phase as the V0.2 Am filter passing phase. About 13 –
60% of dissolved carbon exuded by the diatom Thalassiosira pseudonana was partitioned into the
colloidal phase, and this fraction increased considerably as the diatom cells grew older. A lower
fraction of dissolved 14C (12 – 23%) excreted by the copepods Acartia erythraea was detected in the
colloidal phase compared to carcass (13 – 35%) and feces decomposition (21 – 34%). In contrast to
carbon, a lower fraction of regenerated dissolved Cd (1 – 11%) and Zn (0 – 20%) from copepods and
diatoms was consistently detected in the colloidal phases. Copepod excretion and carcass
decomposition resulted in more colloidal Fe (51 – 91%) than diatom exudation (46 – 62% for
Thalassiosira weissflogii, and 3 – 33% for T. pseudonana) and copepod feces decomposition (16 –
30%). Copepod (Calanus sinicus) grazing reduced the colloidal fraction of dissolved 14C, although
a higher concentration of the diatom’s (T. weissflogii) carbon was regenerated into the dissolved
phase. The grazing of these copepods did not have any influence on the colloidal metal partitioning.
The release of trace metals and carbon was enhanced by a higher density of copepod’s grazing.
Thus, different biological processes (grazing, excretion, exudation, and decomposition) may
* Corresponding author. Tel.: +86-852-23587346; fax: +86-852-23581559.
E-mail address: [email protected] (W.-X. Wang).
1
Present address: Key Laboratory of Marine Ecology and Environmental Science, Institute of Oceanology,
Chinese Academy of Sciences, Qingdao 266071, PR China.
0022-0981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2004.01.016
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W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
contribute differently to the production and dynamics of colloidal carbon and metals in planktonic
systems.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Colloidal organic carbon; Colloidal metals; Iron; Regeneration; Grazing
1. Introduction
Over the past decade, many studies have highlighted the significance of colloidal
nanoparticles/macromolecular in the overall biogeochemical cycling of carbon and metals
in the oceans (Martin et al., 1995; Guo and Santschi, 1997; Gustafsson and Gschwend,
1997; Santschi et al., 1999). Colloids are operationally defined as the size range between 1
nm and 0.2 Am (Buffle, 1990), and are an important intermediary between the truly
dissolved phase and the particulate phase. With the advances of ultrafiltration technology,
significant fractions of dissolved organic carbon (DOC) and metals were found to be
associated with the colloidal materials in diverse marine environments (Martin et al., 1995;
Guo and Santschi, 1997; Wen et al., 1999; Guo et al., 2000). The colloidal organic matter
(COM) was found to have a high C/N ratio and rich in polysaccharides (Benner et al.,
1992; Biddanda and Benner, 1997; Aluwihare and Repeta, 1999; Aluwihare et al., 2002),
and was more labile than low-molecular-weight compounds (Amon and Benner, 1994,
1996; Santschi et al., 1995). Recent studies have demonstrated that the bioavailability of
colloid-bound metals is different from that of their low molecular weight counterparts
(Wang and Guo, 2000; Chen and Wang, 2001). For example, the biological availability of
colloid-bound Fe and Zn was lower than that of their counterparts in the low molecular
weight (LMW) fractions, whereas the uptake of colloidally bound Cd was comparable to
the LMW counterparts due to its relatively weak binding to colloidal particles (Wang and
Guo, 2000; Chen and Wang, 2001) and tendency for repartitioning (Guo et al., 2002).
Therefore, an understanding of the colloidal dynamics will have profound implications for
the prediction of the overall biogeochemical fates of organic carbon and metals in the
oceans.
To gain a better understanding of the fate and the cycling of elements in the ocean, the
association of metals and organic carbon with the colloidal particles from various sources
needs to be determined. It is generally thought that the compositions of colloidal particles
are diverse and includes polysaccharides, humic substances, or even viral particles (Stumm
and Morgan, 1996; Santschi et al., 1999; Guo et al., 2001). Various mechanisms such as
the direct algal biosynthesis (Aluwihare and Repeta, 1999), predatory discharge of the
intracellular (high molecular weight, HMW) component (Wells et al., 1998), terrestrial
organic matter (Santschi et al., 1995; Bianchi et al., 1997), or phytoplankton decomposition (Wang and Guo, 2001) have all been proposed as major sources of colloidal organic
matter. Although many studies have quantified the colloidal fractions of metals and
organic carbon in many marine environments (Guo and Santschi, 1997; Santschi et al.,
1999), it is difficult to identify the contributions of various sources on the basis of these
field studies (Kepkay et al., 1993). In a recent study, Wang and Guo (2001) examined the
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
19
colloidal fraction of organic carbon and trace metals released from decomposing
phytoplankton, and speculated that phytoplankton decomposition may be a major source
for natural colloidal metals in the ocean.
Phytoplankton uptake, zooplankton assimilation and excretion, phytoplankton and
zooplankton debris decomposition, and the re-uptake or coagulation of regenerated metals
are the important steps in the biological controls of trace metal cycling in the oceans. In
this paper we examined the partitioning (expressed as percentage) of dissolved carbon and
metals produced by various biological processes into the colloidal phase under the
laboratory conditions. The biological processes considered in this study included diatom’s
exudation during growth, copepod’s excretion and grazing, and copepod’s carcasses and
feces decomposition. To our knowledge, no previous study has specifically examined the
simultaneous production of colloidal carbon and metals mediated by copepod grazing,
although a few studies had considered the production of surface-active organic matter and
colloidal iron and thorium by protist grazing (Barbeau et al., 2001; Kujawinski et al.,
2001). We considered three oceanographically important metals, including Cd, Fe and Zn,
primarily because of the availabilities of these radiotracers. Among the three metals, Fe
and Zn are essential to marine plankton, and Cd can be essential to marine diatom under
Zn-limited conditions (Cullen et al., 1999). Radiotracer techniques were employed in this
study such that the releases of metals and carbon into the colloidal phase can be monitored
kinetically.
2. Materials and methods
2.1. Diatoms, copepods, carbon and trace metals
Two species of the diatoms Thalassiosira weissflogii (CCMP 1048) and Thalassiosira
pseudonana (clone 3H) were obtained from the Provasoli – Guillard Phytoplankton
Collection Center (Maine, USA), and maintained in clonal cultures in f/2 medium
(Guillard and Ryther, 1962) at 18 jC on a 14:10 h light:dark cycle. Adult copepods,
Acartia erythraea, and Calanus sinicus, were collected by net towing (250 Am mesh size)
from Port Shelter, Clear Water Bay, Hong Kong. The copepods were maintained under
laboratory conditions and fed with mixed diatoms T. weissflogii and T. pseudonana. One
day before the experiments, the LMW water (<1 kDa) was collected using cross-flow
ultrafiltration with a S10Y1 Amicon Spiral Cartridge with a nominal molecular weight
cutoff of 1 kDa. The LMW water was used in all experiments to avoid the influences of the
presence of background colloidal nanoparticles as well as the bacterial activity. All
experiments were carried out at 18 jC and at a 30 psu salinity in a Class-100 clean
bench. All the experimental polycarbonate bottles (with caps) were cleaned thoroughly
with Ultrapure HCl and HNO3 acids before use.
Trace metals (Cd, Fe and Zn) and carbon were studied using the radiotracers 109Cd (in
0.1 N HCl), 59Fe (in 0.1 N HCl), 65Zn (in 0.1 N HCl), and 14C (as bicarbonate). The 14C
and 59Fe were spiked separately while the 109Cd and 65Zn were spiked into the same
cultures. The pH of the labeled diatom culture was adjusted to 8.0 by adding 0.5 N
Suprapur NaOH.
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W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
The percentage partitioning (%) of dissolved radioactive elements into the colloidal
phase was calculated as the ratio of the radioactivity (counts per volume) in the
colloidal fraction (Rcolloidal, 5 kDa – 0.2 Am) to the radioactivity in the dissolved phase
(Rdissolved,<0.2 Am). In all experiments, Rdissolved was determined directly by filtering
aliquot of water onto the 0.2 Am polycarbonate membrane and the radioactivity in the
<0.2 Am filtrate was quantified. The centrifugal ultrafilters (Amicon, Centricon plus-20;
molecular weight cutoff 5 kDa) were used to collect the LMW fraction (<5 kDa).
According to the manufacture’s manual, the ultrafiltration was conducted at 4500 g for
15 min. We did not directly determine the colloidal radioactivity because the centrifugal
ultrafilter was designed to collect the LMW solution (<5 kDa). Therefore, the Rcolloidal
was calculated from the difference between the Rdissolved (<0.2 Am) and the radioactivity
in the LMW solution (RLMW,<5 kDa). The partitioning of dissolved radioactive elements
into the colloidal phase can thus be calculated by:
% in colloidal phase ¼ ðRdissolved RLMW Þ=Rdissolved 100%
ð1Þ
The retention efficiency of the ultrafiltrate membranes was tested using vitamin B12
(with a molecular weight of 1.33 kDa) and 3 and 10 kDa dextrans, and was 16F4%,
45F2%, 98F1% (meanFS.D., n=3), respectively. Sorption of metals onto the ultrafilters
was negligible (Wang and Guo, 2001).
2.2. Diatom exudation
There were a total of four independent experiments to examine the colloidal
partitioning of carbon and trace metals by diatom exudations, including two replicated
experiments for 14C (Expt. 1 and Expt. 2), one experiment for both 109Cd and 65Zn,
and one experiment for 59Fe. In each experiment, the log-phased T. pseudonana cells
were filtered onto a 3 Am membrane and rinsed twice with filtered seawater to remove
the nutrient medium. Rinsed cells were resuspended in three replicated flasks, each
containing 400 ml new media (f/2 levels of macronutrients and vitamins, and f/20
levels of trace metal medium without Cu, Zn, and EDTA). The initial cell concentration of the culture was 105 cells ml1. Each flask was then spiked with 1110 kBq of
14
C, or 148 kBq of 109Cd (corresponding to 31 nM)+65Zn (0.2 nM), or 148 kBq of
59
Fe (12 nM).
In the first 14C experiment, the flasks (500 ml volume) were sampled daily for 5 days,
whereas in the second 14C experiment and all the metal experiments, the flasks were
sampled on days 1, 2, 3, and 5. The diatom concentrations in each flask were first
counted using a haemocytometer under the microscope. A certain volume of the cells
was then filtered onto a 3 Am membrane and re-suspended twice into the LMW water.
After a further count, the cells were added into the glass bottles containing 100 ml LMW
seawater at a cell density of 5105 cells ml1. At different time intervals (1, 2, 3, and 24
h for the first 14C experiment; 1, 3, 5, 8 h for the second 14C experiment and metal
experiments), aliquots of samples (8– 10 ml) were removed from the bottles and filtered
through the 0.22 Am membrane to remove the diatom particles. A subsample (2 –4 ml)
was then counted for Rdissolved. Another 4 ml of the filtrate was ultrafiltered using a
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
21
centrifugal ultrafilter as described above. Two milliliter of the ultrafiltrate was used to
determine RLMW.
2.3. Copepod excretion, carcass and feces decomposition
In these experiments, we used the larger diatom T. weissflogii because they were more
efficiently preyed on by the copepods compared to the smaller diatom T. pseudonana. The
cells in the late log phase were collected onto a 3 Am polycarbonate membrane and resuspended into 75 ml of 0.2 Am filtered seawater enriched with f/2 levels of N, P and Si,
and vitamins, and f/20 levels of trace metals without the additions of Cu, Zn, and EDTA.
In the case of the carbon experiment, f/2 levels of both macronutrients and trace metals
were added to the medium. The initial cell concentration in the media was generally
2104 cells ml1. The amounts of radioisotope additions were 148 kBq for 109Cd
(corresponding to 164 nM), 148 kBq for 65Zn (corresponding to 1.2 nM), 148 kBq for
59
Fe (corresponding to 62 nM), and 1110 kBq of 14C. Relatively high amounts of
radioactivity (thus the resulting concentrations) of 109Cd were employed in these experiments for the purpose to obtain sufficient counts of radioactivity in the copepod’s excreted
products. Earlier experiments demonstrated that the efflux rates of metals were not
affected by different metal concentrations in ingested food (or ambient metal concentrations) (Xu et al., 2001). After 4– 5 days growth, the cell densities reached 1 –2105
cells ml1 (3– 4 divisions).
Before the radioactive feeding, about 1000 individuals of A. erythraea were transferred
into glass-fiber-filtered seawater for about 1 h to evacuate their guts. The copepods were
then transferred into the feeding beaker with 1 l of filtered seawater. The radiolabeled
diatoms were subsequently added every 8 h into the beakers to feed the copepods. The
fecal pellets of the copepods were collected every 2– 4 h. After feeding for 24 h, the
copepods were filtered onto a mesh, washed with LMW seawater three times to remove
any weakly bound radioactive elements, and then evenly transferred into three containers
with 300 ml LMW seawater. At 2, 4, 6, and 8 h, samples were taken to determine Rdissolved
and RLMW as described above.
After the excretion experiments, the copepods were collected by a mesh and were
killed by being briefly exposed to air. The fecal pellets and the carcasses of the
copepods were subsequently rinsed gently with LMW seawater for at least two times.
Each sample was then divided into three replicates in LMW water (100 ml). The bottles
containing the decomposing feces or carcasses were kept in the dark without disturbance. At 1, 3, 5, 7, 9, and 11 days, a 4 ml sample was taken to determine Rdissolved and
RLMW. After the sampling, LMW seawater was added to the bottles to maintain the
volume of the water.
2.4. Copepod grazing on diatoms
T. weissflogii was labeled in 100 ml filtered seawater at an initial cell concentration of
104 cells ml1, using the methods as described above. The amounts of radioisotope
additions were 148 kBq for 109Cd (123 nM), 148 kBq for 65Zn (0.9 nM), 148 kBq for
59
Fe (46.4 nM), and 1110 kBq for 14C. After 5 days of growth, the cell concentration
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W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
reached 105 cells ml1. The cells were then filtered onto a 3 Am polycarbonate
membrane and resuspended twice to remove any loosely bound radioactive materials.
The resuspended cells were added to triplicated 100 ml LMW seawater at a cell
concentration of 1.5 –1.7104 cells ml1. Copepods C. sinicus were added into the
bottles at two different densities (10 and 50 animals, respectively, corresponding to a
copepod density of 0.1 and 0.5 individuals ml1). The control treatment received no
copepod addition. C. sinicus was used in this experiment primarily because this species
of copepods was the dominant copepod when the experiments were conducted during
the winter season.
The bottles containing the copepods and radiolabeled diatoms were kept in dim light for
8 h. At 1, 3, 5, and 8 h, samples were taken to determine Rdissolved and RLMW using
methods described above.
2.5. Radioactivity measurements
The radioactivity of the gamma-emitting isotopes was measured with a Wallac 1480
automatic gamma counter (Turku, Finland). All measurements were related to appropriate
standards and calibrated with spillover. The gamma emission of 109Cd was determined at
Fig. 1. Percentages of dissolved carbon in the colloid phase exuded by different growing phases of the diatom T.
pseudonana (top panel), and the relative concentrations of dissolved carbon as compared to the dissolved
concentration at 1 h of resuspension (bottom panel). Two replicated experiments were conducted. Days in the
legends are the days at which the diatoms were harvested for experiments. Values are meansFS.D. (n=3).
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
23
88 keV, of 65Zn at 1115 keV, and of 59Fe at 1092 keV. To count the 14C activity in the
samples, cocktail (Fisher Chemicals, UK) was added and well mixed with the samples.
The radioactivity of 14C was measured by a Wallac 1414 liquid scintillation counter using
the external standard ratio method. Counting times were adjusted to yield a propagated
counting error of less than 5% (typically 2 –3%).
Fig. 2. Percentages of dissolved Cd, Zn, and Fe in the colloid phase exuded by different growing phases of the
diatom T. pseudonana, and the relative concentrations of dissolved metals as compared to the dissolved
concentration at 1 h of resuspension (bottom panel). Days in the legends are the days at which the diatoms were
harvested for experiments. Values are meansFS.D. (n=3).
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W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
3. Results
3.1. Diatom exudation
The diatom T. pseudonana reached the maximum cell density (1.4 –1.8106 cells ml1)
on Day 4 in the 14C and metal experiments. The percentage of 14C activity in the colloidal
phase decreased sharply over 8 and 24 h in the two replicated incubation experiments (Fig.
1). However, more 14C was partitioned into the colloidal phase as the diatom culture aged.
Within the first hour of resuspension into LMW DOC water in Expt. 1, the percentage of
Fig. 3. Percentages of dissolved carbon and metals in the colloid phase excreted by the copepod A. erythraea (top
two panels), and the relative concentrations of dissolved carbon and metals excreted by copepods as compared to
the dissolved concentration at 2 h of excretion (bottom panel). Two replicated experiments were conducted for
carbon excretion. Values are meansFS.D. (n=3).
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
25
colloidal carbon was 34%, 33%, 44%, 52%, and 60% for the Day 1, 2, 3, 4, and 5 cells,
respectively. After 24 h of incubation in LMW DOC water, this percentage decreased to
13%, 19%, 16%, 16%, and 22%, respectively. In Expt. 2, the percentage of colloidal 14C
was 43%, 44%, 50%, 55% in the first hour for the Day 1, 2, 3, and 5 cells, respectively.
After 8 h of incubation, this percentage decreased to 16%, 17%, 32%, and 32%. In contrast
to the colloidal organic carbon (COC), the percentage of colloidal metals generally showed
no difference over the 8-h incubation period, with a few exceptions (Fig. 2). There was no
major difference among the differently aged cells. The ranges of Cd, Zn and Fe in the
colloidal phase were 1– 11%, 0 –20%, 3– 33%, respectively.
The radioactivity in the dissolved phase (<0.2 Am) was calculated as the ratio of the
initial radioactivity in the dissolved phase measured at 1 h. The release patterns varied
among C, Cd, Zn and Fe (Figs. 1 and 2). In general, there was an increase in the release of
dissolved 14C and Cd by the diatoms during the course of resuspension. Very little change
in the concentration of dissolved Fe was found. However, the dissolved Zn concentrations
decreased after 1 h, except for the Day 1 cells, which showed an increase in dissolved Zn
after 3 h and then the concentration decreased again. The release of carbon into the
dissolved phase was higher for exponentially growing cells (e.g., Day 2 and 3 in Expt. 1
and Day 2 in Expt. 2) than cells in the stationary phase (Fig. 1). For metals, the releases
were comparable for different aged cells (except for the Day 1 cells, Fig. 2).
Fig. 4. Percentages of dissolved carbon and metals in the colloid phase regenerated by the decomposition of
copepod A. erythraea carcass (left panel) and feces (right panel). Values are meansFS.D. (n=3).
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W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
3.2. Copepod’s excretion, carcass and feces decomposition
The percentages of copepod’s excreted colloidal 14C in the two replicated experiments
and metals are shown in Fig. 3. Generally, 12 –23% of excreted dissolved 14C and small
fractions of excreted Cd and Zn (1– 7% and 1– 5%, respectively) were detected in the
colloidal phase. The percentages of excreted Fe in the colloidal phase (61 – 84%) were
much higher than those of colloidal carbon, Cd, and Zn. The fractions of colloidal C, Cd,
and Zn were rather constant throughout the 8-h incubation period, but decreased for Fe.
The radioactivity in the dissolved phase at different sampling periods was also
calculated as the ratio of the radioactivity in the dissolved phase measured to that at 2
h (Fig. 3). This ratio increased linearly with increasing incubation time, and was highest
for Cd and Zn, followed by carbon and Fe.
The colloidal fractions of carbon and metals during the 11 days of feces and carcass
decomposition are shown in Fig. 4. Over the 11-day period of carcass decomposition, the
colloidal Cd and Zn were consistently lower than 7%. The ranges of colloidal carbon and
Fe were 13– 35% and 51– 91%, respectively. Furthermore, both fractions of carbon and Fe
decreased in the colloidal phase during the decomposition period. Similar to the carcass
decomposition experiments, very little Cd and Zn was released into the colloidal phase by
Fig. 5. Percentages of dissolved carbon in the colloid phase regenerated from the diatom T. weissflogii by copepod
C. sinicus grazing (top panel) at two different copepod’s densities, and the relative concentrations of dissolved
carbon as compared to that in the controlled treatment at 1 h (bottom panel). Two replicated experiments were
conducted. Values are meansFS.D. (n=3).
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
27
feces decomposition (1– 9% for Cd, and 1 –7% for Zn). A much smaller percentage of
59
Fe was found in the colloidal phase (16 – 30%) during the feces decomposition
experiment compared with the carcass experiment. The percentages of both colloidal
carbon (21 – 34%) and Fe released by feces decomposition also decreased slightly over the
period of incubation.
3.3. Copepod’s grazing
The releases of dissolved carbon from the radiolabeled diatoms (T. weissflogii) into the
colloidal phase were quantified in the presence of copepod’s grazing over an 8-h incubation
period (Fig. 5). In the control, about 49 – 52% of the released carbon was detected in the
Fig. 6. Percentages of dissolved Cd, Zn, and Fe in the colloid phase regenerated from the diatom T. weissflogii by
copepod C. sinicus grazing at two different copepod’s densities (left panel), and the relative concentrations of
dissolved metals as compared to that in the controlled treatment at 1 h (right panel). Values are meansFS.D.
(n=3).
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W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
colloidal phase within the first hour of the two replicated experiments. This fraction then
decreased to 37 –38% after 8 h of incubation. At higher densities of copepods, the
percentage of colloidal carbon was lower than the percentage in the control treatment. For
example, at the low copepod density treatment (0.1 ind. ml1), the colloidal carbon
fraction was 30% and 25% at 8 h in Expt. 1 and Expt. 2, respectively. This fraction
decreased to 19% at the higher copepod density of 0.5 ind. ml1.
To examine the influence of copepods on the release of dissolved 14C, the radioactivity
was calculated as the ratio of the radioactivity in the dissolved phase to the one measured
in the control treatment at 1 h. A higher fraction of dissolved 14C was found in the
presence of copepods after 1 h of incubation. The dissolved pool increased at a higher rate
at the high copepod density (0.5 ind. ml1). After 8 h of incubation, the dissolved 14C
concentration in this treatment was 2.2 –2.6 higher than the concentration in the control
treatments. At a low copepod density, however, there was no increase in the dissolved pool
as compared to the control treatment over the course of incubation.
In contrast to carbon, there was no major difference in the percentage of colloidal
metals among the different treatments (Fig. 6). The percentages of colloidal Cd and Zn
were consistently low (1– 6% for Cd and 3– 15% for Zn), while 46– 62% of dissolved Fe
was in the colloidal phase. Similarly, the radioactivity of the dissolved metals was
calculated as the ratio of the radioactivity in the dissolved phase to that in the control
treatment at 1 h. A higher fraction of dissolved metals was only found for Zn and Fe at the
high copepod density treatment (0.5 ind. ml1). At the end of the experiments, the
concentrations of dissolved Zn and Fe in this treatment were 2.0 and 1.4 higher than the
concentrations in the control treatments, whereas the concentration of dissolved Cd was
only 0.85 of that. No major difference was found between the low copepod density
treatment and the control treatment.
4. Discussion
In this study, we considered the production of dissolved carbon and metals into the
colloidal phase from several sources of biological activity, including diatom exudation,
copepod’s excretion and grazing, and feces and carcass decomposition. We employed a
radiotracer technique to examine the kinetics of colloidal element production instead of
measuring the stable concentrations of carbon and metals. Typical concentrations of the
dissolved Cd, Fe, Zn, and DOC in the studied waters were 0.2– 0.6 nM, 50 – 59 nM, 4– 38
nM, and 98– 130 AM, respectively (Wang and Dei, 2003; Wang, W.-X., unpublished data).
In our experiments, we used the freshly collected LMW (<1 kDa) filtered seawater to
minimize the influences of background colloidal nanoparticles as well as the bacterial
growth, which may significantly affect the interpretation of our experimental data.
However, bacterial growth may be a potential problem in the long-term experiments such
as the copepod’s carcass and feces decomposition experiments, during which the bacterial
numbers were not quantified. In addition, since the dissolved carbon may include dissolved
organic and inorganic carbon (e.g., CO2) in the experimental waters, our estimates of the
COC in the total dissolved carbon pool may underestimate the fraction of COC in the DOC
pool. Sources of CO2 in the experimental waters included bacterial activity as well as the
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29
respiration by the experimental plankton themselves. Recent study showed that the respired
carbon represented about 1/4 of the total carbon regenerated by the marine copepod Acartia
spinicauda (Xu and Wang, 2003). Since the experiments were conducted with 14C-tagged
plankton over a short period of time, the percentage of H14CO3 produced was assumed to
be relatively small, but this remains to be further tested.
4.1. Colloidal carbon production
In our study, about 13 –60% of diatom exuded dissolved 14C was detected in the
colloidal phase when the diatoms were suspended in the LMW seawater. Furthermore, the
production of COC was dependent on the growth stage of the diatoms, with aged diatoms
producing more COC compared to the cells in the exponentially growing phase. This
increase is in accordance with that of Kepkay et al. (1993), even though the relative
magnitude is different. They found that the percentage of COC/DOC (calculated by
absolute concentration) increased from 4.8% from the early spring bloom stage to 16.1%
during the late bloom stage.
The DOC consists of all types of biochemical products including carbohydrates
(mono-, oligo-, and polysaccharides), nitrogenous compounds (amino acids, proteins and
polypeptides), lipids (fatty acids), and organic acid (glycollate and tricarboxylic acids).
Carbohydrates are one of the main components of DOC released by phytoplankton, along
with amino acids and fatty acids (Handa, 1970). Polysaccharides constituted the dominant
fraction of the exuded dissolved carbohydrates in phytoplankton culture (70 – 94%,
Biddanda and Benner, 1997) and in the surface water (71%, Pakulski and Benner,
1994). Aluwihare and Repeta (1999) and Aluwihare et al. (2002) found that acyl
polysaccharides, composed of carbohydrates, acetates and lipids account for approximately 70 – 75% of the total carbon in the HMW DOC. Morris and Seka (1978) showed
that about half of the incorporated carbon by natural phytoplankton was in the cellular
polysaccharide fraction. The relative abundance of carbohydrate in phytoplankton organic
matter increases as phytoplankton grow older (Barlow, 1982; Biddanda and Benner,
1997). For example, McAllister et al. (1961) reported that carbohydrates in phytoplankton
cells increased from 20% to 70% of cellular carbon when phytoplankton was cultured for
20 days.
Aluwihare and Repeta (1999) isolated the colloidal or HMW (1 kDa –0.2 Am) organic
carbon from different phytoplankton cultures. For T. weissflogii, Emiliania huxleyi and
Phaeocystis sp., they found that the HMW DOC comprised 27%, 25%, and 21% of the
total DOC, respectively, at the time of harvesting. Biddanda and Benner (1997) also
determined the HMW (1 kDa – 0.1 Am) COC fraction in different phytoplankton cultures.
Their results were 36%, 31%, 36% and 38% for Synechococcus, Phaeocystis, Emiliania,
and Skeletonema, respectively. Wang and Guo (2001), using the radiolabeling technique,
quantified the production of COC by the decomposition of T. pseudonana and dinoflagellate Prorocentrum minimum over 21 days. About 31 – 41% of DOC was in the colloidal
phase (5 kDa – 0.22 Am). In our study, the percentages of COC produced by T. pseudonana
exudation were 13– 22% at 24 h in Expt. 1, and 16 –32% at 8 h in Expt. 2. For another
diatom T. weissflogii, the percentage was about 38% after 8 h of suspension. Our results
are thus generally consistent with the results from previous studies.
30
W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
A marked difference in the dynamics of COC release was also found among the different
experiments. In the diatom exudation experiment, this fraction decreased significantly
during the incubation period, which may be due to re-equilibrium after the diatom cells were
transferred into the LMW seawater. It is unknown whether the significant decrease of
colloidal carbon was due to breakdown of HMW carbon into LMW compounds. In contrast
to diatom exudation, a relatively smaller and constant percentage (12 – 23%) of COC was
excreted by the copepods. We are not aware of any previous measurements of the COC
produced by copepod excretion. The percentage of colloidal 14C excreted by the copepods
remained somewhat constant, suggesting that the COC released by the copepods was at
steady-state. In the carcass and feces decomposition experiments, this fraction decreased
over time, thus the colloidal particles might have been continuously decomposed to the low
molecular carbon and dissolved inorganic carbon, or removed by coagulation. In this regard,
the COC acts more or less as an intermediary phase between the LMW carbon and the
particulate organic carbon. In the copepod grazing experiments, the colloidal 14C fraction
appeared to be reduced more drastically in the presence of copepods. Whether COC can be
directly utilized by the copepods leading to a decrease of colloidal fraction is a matter of
speculation. All these data demonstrated that the production of COC is a very dynamic
process. Barbeau et al. (2001) and Kujawinski et al. (2001) also showed the production of
surface-active COC by the protist grazing on phytoplankton cells.
4.2. Dissolved carbon production
In diatom exudation experiments, the exuded carbon was calculated as the ratio of
exuded carbon to that at 1 h. The dissolved 14C production rates were somewhat higher for
exponentially growing cells (Days 2 and 3). There were generally conflicting reports on
the timing of the highest DOC production rates by phytoplankton. Guillard and Wangersky
(1958) found that the stationary cells released more DOC than exponentially growing
cells, but newer studies suggested that the fluxes of DOC were the highest under
conditions of rapid growth (Anderson and Zeutschel, 1970; Zoltnik and Dubinsky,
1989; Biddanda and Benner, 1997). In some species such as Phaeocystis and Skeletonema,
the high DOC production rates prevailed throughout the different growth stages (Biddanda
and Benner, 1997).
In the copepod excretion experiments, the excreted 14C was also calculated as the ratio
of excreted carbon to that at 2 h. We found that this pool of dissolved carbon continued to
increase over the 8 h period. The release of carbon by zooplankton has been discussed in
a few previous studies (Gerber and Gerber, 1979; Xu and Wang, 2003). Gerber and
Gerber (1979) showed that the daily carbon losses were 63% for copepods Undinula
vulgaris, and 88% for mixed small copepods. In a recent study, Xu and Wang (2003)
partitioned the ingested diatom carbon by copepods into different compartments (excretion, feces, and respiration) and showed that 68 –75% of the copepod’s metabolic carbon
loss was in the form of excretion products. Strom et al. (1997) showed that phytoplankton
on average released 3 – 7% of cellular carbon on a daily basis, but 16 – 37% of
phytoplankton carbon was lost in the form of DOC during an ingestion event. In our
study, copepod’s grazing significantly enhanced the release of diatom 14C only at a high
copepod density (0.5 ind. ml1).
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
31
4.3. Colloidal metal production
In contrast to COC, very little Cd (1– 11%) and Zn (0 –20%) was consistently detected
in the colloidal phase in all the experiments. Differently aged cells did not significantly
produce different amounts of colloidal metals. These data strongly highlight the contrasting behavior of the colloidal Cd and Zn production as compared to the COC production.
Wang and Guo (2001) also showed no significant relationship between the colloidal Cd
and Zn and carbon production by phytoplankton decomposition.
In Rhone delta, colloidal Cd (10 kDa – 0.7 Am) constituted 0 – 38% of the total
dissolved Cd (<0.7 Am), with most values <10% (Dai et al., 1995). Powell et al. (1996)
found that the percentage of colloidal Cd (1 kDa –0.45 Am) in a Southeastern US estuary
was 6%. The fraction of dissolved Cd and Zn in the colloidal phase was <10% in San
Francisco Bay (Sanudo-Wilhelmy et al., 1996) and Narragansett Bay, USA (1 kDa –0.2
Am, Wells et al., 1998). Wells et al. (2000) also found that 2– 14% of the dissolved Zn
occurred within the colloidal phase (1 kDa –0.2 Am) in Narragansett Bay. Other studies
reported a higher colloidal Cd and Zn fraction, e.g., 1 –63% (on average 34%) of the
dissolved (<0.4 Am) Cd was in the colloidal phase (10 kDa – 0.4 Am) in Venice Lagoon
(Martin et al., 1995), and 45% of Cd and 91% of Zn in the colloidal fraction (1 kDa –0.45
Am) in Galveston Bay, TX (Wen et al., 1999). For phytoplankton decomposition, Wang
and Guo (2001) reported that 5– 10% of Cd and 14 –30% of Zn was detected in the
colloidal phase (5 kDa – 0.2 Am).
In contrast to Cd and Zn, a larger fraction of Fe was associated with the colloidal
particles in our study, consistent with several field measurements. For example, colloidal
(0.02 –0.4 Am) Fe represented 80 –90% of the dissolved (<0.4 Am) Fe in the near-surface
waters of the oligotrophic North Atlantic and North Pacific, and this fraction was reduced
to 30 –70% in deeper waters (Wu et al., 2001). About 93 – 99% of the total dissolved (<0.2
Am) Fe resided in the colloidal (1 kDa –0.2 Am) fraction in Narragansett Bay (Wells et al.,
2000). In Venice Lagoon, Italy, 68 – 100% (on average 87%) of the dissolved (<0.4 Am) Fe
was in the colloidal phase (10 kDa – 0.4 Am) (Martin et al., 1995). Wen et al. (1999) found
that 79% of Fe was in the colloidal phase (1 kDa –0.45 Am) in Galveston Bay, TX, USA.
Large variations were documented in the percentage of Fe in the colloidal phase among
the different experiments. Excretion by copepods and carcass decomposition produced
more colloidal Fe than diatom excretion and copepod feces decomposition. The diatom T.
weissflogii also exuded a larger percentage of colloidal Fe (46 – 62%) than T. pseudonana
(3– 33%). It appears that a larger fraction of COC originated from the copepods binds with
Fe than the fraction originated from the diatoms.
4.4. Dissolved metal production
Considerable variations in the release of Cd, Zn and Fe into the dissolved phase were
observed during the diatom exudation experiment. The increasing concentration of
dissolved Cd may have been due to the rapid efflux from the diatom cells (Sunda and
Huntsman, 1998), which released Cd into the dissolved phase. The regenerated Zn may
have been subsequently re-absorbed by the diatoms during the 8-h exposure period,
leading to a decrease in the dissolved Zn pool. For Fe, the dissolved pool remained
32
W. Zhang, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
relatively constant, implying that the rate of Fe released from the diatoms was comparable
to the rate of Fe re-uptake.
In the copepod excretion experiment, dissolved Cd and Zn concentrations built up
much faster than those of dissolved 14C and Fe, suggesting that these two metals were
regenerated by the copepods at a faster rate than 14C and dissolved Fe. In recent
studies, we also quantified the biological excretion of metals and carbon by the copepod
A. spinicauda. The efflux rate constants of Cd and Zn were 0.52 –0.83 and 0.41– 0.65
day1 (Xu et al., 2001) and were higher than that of carbon (0.13 –0.37 day1, Xu and
Wang, 2003). We have not quantified the efflux rate of Fe in the copepods, but Schmidt
et al. (1999) showed that the calanoid copepods lost carbon more rapidly than Fe,
consistent with our observation that Fe released by copepod excretion was slightly
lower than that of carbon. Thus the higher efflux rate of Cd and Zn as compared to
carbon and Fe may lead to rapider increase in their dissolved pools by copepod
excretion.
As in the case of 14C, copepod’s grazing enhanced the release of dissolved Zn and Fe at
a high copepod density (0.5 ind. ml1). There was no appreciable difference in the
dissolved metal pools at the low copepod density treatment (0.1 ind. ml1). Hutchins and
Bruland (1994) investigated the grazer-mediated regeneration of Fe and Zn using the
copepod Acartia tonsa and the diatom T. weissflogii as the grazer and prey system. At the
end of a 9– 10 h grazing period, the concentration of dissolved Fe and Zn were 3 to 7
higher in bottles with grazers than in the control bottles without grazers. However, it seems
that copepod grazing did not enhance the release of Cd because its concentration was
comparable or slightly lower than that in the controls.
In conclusion, our study demonstrates that a significant fraction of dissolved carbon
exuded by the diatoms existed in the colloidal phase, and this fraction increased
considerably as the cells grew older. Lower fraction of dissolved C excreted by the
copepods was detected in the colloidal phase compared to carcass and feces decomposition. In contrast to carbon, a much lower fraction of regenerated dissolved Cd (1– 11%)
and Zn (0 –20%) from copepods and diatoms was in the colloidal phase. Copepod
excretion and carcass decomposition resulted in more colloidal Fe (51 –91%) than diatom
exudation (46 – 62% for T. weissflogii, and 3 – 33% for T. pseudonana) and copepod feces
decomposition (16 – 30%). Although copepod grazing reduced the colloidal fraction of
dissolved 14C, colloidal fractions of dissolved Cd, Zn and Fe were not influenced by
copepod grazing. The release of trace metals and carbon was enhanced by a higher
density of copepod grazing. Different biological activities may thus contribute differently
to the production and dynamics of colloidal carbon and metals in marine planktonic
systems.
Acknowledgements
We are extremely grateful to Prof. Peter Santschi for his very thorough and detailed
comments on this paper. This study was supported by Competitive Earmarked Research
Grants from the Hong Kong Research Grants Council (HKUST6118/01M and
N_HKUST603/01) to W.-X. Wang. [RW]
W. Zhang, W.X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 17–34
33
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