FEMS Microbiology Ecology 27 (1998) 85^102
Measurement of bacterivory by protists in open ocean waters
Mikhail V. Zubkov a , Michael A. Sleigh a; *, Peter H. Burkill
a
b
Division of Biodiversity and Ecology, School of Biological Sciences, University of Southampton, Southampton, SO16 7PX, UK
b
Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, UK
Received 23 April 1998; accepted 9 June 1998
Abstract
Protozoa are the main consumers of heterotrophic bacteria in aquatic habitats. The numbers of these bacteria and protozoa
in oligotrophic areas of the open ocean are low, and current methods lack the sensitivity to assess rates of bacterivory in such
waters. A new method is proposed for estimating bacterivory on dual radioactively labelled natural bacteria using living
ambient prey bacteria and separation of predators from prey by fractionation. This approach is sufficiently sensitive to
measure the consumption of less than 1% of the labelled bacteria during a 13-h incubation period. When tested on samples
collected from 27 stations in mesotrophic and oligotrophic regions of the North and South Atlantic Oceans, about 17% of
metabolically active bacteria were grazed per day and about 60% of consumed prey biomass labelled with 14 C-leucine was
retained by the predators. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All
rights reserved.
Keywords : Microbial loop; Heterotrophic nanoplankton; Grazing on bacteria ; Radioactive labelling; Size fractionation
1. Introduction
In the geological past, Prokaryotes, the Bacteria
and Archaea, were undoubtedly the key organisms
in biogeochemical £uxes on our planet, and they
remain principal promoters of these £uxes today.
They are dominant primary producers of organic
matter in about one third of the World Ocean, the
so-called oligotrophic part, and are believed to be
the main consumers of the pool of dissolved organic
matter in all parts of the World Ocean. Nevertheless,
our present knowledge about the rates of their actual
biogeochemical activities ^ in the present context,
* Corresponding author. Tel.: +44 (1703) 594425;
Fax: +44 (1703) 594269; E-mail: [email protected]
their role in the carbon cycle ^ remains imprecise
and limits our understanding of global biogeochemical processes. It is necessary to determine three main
parameters in order to evaluate the biogeochemical
role of prokaryotes in carbon cycling: their standing
stock and the rates of their production and mortality. Established methods exist to measure the standing stock and production of prokaryote populations,
and we used these in work related to this study, but
methods for determining the causes and extent of
bacterial mortality have been more controversial.
In plankton ecosystems two other groups of microorganisms ^ viruses and protists ^ are thought to be
the main agents responsible for bacterial mortality;
this study concentrates on the assessment of mortality of bacteria due to grazing by protists.
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 5 9 - 2
FEMSEC 945 20-8-98
86
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
Three principal approaches have been used to
study the grazing of protists on prokaryotes: (i)
monitoring the concentrations of both groups during
a period of grazing; (ii) experimental reduction of
the grazing pressure exerted by protists by di¡erential dilution, followed by monitoring the concentration of prokaryotes to compare their growth at different dilutions; (iii) tracing the fate of labelled
bacteria or bacteria-sized particles in a water sample.
All of these approaches have their advantages and
disadvantages, which were comprehensively reviewed
in the recent past (e.g. [1^3]). The ¢rst approach
provides information about the strength of the
trophic link, and has been extensively explored using
laboratory and ¢eld experiments and mathematical
models since the pioneering work of Gause [4], but
cannot generally be applied to ¢eld samples. The
second approach, exempli¢ed in the dilution experiments of Landry and Hassett [5], proved to be fruitful when the grazing pressure on phototrophic organisms was studied, but is of more limited
applicability in studies of grazing on heterotrophic
bacteria [3]. Lately the third approach has been
most often employed in ¢eld studies of bacterivory
and recognised as an indispensable tool to study
trophic links.
Techniques for labelling bacteria can be divided
into two classes, depending on the kind of label
used: £uorescent or isotopic. The £uorescently labelled bacteria (FLB) technique [6] is now accepted
as a most important method for estimating bacterivory. It is based on microscopical or £ow cytometric
examination of samples after incubation with FLB
added in a known proportion to the naturally occurring bacteria, and either the number of FLB ingested
by protists or the decrease of FLB in the water can
be counted. The major attractions of the microscopic
version of this method are its conceptual simplicity
and the direct observation of bacterial consumption
by identi¢able species of protists. However, this
method is laborious and in order to obtain reliable
quantitative results a signi¢cant number of ingestion
events needs to be counted. Therefore it is not practicable to estimate bacterivory accurately in oceanic
waters with low abundances of protists using this
method [2]. This led us to look for an alternative
approach to estimate bacterivory at sea.
The employment of radioactively labelled bacteria
to study bacterivory has attracted considerable interest almost since radioactive isotopes were ¢rst introduced in hydrobiology ([7], and references therein).
The loading of bacteria with a radioactively labelled
precursor never presented serious di¤culties, but
variation in retention of the label by the living, metabolically active, bacterial cells has been a problem.
The rapid killing of bacteria by heating or freezing at
the end of the labelling period was proposed to resolve this problem [8^10]. However, in solving one
problem, another emerged, since feeding selection by
protists between living and dead or surrogate prey
was observed [11^15].
An alternative, less radical, way to preserve the
level of a label in bacterial cells is by means of
pulse-chase labelling [7,16]. In principle the method
involves short-term (pulse) incubation of bacterial
cells with an actively incorporated precursor of
high speci¢c radioactivity and subsequent addition
of a large excess of non-labelled precursor. As a
result the speci¢c radioactivity of the precursor is
drastically reduced and uptake of the radioactive label then falls to a negligibly low level. The result is a
quantitative marking of living bacterial cells with a
radioactive label and pulse-chase-labelled bacteria retain all the features of unlabelled bacteria. If a precursor is speci¢cally incorporated in a particular
pool of macromolecules of bacterial cells, the fate
of these macromolecules in protist predators can be
traced. For example, when Vibrio natriegens bacteria, simultaneously pulse-chase-labelled with 3 H-thymidine and 14 C-leucine (incorporated preferentially
in DNA and proteins, respectively [16]), were grazed
by protists, the latter retained less than 10% of the
3
H label and 20^80% of the 14 C label [17,18]. Ciliates
and £agellates digested these dual pulse-chase-labelled bacteria similarly. Perhaps the low assimilation of the 3 H label is a result of the limited metabolic usefulness of 3 H-thymidine-labelled bacterial
macromolecules compared with 14 C-leucine-labelled
macromolecules.
One experimental bene¢t of the di¡erential digestion of bacterial macromolecules is that it is possible
to estimate grazing and assimilation rates of protists
without separating prey from predator, which was
necessary when a single label was used [9,19]. The
decrease in the amount of 3 H label in the presence of
protists compared with the change in amount of la-
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
bel in the absence of protists can be used as an estimate of grazing, assuming that the assimilation of
3
H is negligibly small. The di¡erence between the
decrease of 3 H and 14 C labels can give an estimate
of the assimilation of 14 C (bacterial protein) by protists [17,18] under the same assumption. Even if the
assimilation of 3 H is not so low as to be negligible,
the di¡erence between 14 C and 3 H assimilation can
still remain an index of prey assimilation e¤ciency.
Therefore the application of the dual radioactive labelled bacteria (DRLB) method to grazing permits
the e¤ciency of digestion of bacterial prey (i.e. the
gross growth e¤ciency (GGE) of bacterivorous protists) to be estimated directly at the time of grazing,
in comparison with the FLB method which can only
estimate length of time required for digestion of bacteria [20].
Methods using bacteria labelled with a single radioactive isotope to estimate bacterivory have involved substantial assumptions. For example, some
studies assumed that during the time of the grazing
experiment there is no respiratory metabolism of labelled molecules from the consumed prey (100% assimilation e¤ciency [9]) or, on the contrary, others
assumed that there is no assimilation of molecules
from prey biomass by protists (0% assimilation e¤ciency, e.g. following ingestion of minicells [10]).
Dual labelling allows these assumptions to be tested.
There are comparable conceptual problems when
the FLB method is used. The concentration of added
£uorescently labelled bacteria should be chosen so
that the food vacuoles of bacterivorous protists will
mostly contain only one FLB, because, when several
£uorescent bacteria are present in a single shrunk
vacuole, they cannot be enumerated accurately. The
proportion of added FLB should be relatively small
in order to minimise the increase in available prey
and avoid an arti¢cial enhancement of predation.
When protists, such as ciliates, which engulf several
bacteria in a single food vacuole, are the subject of
grazing studies, the ratio between FLB and natural
bacteria can indeed be kept low, about 1:10. But, in
studies of grazing by £agellates, where food vacuoles
contain single bacteria, and grazing events are less
common, the ratio can be (and usually is) increased
to provide adequate counts of ingested bacteria; then
the number of added FLB may become comparable
to the number of natural bacteria. Another draw-
87
back is that labelling bacteria with a £uorescent
dye usually involves killing the cells.
Our ¢rst experience with using the dual radioactive labelling method employed laboratory cultured
bacteria (Vibrio natriegens) and cultured protists [17].
Attempts to use radiolabelled Vibrio to study bacterivory in ¢eld samples showed that cultured bacteria
can be successfully used to estimate bacterivory only
in relatively rich, eutrophic waters [18]. An obvious
limitation was that cultured bacteria are larger than
natural bacterioplankton cells, and therefore sizebased grazing selection could distort the results [7].
A second problem was that cultured bacteria experienced a drastic down-shift in nutrients when inoculated into oceanic water, and, while they were adjusting their metabolism in a new environment, they
released considerable amounts of labels. However,
we found that the loss of labels was remarkably similar in parallel replicates, and this particular reproducibility encouraged us to attempt pulse-chase labelling of natural bacterioplankton.
Thus the aim of the present study was two-fold: to
develop a procedure of quantitative dual pulse-chase
labelling of natural bacteria, preferably without
physical manipulation of the original sample, and
to use these living natural, but labelled, bacteria to
estimate rates of bacterivory and assimilation of bacterial biomass by natural protist communities.
The design of the experiment used established
methods where possible. For pulse-chase labelling
of natural heterotrophic bacteria a sample of the
original sea water was inoculated with radioactive
precursors and incubated for 4 h in just the same
manner as when uptake rates of heterotrophic bacteria are measured to estimate production. But then,
instead of ¢xing samples at the end of incubation, an
excess (500^1000 times) of non-radioactive precursors was added and the incubation continued for
another hour to stabilise the levels of labels in bacterial cells, in the same way as during pulse labelling
of a bacterial culture (e.g. [17]). There are possibilities that other groups apart from bacteria could take
up labels directly. For example, eukaryotic algae
[21,22] and Synechococcus [23] are able to incorporate organic molecules to a certain extent, but generally the uptake by phytoplankton is slow and does
not obscure the uptake by heterotrophic bacteria
[24]. Most free living heterotrophic £agellates and
FEMSEC 945 20-8-98
88
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
ciliates cannot survive saprotrophically [25,26] and
direct protozoan incorporation of precursors is negligibly small [9,17]. Therefore the bulk of incorporated pulse-chase labels should be found in heterotrophic bacteria. A more important interference with
predictable precursor uptake by heterotrophic bacteria can result from protozoan grazing in the natural
sample during pulse-chase labelling; this was accounted for in designing the protocol of bacterivory
experiments.
There are two major potential problems with
pulse-chase labelling of heterotrophic bacteria.
Firstly, the bacteria can respire part of a pulse during incubation; this can be accounted for by monitoring bacterial metabolism in control incubations.
Secondly, the excessive surplus of non-radioactive
precursors can stimulate arti¢cial bacterial growth.
However, it was shown that natural bacteria respond
to an addition of various organic substrates and increased precursor uptake rates more by growth in
size than by increasing cell numbers, within the ¢rst
24 h of incubation [27^29]. Therefore we designed
experiments to be completed within a 24-h period,
used precursors with high speci¢c radioactivity so as
to decrease the amount of unlabelled excess to the
minimum required, and subsequently diluted the
pulse-chase bacterial inoculum by mixing one part
with four parts of the studied water sample. The
successive up- and down-shifts tend to stabilise the
label [17], but in any case controls were always used
to monitor the radioactivity of ungrazed labelled
bacteria.
When a pulse of labels is stabilised in bacterial
cells, they, similar to bacteria labelled in other
ways, can be used as probes in bacterivory experiments. The grazing can be measured either as the
metabolic release (resulting from predators' respiration) of labels from the total particulate fraction of a
sample or as the assimilation of labels within protists. The ¢rst approach can be used when the grazing rate is high, because release must be su¤cient to
be registered as a reduction in radioactivity of the
sample particulate fraction. Such metabolic release
of labels has been observed when bacterivory is
high, but generally we were unable to detect metabolic release in oceanic samples. The second approach is more sensitive because an increase in radioactivity from zero is measured instead of a decrease
from a high level, but some method of separation of
predators from their prey is required. If the size of
predators permits they can be picked out manually
[17,19] or fractionated from bacteria using a `coarse'
¢lter [9,30]. In the present study we combined both
the metabolic release and assimilation approaches.
There are opposing views on whether ¢xation
should precede or follow size fractionation. On one
side ¢xation enables incubation to be stopped instantly and preserves the cells, and therefore prevents
cell disruption during fractionation [9]. On the other
side protists can release food vacuole contents when
¢xed [31], change their shape and surface properties,
become sticky, and can then behave unpredictably
during size fractionation. We decided to size-fractionate un¢xed samples, following the protocol
adopted for size fractionation of phytoplankton
(e.g. [32]), keeping in mind that we may have some
inevitable losses. Therefore we decided to use two
types of `coarse' ¢lters in parallel, traditional polycarbonate ¢lters of 0.8 or 1.2 Wm pore size and
`coarse' glass ¢bre ¢lters (GF/A). The latter should
retain fragile cells better in the three-dimensional
mesh of glass ¢bres [33]. In order to estimate the
extent of bacterivory the measured radioactivity retained on a coarse ¢lter can be compared with the
total radioactivity of the particulate fraction of the
sample retained on a 0.2-Wm polycarbonate ¢lter. If
samples are not ¢xed for size fractionation purposes,
an additional preparatory study is needed to estimate
the size of the pool of labelled small molecules in
bacterial cells. This can be done by comparing un¢xed samples retained on 0.2-Wm polycarbonate ¢lters with similar samples ¢xed with 5% trichloroacetic acid (which precipitates macromolecules, but
solubilises small molecules) before retention on ¢lters
of the same type.
If labelled cells are to be reliable in quantitative
studies, it is important that the cells be homogeneously labelled. One way to check the homogeneity of
pulse-chase labelling of heterotrophic bacteria of different size is to size fractionate them through a series
of polycarbonate ¢lters, of say 0.4, 0.6, 0.8, and 1.2
Wm pore size. The radioactivity retained on these
¢lters can be compared with the radioactivity retained on a 0.2-Wm polycarbonate ¢lter, assuming
that the latter ¢lter retained most of the bacterial
cells. These results can also be compared with direct
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
counts of heterotrophic bacteria in ¢ltrates passed
through the same series of ¢lters. Counts in the original un¢ltered water sample can be used as a reference.
A basic measure of community bacterivory can be
obtained from the ratio between the radioactivity
retained on a coarse ¢lter, which is enriched with
predators, and that counted on a ¢ne ¢lter, which
retains most particles. However, this is an idealised
situation, which can only be approached if a considerable number of `controls' are performed. These
controls must be accommodated in the experimental
protocol. A coarse ¢lter will unavoidably retain not
only predators but also some labelled bacterial cells,
which will increase the apparent bacterivory measured. Because bacteria may grow in size during incubation (see above) a control fractionation at the
beginning and at the end of incubation is insu¤cient
to make allowance for these retained bacteria. Hence
it is necessary to include another form of control by
making measurements of di¡erent levels of predation
on the same stock of bacteria. This can be provided
if the labelled inoculum is mixed either with intact
original water or with the same water that has been
¢ltered through a 0.8- or 1.2-Wm polycarbonate ¢lter.
This ¢ltrate should contain most of the individual
bacteria but there would be almost no predators or
other particles of larger size. The former mixture
would represent 100% predator pressure and the latter a considerably lower predator pressure (e.g. 20%,
if the labelled inoculum is mixed with 4 times its
volume of ¢ltrate). Because the inoculum and bacterial content in both mixtures is identical the di¡erence between the radioactivity of the two mixtures
retained on coarse ¢lters should correspond to the
di¡erence in bacterivory pressure, ful¢lling the aim
of the experiment.
One more control is required to allow for `background' changes in bacterial labels which are consequences of the methodology. Firstly, the microorganisms and their environment in the ¢ltrate (0.8
or 1.2 Wm) used as a diluent are not exactly the
same as in the unlabelled sample. The level of organic matter, and hence bacterial metabolism, in the
¢ltrate may be reduced by removal of nanoplankton
which normally continually release organic matter,
or may be increased by some breakage of nanoplankton cells during ¢ltration. A second conse-
89
quence is that the surplus of non-radioactive labelled
precursors does not completely abolish the uptake of
labelled molecules during incubation, especially uptake by the excess of unlabelled bacteria in the mixtures. This additional control must monitor the extent of background labelling at the speci¢c
radioactivity levels present in the mixtures during
the ¢nal incubation period.
The outcome of the described technique is intended to be an estimate of community bacterivory
of natural heterotrophic nanoplankton on natural
heterotrophic bacteria at the original concentrations
of both groups. To our knowledge there are no existing methods which allow this to be done directly,
since alternative methods are indirect (e.g. [19]) and
based on the three compartment model of Daro [34].
The amounts of radioactivity retained on coarse ¢lters will be a measure of grazing rate only if the
gross growth e¤ciency of predators is 100%, which
is most unlikely. In reality this radioactivity indicates
the amount of bacterial biomass accumulated or assimilated by protists. By simultaneously using two
radioactively labelled precursors which are handled
di¡erently by predators, it is possible to estimate the
grazing and assimilation of two di¡erent pools of
bacterial macromolecules preferentially labelled
with these precursors.
2. Methods
2.1. Sampling and determination of the concentration
of natural bacteria
After development on laboratory cultures, ¢eld
studies were performed to test the technique using
natural bacteria on cruises 15 and 21 of the R.R.S.
James Clark Ross along the Atlantic Meridional
Transect between the British Isles and the Falkland
Islands between 22 September and 25 October, 1996
and between 22 April and 26 May, 1997, respectively. Seawater was collected from 3 to 5 depths
through the upper mixed water layer by CTD casts
with a cassette of Niskin bottles. Water samples representing all main regions traversed by both cruises
were collected at 9 stations on the ¢rst cruise and at
18 stations on the second cruise. Subsamples of
equal volumes, of between 200 and 500 ml, from
FEMSEC 945 20-8-98
90
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
these sampled depths were pooled together to obtain
a combined sample which had average concentrations of organisms representative of the surface
mixed layer. In the description of bacterivory experiments we shall refer to these pooled samples as `original' samples.
The concentrations and size of heterotrophic bacteria and cyanobacteria were measured by £ow cytometry and di¡erential ¢ltration, as described in
more detail elsewhere [35]. Brie£y, 3-ml samples
were ¢xed with 0.2% glutaraldehyde and kept below
330³C before being analysed. The total concentration of bacteria was measured in samples stained
with the £uorochromes TOTO-I iodide or SYBR
Green I, which have strong binding a¤nities to double-stranded DNA [36,37].
2.2. Grazing experiments
In all experiments seawater from the original sample was gravity ¢ltered through a 0.8-Wm polycarbonate ¢lter (replaced by a 1.2-Wm ¢lter in temperate
waters at the two ends of the transect) and was used
to dilute the original sample and reduce the concentration of bacterivorous predators without signi¢cantly changing the heterotrophic bacterial concentration. Experiments, described in detail below, were
based on pulse-chase labelling of metabolically active, natural heterotrophic bacteria, which were
able to incorporate small organic molecules, and
comparing bacterial fate during a period of incubation in bottles containing 100% original sample
(100% Grazers) with that in bottles containing 20%
original sample plus 80% of 0.8- or 1.2-Wm ¢ltered
seawater (20% Grazers), which reduced the predator
pressure to 20%. After a selected period of incubation, un¢xed samples from 100% Gr and 20% Gr bottles
were ¢ltered onto `coarse' 0.8- or 1.2-Wm polycarbonate and GF/A ¢lters, retaining the protists but
passing most of the bacteria, and `¢ne' 0.2-Wm polycarbonate ¢lters, retaining both bacteria and protists. Di¡erences between the 100% Gr and 20% Gr bottles in the retention of each of the labels on ¢lters
were interpreted to indicate the extent of assimilation
of radioactive labelled compounds from bacteria by
protozoa.
2.3. Experimental protocol (see Fig. 1)
2.3.1. Pulse-chase labelling
I: Pulse, 13 Wl of 22.7 WM 3 H-thymidine (44 Ci/
mmol, Amersham), ¢nal concentration 2 nM, and 21
Wl of 286 WM 14 C-leucine (350 Ci mol31 , DuPont),
¢nal concentration 40 nM, were inoculated into 150
ml of newly collected original sample and this subsample was incubated for 4 h.
II: Chase, 150 Wl of 2 mM non-labelled thymidine,
¢nal concentration 2 WM, and 150 Wl of 20 mM nonlabelled leucine, ¢nal concentration 20 WM, were
subsequently inoculated into the same subsample
and it was incubated for one more hour to stabilise
radioactive label pulses.
2.3.2. Background labelling (parallel with pulse-chase
labelling)
I: `Pulse', 10 Wl of 20 WM non-labelled thymidine,
¢nal concentration 2 nM, and 20 Wl of 200 WM nonlabelled leucine, ¢nal concentration 40 nM, were inoculated into 100 ml of original seawater and this
subsample was incubated for 4 h.
II: `Chase', 100 Wl of 2 mM non-labelled thymidine plus 8.8 Wl of 22.7 WM 3 H-thymidine (44 Ci
mmol31 , Amersham), total ¢nal concentration
2.002 WM, and 100 Wl of 20 mM non-labelled leucine
plus 14 Wl of 286 WM 14 C-leucine (350 Ci mol31 ,
DuPont), total ¢nal concentration 20.04 WM, were
subsequently inoculated into the same subsample
and it was incubated for one more hour.
2.3.3. Bacterivory experiment (III)
In preparation, 0.5 l of the same original sample
were gravity ¢ltered through a 0.8-Wm (or 1.2-Wm in
mesotrophic waters) polycarbonate ¢lter. 100-ml
subsamples of the original sample (4 bottles) and
gravity ¢ltered sample (4 bottles) were dispensed
into 250-ml polycarbonate bottles.
At the end of the chase incubations 25 ml of either
pulse-chased or background-labelled subsamples
were dispensed into replicated bottles with
unscreened and screened seawater and the mixtures
were incubated for 13 h. All incubations were done
in the dark at the water temperature of the ambient
surface mixed layer.
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
2.3.4. Filtration (IV)
At the end of incubation 25-ml subsamples from
each bottle were ¢ltered onto a `¢ne' 0.2-Wm polycarbonate ¢lter, and 50-ml subsamples onto a
`coarse' 0.8-Wm (or 1.2-Wm) polycarbonate ¢lter
(at all 27 stations). In addition, 50-ml subsamples
were collected on GF/A ¢lters at 15 stations on the
second cruise and on both 0.8- and 1.2-Wm polycarbonate ¢lters in parallel at 3 stations on the second
cruise.
The full protocol (Fig. 1) was executed with 13 h
incubation at 27 stations, and additional subsamples
were taken at 4 h at four of the stations. At 7 stations on the ¢rst cruise 25-ml un¢xed subsamples
were ¢ltered through 0.4-Wm and 0.6-Wm polycarbonate ¢lters at the end of the bacterivory experiment to estimate the homogeneity of labelling of
heterotrophic bacteria of di¡erent size fractions. To
estimate the size of any pool of labelled small molecules in bacterial cells, supplementary 25-ml subsamples were ¢xed at 9 stations on the ¢rst cruise
by mixing with equal volumes of 10% w/v trichloroacetic acid (TCA) and subsequently ¢ltered onto 0.2Wm polycarbonate ¢lters. All ¢lters were washed with
equal volumes of 0.2-Wm ¢ltered seawater.
2.3.5. Radioassay (V)
The ¢lters with retained material were put into
pony scintillation vials and dried overnight at
60³C. The vials were then ¢lled with 5 ml of scintillation cocktail (Optiphase, Wallac) and several days
were allowed to solubilize material. Radioactivity
was measured with a Pharmacia (Turku, Finland)
RackBeta 12809 liquid scintillation counter, using
the installed three-over-two counting method for effective dual-label counting.
All plastic- and glass-ware used for handling and
incubation of seawater samples was given a preliminary wash with 1 N HCl and rinsed with 0.2-Wm
¢ltered seawater from a depth of 200 m. The grazing
experiments were conducted in 250-ml polycarbonate
bottles (Nalgene). All non-radioactive chemicals
were purchased from Sigma.
2.4. Calculations and statistical analysis
F-tests were used to compare the variances of sample sets based on comparable measurements, Bart-
91
lett's test was used to check for homogeneity of variance, a t-test was used to compare the means, and
linear regression was employed to compare data sets.
All measurements were at least duplicated, and all
statistical tests were signi¢cant at levels above the
95% con¢dence limit, so that individual P values
are not given. The numbers are presented as
means þ S.D., or as regression slopes þ S.E.
Calculations were performed in the following sequence: (i) All values of pulse-chase labelling were
corrected for corresponding background labelling
(Fig. 1)
x
vxxL ˆ …x PxxL3x BxxL†=V;
where v = net radioactivity of a pulse (dpm per sample); P = measured radioactivity (dpm per sample) of
a sample with pulse-chase labelled bacteria; B =
measured radioactivity (dpm) of a sample with background labelled bacteria; x = percentage of grazers,
either 20% or 100%; xx = ¢lter quality, coarse (cf)
or ¢ne (¡); L = type of label, either 3 H or 14 C;
V = sample volume, ml.
(ii) The daily assimilation of label by protists from
a standing stock of metabolically active heterotrophic bacteria (Assim.L) was calculated as a percentage from the di¡erence between the radioactivity
(corrected for background) retained on coarse ¢lters
from 100% and 20% grazed samples, divided by the
radioactivity of a 20% Gr sample retained on a ¢ne
¢lter
Assim:L ˆ CU…100% vcfL320% vcfL†=20% vffL;
where C = 100/(100320)U(24/13)/2U100%w115% is
a coe¤cient to correct for dilution, incubation time
and di¡erence of ¢ltered volumes; other symbols are
the same as before.
(iii) Metabolic release, as a result of respiration of
bacterial biomass (Resp.3 H), was calculated as a percentage from the di¡erence between the radioactivity
retained on ¢ne ¢lters from 20% Gr and 100% Gr divided by the radioactivity of the 20% Gr ¢ne ¢lter
Resp:3 H ˆ 2UCU…20% vff 3 H3100% vff 3 H†=20% vff 3 H:
Respiration of 14 C label by protozoa was undetectable.
(iv) Consumption (Cons.3 H) was calculated as the
FEMSEC 945 20-8-98
92
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
Fig. 1. Flow chart to describe the method of pulse-chase labelling of heterotrophic bacteria in natural seawater samples with 3 H-thymidine and 14 C-leucine and the subsequent assessment of bacterivory (see text for details).
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
sum of assimilation and metabolic release
Cons:3 H ˆ Assim:3 H ‡ Resp:3 H:
(v) Assimilation e¤ciency of labels (AE) was calculated for 3 H or 14 C as the ratio between assimilation of a label and consumption
AE ˆ Assim:L=Cons:3 H:
All bacterivory results were converted into percentages of the standing stock of heterotrophic bacteria
processed per day by the full standing stock of protists. If the di¡erence between 100% Gr and 20% Gr was
less than the average S.D. of measured radioactivity
retained on the corresponding ¢lters, either assimilation or metabolic release was considered to be below
the sensitivity of the method, and sum average values
were used in calculations.
93
(Fig. 3). In this comparison the slopes were close to
unity; for the 3 H label it was 0.971 þ 0.013, and for
the 14 C label it was 1.032 þ 0.013. Therefore, only
about 3% of 3 H-labelled molecules became mobile
upon ¢xation and about 3% more 14 C-labelled molecules were precipitated by 5% TCA than were retained in un¢xed cells. Since the bulk of the pulsed
radioactive labels retained by bacteria were consistently incorporated in TCA-insoluble macromolecules, comparisons between labels measured in the
un¢xed samples can reasonably be used to assess
bacterivory. This result permits the use of fractionation of living samples which is preferred to the use of
¢xed samples, since ¢xed cells usually change shape
and surface properties, e.g. become arti¢cially adhesive.
One could reasonably expect that when a laboratory culture of a particular species of bacteria is
3. Results
The retention of labels by heterotrophic bacteria
prepared by incubation with radiolabelled thymidine
and leucine, as described in Fig. 1, was routinely
followed over a period of 13 h in all grazing experiments; mean values from the monitoring of these
labels in all 18 experiments of the second cruise are
shown in Fig. 2. The increase of total radioactivity of
samples was due to the `background' incorporation
of labels referred to earlier, and showed the importance of measuring this background uptake of labels.
When background incorporation was subtracted
from total radioactivity, no metabolic release of 3 H
label was found and about 17% of 14 C label was
metabolised by pulse-chase labelled bacteria during
a 13-h incubation. The metabolic release of 14 C label
showed ¢rstly that labelled bacteria were alive and
metabolically active, and secondly it con¢rmed the
necessity (as discussed in Section 1) of running experimental and control series (100% Gr and 20% Gr) in
parallel to account for this microbial process, rather
than simply comparing the initial and ¢nal radioactivity of pulse-chase labelling.
Another preparatory study was a comparison of
the amounts of incorporated labels by heterotrophic
bacteria in un¢xed samples and TCA-¢xed samples
Fig. 2. Radioactivity retained during incubation for up to 13 h
by natural bacteria from surface mixed layer samples, pulse-chase
dual labelled with 3 H-thymidine and 14 C-leucine and inoculated
in a 1:4 ratio into the same surface seawater that had been
screened through 0.8- or 1.2-Wm polycarbonate ¢lters. Values are
plotted as percentages of the radioactivity retained by the bacteria after the ¢rst hour of incubation, and are means of 18 sets of
duplicated samples. Error bars at 4 and 13 h show corresponding
coe¤cients of variation. In the case of surface samples at the ¢rst
hour (100%) error bars show average coe¤cients of variation of
replicates. The blank part of the bars shows the extent of the
correction required due to background labelled bacteria (see text
for details).
FEMSEC 945 20-8-98
94
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
Fig. 3. Comparison of radioactivity (3 H in (a) and 14 C in (b)) of un¢xed 25-ml samples retained on 0.2-Wm polycarbonate ¢lters with
identical samples ¢xed in 5% TCA before ¢ltration (dotted lines show 99% con¢dence intervals).
dual-labelled, each cell will incorporate similar proportions of the two labels. The same could not necessarily be expected when labelling bacteria of a natural community, since these will consist of many
species, with cells in di¡erent physiological states.
The size-related uniformity of labelling of bacteria
from the natural communities used in these experiments was checked by size fractionation through ¢lters of di¡erent pore size and compared with direct
counts of the fractionated heterotrophic bacteria
(Fig. 4). The proportion of labels which were retained on ¢lters of di¡erent pore sizes agreed reasonably well with the proportions of heterotrophic bacteria retained on ¢lters of the same pore sizes, as
measured by £ow cytometry. Some small bacteria
that were not retained by 0.4-Wm ¢lters may not incorporate labels. The amounts of 3 H label found in
the di¡erent fractions closely followed the bacterial
counts in these fractions. In each size fraction, higher
counts of 14 C label than of 3 H label or of bacterial
cells were retained on the ¢lters, suggesting that the
larger cells, which were presumably more metabolically active, had incorporated more 14 C-leucine.
The aim of bacterivory experiments was to measure the assimilation of labelled bacterial prey by pro-
Fig. 4. Results of fractionation of heterotrophic bacteria on polycarbonate ¢lters of di¡erent pore size. Bacteria were counted in
¢ltrates by £ow cytometry and ¢lters were radio assayed for heterotrophic bacteria pulse-chase-labelled with 3 H-thymidine and
14
C-leucine. The values are either percentages of numbers of bacteria retained on ¢lters compared with original un¢ltered samples
or percentages of radioactivity retained on ¢lters compared with
radioactivity retained on 0.2-Wm polycarbonate ¢lters. Percentages are means of 7^30 replicates, error bars show corresponding
coe¤cients of variation.
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
95
Fig. 5. Two examples described in the text to illustrate the experimental data used in estimation of bacterivory on dual radioactive labelled natural heterotrophic bacteria. a: The retention of labels on coarse and ¢ne ¢lters after 13 h incubation. b: The retention of radioactivity as 3 H and 14 C from 100% Gr and 20% Gr bottles on 0.8-Wm ¢lters after 4 and 13 h incubation. The columns in (a) show the average
radioactivity of 3 H (a1 ) and 14 C (a2 ) labels of 50-ml duplicated subsamples collected on either 1.2-Wm polycarbonate ¢lters (left), glass ¢bre ¢lters (centre), or 0.2-Wm polycarbonate ¢lters (right). The left column in each pair shows counts from 100% Gr bottles and the right
column of each pair shows counts from 20% Gr bottles. Upward columns show the net radioactivity of a pulse (x vxxL), downward columns show radioactivity of background labelling (x BxxL) and the whole height of a column is the total radioactivity of pulse-chase labelling (x PxxL). Error bars show S.D.
tozoa, size-fractionated from the bulk of bacteria on
coarse, 0.8- or 1.2-Wm polycarbonate-membrane or
GF/A ¢lters. These polycarbonate membranes retained only a small fraction of the bacteria (Fig. 4).
The di¡erence of retention on these ¢lters for 100% Gr
and 20% Gr bottles was assumed to represent the assimilation of radiolabels by protozoa. Examples of
original data sets for experiments conducted following the protocol outlined in Fig. 1 are presented in
Fig. 5a, showing how data for pulse-chase labelled
FEMSEC 945 20-8-98
96
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
Fig. 6. The results of bacterivory experiments on natural heterotrophic bacteria pulse-chase labelled with 3 H-thymidine and 14 C-leucine
(left and right columns, respectively ; 13 h incubation, 50-ml samples, data corrected for background). In (a) the retention of radioactivity
on coarse 0.8- or 1.2-Wm polycarbonate or GF/A glass ¢bre ¢lters of subsamples from 100% Gr bottles is plotted against the corresponding
set derived from 20% Gr bottles ; (b) shows the equivalent comparison of retention on ¢ne 0.2-Wm polycarbonate ¢lters. Error bars show
S.D., solid lines show linear regressions and dotted lines show 99% con¢dence intervals.
and background-labelled samples were combined.
Comparisons of the retention of labels on 0.8-Wm
polycarbonate ¢lters after 4 and 13 h incubation
are presented in Fig. 5b to show time changes of
label retention. The number of labelled bacteria is
initially the same in both 100% Gr and 20% Gr bottles,
but the number of protozoa and algae bigger than
about 1 Wm which might interfere with the passage
of bacteria through the ¢lter is ¢ve times as high in
100%
Gr bottles as in 20% Gr bottles, and is the same
after 13 h as after 4 h. The similarity of radioactive
counts in the two bottles after 4 h, and the increased
di¡erences between them by 13 h, support the assumption that when low concentrations of protozoa
and algae are present in oceanic waters, these larger
cells do not physically interfere with the passage of
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
97
Fig. 7. Rates of daily assimilation of bacterial biomass by bacterivores (upward arrows) as percentages of standing stocks of metabolically
active bacteria in the top mixed layer at stations along the Atlantic Meridional Transect, estimated by retention of 3 H label (a), and by
retention of 14 C label (b). The dotted lines show the corresponding mean values.
bacteria through coarse ¢lters. Clearly, the larger
numbers of grazers present in 100% Gr bottles accumulated some additional labels of both types.
The results of all experiments where bacterivory
was estimated (including duplicate experiments at
some stations) are summarised in Fig. 6, where
data for the retention of labels for each 100% Gr set
has been plotted against the corresponding 20% Gr
set, after correction for background labelling. The
retention of labels on coarse ¢lters, either 0.8- or
1.2-Wm polycarbonate ¢lters or GF/A ¢lters, was
generally higher in subsamples from 100% Gr bottles
compared to 20% Gr bottles for both the 3 H label and
the 14 C label (Fig. 6a, slope 0.978 þ 0.02 and slope
0.905 þ 0.018, respectively). On average the retention
of radioactivity on 0.2-Wm ¢lters from 100% Gr bottles
and 20% Gr bottles was nearly the same for both the
3
H label (Fig. 6b1 : slope 0.997 þ 0.016) and the 14 C
label (Fig. 6b2 : slope 1.008 þ 0.016); therefore the
metabolic release of labels upon grazing was typically below the accuracy of measurements and normally could not be detected. However, in three ex-
periments 6^13% (20 þ 8% per day) lower
radioactivity of 3 H was retained on 0.2-Wm ¢lters
for subsamples from 100% Gr bottles compared to
20%
Gr bottles (e.g. Fig. 5a1 ); this di¡erence was interpreted as the result of metabolic release of labels
by protozoa in these cases. The overall reduction in
bacterial label (i.e. the extent of grazing) can be estimated as the sum of the assimilation and the metabolic release of labels during grazing. On average the
total bacterivory estimated in this way in these three
cases was 24 þ 9% of the bacterial standing stock per
day. Although the labelling of natural bacteria is not
quite uniform, it is possible in these cases to make an
overall estimate of the assimilation e¤ciency of labelled bacterial biomass by bacterivores, given by the
percentage of the label retained on large pore size
¢lters divided by an estimate of the total bacterivory.
The mean assimilation e¤ciency in these three cases
for pooled data of two types of ¢lter was 16 þ 13%
for the 3 H label and 58 þ 29% for the 14 C label
(n = 6).
Di¡erences in radioactivity retained on coarse ¢l-
FEMSEC 945 20-8-98
98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
ters were undetectable on the second cruise at two
stations for the 3 H label and at ¢ve stations for the
14
C label. This disparity is probably a result of lower
speci¢c radioactivity of 14 C-leucine compared with
3
H-thymidine, which ultimately reduced the sensitivity of 14 C measurements. The assimilation of each
label within bacterivorous protists was calculated
for all of the other stations; the summarised results
are presented in Fig. 7. These data illustrate the spatial variability of assimilation rate as probably the
main feature of a tightly coupled trophic link;
more detailed analysis of these data is not appropriate in this paper and will be presented elsewhere in
conjunction with results on standing stocks and production of heterotrophic bacteria and biomass of
heterotrophic nanoplankton. The average assimilations were slightly, but not signi¢cantly, higher on
the ¢rst cruise than on the second cruise. The mean
daily assimilation for both cruises was 4.8 þ 2.7%
(n = 25) of the standing stock of metabolically active
bacteria for the 3 H label and 10.1 þ 5.3% (n = 22) for
the 14 C label.
The sensitivity of the DRLB method at the present
stage allows determination of assimilations as low as
0.5^1% of total bacterial stock after 13 h incubation.
Sensitivity can be increased further by reducing variance through the use of larger sample volumes and
larger numbers of replicates.
4. Discussion
The approach employed here to study grazing was
derived from the technique developed for dual radioactive labelling of cultured bacteria [17,18]. Its design
followed the framework of the dilution technique
[5,38] and the labelling approach of Hollibaugh et
al. [7]. Radioactive labelling of bacteria was chosen
as a more sensitive way of tracing bacterial fate than
£uorescent labelling or enumeration of bacterial concentration. The latter is still not sensitive enough
even when counting was done by £ow cytometry
(our unpublished data). The major di¡erence from
the other methods used to radiolabel bacteria, e.g.
[7^9,39], was to employ pulse-chase labelling of natural bacteria of the original sample for a short time
and to avoid any preliminary or supplementary ¢ltrations, washing or killing of the labelled bacteria.
There was minimal handling of the original sample
(Fig. 1). The dual labelling increased con¢dence in
the results, and enabled us to trace the fate of two
pools of bacterial macromolecules, DNA and proteins, e.g. [16], which are digested di¡erently by cultured protozoa [17,40].
Size fractionation was used to separate the predators from the bulk of the prey as a way of improving
the sensitivity of bacterivory measurements. The ¢lters used to collect the predators certainly also retained some of the uneaten pulse-labelled bacteria.
Since there were the same numbers of pulse-labelled
bacteria in both 100% Gr and 20% Gr incubations, similar numbers of labelled bacteria should be retained
on the respective coarse ¢lters, and subtraction of
20%
Gr from 100% Gr counts should eliminate that proportion of counts which was due to uneaten bacteria.
In fact, only a small proportion of the bacteria in the
incubated samples were retained on 0.8- or 1.2-Wm
¢lters (see Figs. 4 and 5), and only one ¢fth of these
would be pulse-labelled bacteria. However, the presence of additional particles of larger size in 100% Gr
compared with 20% Gr could hold back more bacteria
on the 100% Gr coarse ¢lter than on the 20% Gr coarse
¢lter and arti¢cially increase retention of radioactivity in the former case. In order to minimise this
interference with ¢ltration, the ¢ltration area was
increased so that only 5 ml of water was ¢ltered
through an area of 1 cm2 , and no, or very little,
di¡erence of retention between 100% Gr and 20% Gr
¢lters was detected after 4 h incubation (Fig. 5b).
In addition, three di¡erent ¢lters were used, with
two types of ¢lters being used in each experiment
on the second cruise, either polycarbonate ¢lters of
two di¡erent pore sizes or polycarbonate and glass
¢bre ¢lters. It is our experience that as much as 1 l of
oceanic water could be passed through a 1-cm2 GF/
A ¢lter without clogging. At the same time, the passage of whole cells of predators through the coarse
¢lters used here can be assumed to be negligible, e.g.
[18,24] and personal observations, although some
protists may be damaged or broken by ¢ltration
and their debris can be washed through the ¢lter.
It is more likely that such damage would occur on
the £at surface of a polycarbonate ¢lter than in the
mesh of a glass ¢bre ¢lter [33]. The drawback of
using glass ¢bre ¢lters is that they retain a larger
proportion of the labelled bacteria, which decreases
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
the sensitivity of the method. There are advantages
and disadvantages in the use of both types of ¢lter,
therefore both ¢lters were used in parallel to add
con¢dence to the combined results. Substantial numbers of predators, probably enriched with the more
robust protozoan cells, e.g. dino£agellates, were retained even on polycarbonate ¢lters, and results
from these can probably be interpreted as being at
the lower end of the range of assimilation of grazed
bacteria by predators.
There are several unavoidable problems associated
with the employment of size fractionation for separation of protozoan predators from their prey. It is
known from laboratory studies that bacteria can
£occulate in the presence of predators and clumping
reduces the e¤ciency of fractionation. Flocculation
is unlikely to be signi¢cant, however, in oceanic
waters with low abundances of microorganisms,
although it should be taken into consideration
when more productive, especially coastal, waters
with high concentrations of detritus are studied. Another problem is associated with the egestion of bacterial debris by protists. The size of these egested
particles is comparable with the size of bacteria,
and therefore they can be retained on ¢ne, 0.2-Wm,
¢lters and can increase estimates of bacterial standing stock. Because some of the bacterial biomass is
assimilated by predators, the radioactivity of egested
debris should be lower than the radioactivity of intact bacteria and we assume that the impact of debris
should be negligibly small. A third problem is associated with the release of radioactively labelled dissolved organic molecules by protozoan grazers, because these molecules could be scavenged by
heterotrophic bacteria. Considering all these listed
problems one has to keep in mind the scale of the
process being studied. When only a few percent of
the bacterial standing stock is consumed per day, the
amount of released debris and dissolved molecules
should be even smaller, and probably undetectable
by current methods. In addition, the uptake of released labelled dissolved molecules was most likely
sharply reduced by the pool of non-labelled precursors in the medium. The metabolic release of labels
by pulse-chase labelled bacteria was insigni¢cant for
3
H but was about 17% for the 14 C label, and was
closely monitored throughout the experiment.
The proposed method certainly cannot resolve all
99
problems, but made it possible to estimate bacterivory on natural populations of heterotrophic bacteria in oligotrophic ocean waters, with a sensitivity
which enabled daily assimilation rates of below 1%
of the standing stock of bacteria to be measured. The
estimated average daily assimilation of labels from
heterotrophic bacteria by protozoa in samples from
two cruises in oceanic waters of widely di¡erent
trophic status was about 5% and 10% of the bacterial standing stock, respectively, for the 3 H label and
the 14 C label. Natural protozoa retained more 14 C
than 3 H in the same way as cultured protozoa
have been shown to do [17,18]. However, the retention of 3 H was signi¢cantly higher than found for
cultured protozoa, which could be explained by more
e¤cient utilisation of prey biomass by natural heterotrophic protists. In some oligotrophic conditions
where bacterial and predator concentrations were
very low, bacterivory was undetectable during 13 h
experiments.
Generally there was no detectable metabolic release of labels from the particulate fraction by predators, at least at the resolution of the method applied
(coe¤cient of variation of these measurements 4^5%)
(e.g. Fig. 6b). However, in three cases of measurements made in mesotrophic temperate waters at the
ends of the transects, the daily release of 3 H label
was relatively high at 20 þ 8%, and this was interpreted as a result of metabolism of the ingested bacterial compounds by protozoa in these cases. The
mean assimilation e¤ciency in these cases was estimated as about 60% for the 14 C label and 16% for
the 3 H label. The assimilation e¤ciency of the 3 H
label seems to be actually higher in oligotrophic
waters than in mesotrophic waters, because, as mentioned above, the overall average assimilation of the
3
H label is about half of the assimilation of the 14 C
label.
Assuming that the protozoan assimilation e¤ciency of the 14 C label (protein) is more conservative
at 60%, and since the rate of assimilation for carbon
is 10% of the bacterial standing stock per day, the
average daily grazing rate of protozoa from the
pooled data set of two cruises can be estimated as
nearly 17% of the bacterial standing stock. This estimate of the grazing loss of bacteria is based on
those bacteria which can incorporate radioactive precursors and which therefore represent the actively
FEMSEC 945 20-8-98
100
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
growing fraction of the total bacterial community.
We have no evidence that less active bacteria are
grazed di¡erently, although some bacteria counted
either on ¢lters or by £ow cytometry can be dormant
or even dead [41,42]. Our average estimates of grazing rate are comparable with the grazing rates of 3^
12% of bacterial standing stock per day, reported for
coastal Antarctic waters [43], and they are very close
to the reviewed average grazing rate of 17%, obtained with the uptake of £uorescent particles and
epi£uorescence counting of bacteria [3]. At the
same time, our estimates are signi¢cantly lower
than the high average grazing rate of 76% per day,
obtained when rates were determined by dilution,
inhibition and ¢ltration techniques or by the uptake
of radioactively labelled particles, all of which required undesirably long (24^48 h) incubations [3].
Daily production of heterotrophic bacteria in mesotrophic and oligotrophic waters is about 12% of the
standing stock of total enumerated heterotrophic
(non-phototrophic) bacteria, assessed by uptake of
radioactively labelled amino acid, leucine (paper in
preparation) and is signi¢cantly lower than claimed
by the latter values of predatory pressure. According
to our estimates protozoa should assimilate about
90% of daily production of heterotrophic bacteria
and therefore their grazing rate could exceed estimated bacterial production. As mentioned above,
some of the enumerated bacteria can be metabolically inactive or even dead [41,42] and inevitably
the estimated relative rate of production of heterotrophic bacteria would be underestimated if production is divided by the standing stock of all enumerated bacteria.
There are many reports that bacterivorous protozoa can control bacterial concentration in eutrophic
and mesotrophic marine and freshwater communities
[41,43^47]. However, these reports are based on
methods using £uorescent particles which are very
time consuming and laborious and are not satisfactory for use in oligotrophic waters [2]. The present
method has su¤cient sensitivity for measuring bacterivory in oligotrophic waters, and produces results
close to those obtained by following the uptake of
£uorescently labelled bacteria. It does not require
microscopical examination of samples and allows estimation of predatory control over growing, metabolically active bacteria. In mesotrophic waters, it
is also possible to estimate the assimilation e¤ciency
of protozoa consuming bacterial biomass, an analog
of their gross growth e¤ciency, which was estimated
to be about 60% for bacterial macromolecules labelled with 14 C-leucine, probably proteins.
Bacterivorous protists tightly control the abundance of heterotrophic bacteria, being able to consume as much as these bacteria can produce, and it is
most probable that spatial variability (e.g. Fig. 7)
gives an indication of how the oceanic microbial
loop is equilibrated. It is likely that heterotrophic
bacteria represent only a part of the diet of bacterivorous protozoa. We have no reason to doubt that
autotrophic bacteria, Prochlorococcus spp. and Synechococcus spp., are also consumed by planktonic
protists, and consider it likely that the diet of protists
includes both heterotrophic and autotrophic bacteria
in the proportions in which they occur. Further studies are needed to reveal the whole extent of the role
of bacterivorous protozoa in oligotrophic oceanic
waters, which cover about 30% of the surface of
the Earth.
Acknowledgments
We are pleased to thank Anthony Bale for leading
the cruises of the R.R.S. James Clark Ross, and the
crews of the ship on these cruises for their support.
This research was supported by grants GR9/02569
and GST/02/1062 from the Natural Environment
Research Council, and is a component of Strategic
Research Project 1 of Plymouth Marine Laboratory.
It is PRIME contribution number 42 and Atlantic
Meridional Transect contribution number 09.
References
[1] Landry, M.R. (1994) Methods and controls for measuring the
grazing impact of planktonic protists. Mar. Microb. Food
Webs 8, 37^57.
[2] Sherr, E.B. and Sherr, B.F. (1994) Bacterivory and herbivory:
Key roles of phagotrophic protists in pelagic food webs. Microb. Ecol. 28, 223^235.
[3] Vaqueè, D., Gasol, J.M. and Marraseè, C. (1994) Grazing rates
on bacteria: the signi¢cance of methodology and ecological
factors. Mar. Ecol. Prog. Ser. 109, 263^274.
[4] Gause, G.F. (1935) Experimental demonstration of Volterra's
FEMSEC 945 20-8-98
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
periodic oscillations in the number of animals. J. Exp. Biol.
12, 44^48.
Landry, M.R. and Hassett, R.P. (1982) Estimating the grazing
impact of marine micro-zooplankton. Mar. Biol. 67, 283^
288.
Sherr, B.F., Sherr, E.B. and Fallon, R.D. (1987) Use of monodispersed, £uorescently labeled bacteria to estimate in situ
protozoan bacterivory. Appl. Environ. Microbiol. 53, 958^
965.
Hollibaugh, J.T., Fuhrman, J.A. and Azam, F. (1980) Radioactive labelling of natural assemblages of bacterioplankton for
use in trophic studies. Limnol. Oceanogr. 25, 172^181.
Caron, D.A., Lessard, E.J., Voytek, M. and Dennett, M.R.
(1993) Use of tritiated thymidine (TdR) to estimate rates of
bacterivory: implication of label retention and release by bacterivores. Mar. Microb. Food. Webs 7, 177^196.
Nygaard, K. and Hessen, D.O. (1990) Use of 14 C-protein-labelled bacteria for estimating clearance rates by heterotrophic
and mixotrophic £agellates. Mar. Ecol. Prog. Ser. 68, 7^14.
î.
Wikner, J., Andersson, A., Normack, S. and Hagstroëm, A
(1986) Use of genetically marked minicells as a probe in measurements of predation on bacteria in aquatic environments.
Appl. Environ. Microbiol. 52, 4^8.
Gonzalez, J.M., Sherr, E.B. and Sherr, B.F. (1990) Size-selective grazing on bacteria by natural assemblages of estuarine
£agellates and ciliates. Appl. Environ. Microbiol. 56, 583^589.
Landry, M.R., Lehner-Fournier, J.M., Sundstrom, J.A., Fagerness, V.L. and Selph, K.E. (1991) Discrimination between
living and heat-killed prey by a marine zoo£agellate, Paraphysomonas vestita (Stokes). J. Mar. Biol. Ecol. 146, 139^151.
Monger, B.C. and Landry, M.R. (1992) Size-selective grazing
by heterotrophic nano£agellates : an analysis using live-stained
bacteria and dual-beam £ow cytometry. Arch. Hydrobiol. 37,
173^185.
Síimek, K. and Chrzanowski, T.H. (1992) Direct and indirect
evidence of size-selective grazing on pelagic bacteria by freshwater nano£agellates. Appl. Environ. Microbiol. 58, 3715^
3720.
Epstein, S.S. and Rossell, J. (1995) Methodology of in situ
grazing experiments : evaluation of a new vital dye for preparation of £uorescently labeled bacteria. Mar. Ecol. Prog. Ser.
128, 143^150.
Ingraham, J.L., MaalÖe, O. and Neidhardt, F.C. (1983)
Growth of the Bacterial Cell. Sinauer, Sunderland, MA.
Zubkov, M.V. and Sleigh, M.A. (1995) Ingestion and assimilation by marine protists fed on bacteria labeled with radioactive thymidine and leucine estimated without separating
predator and prey. Microb. Ecol. 30, 157^170.
Zubkov, M.V. and Sleigh, M.A. (1997) Bacterivory in seawater samples estimated by a dual radioactive-labelling technique. J. Plankton Res. 19, 209^219.
Lessard, E.J. and Swift, E. (1985) Species-speci¢c grazing rates
of heterotrophic dino£agellates in oceanic waters, measured
with a dual-label radioisotope technique. Mar. Biol. 87,
289^296.
Sherr, B.F., Sherr, E.B. and Rassoulzadegan, F. (1988) Rates
of digestion of bacteria by marine phagotrophic protozoa:
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
101
temperature dependence. Appl. Environ. Microbiol. 54,
1091^1095.
Rivkin, R.B. (1986) Incorporation of tritiated thymidine by
eucaryotic microalgae. J. Phycol. 22, 193^198.
Rivkin, R.B. and Voytek, M.A. (1986) Cell division rates of
eucaryotic algae measured by tritiated thymidine incorporation into DNA: coincident measurements of photosynthesis
and cell division of individual species of phytoplankton isolated from natural populations. J. Phycol. 22, 199^205.
Cuhel, R.L. and Waterbury, J.B. (1984) Biochemical composition and short term nutrient incorporation patterns
in a unicellular marine cyanobacterium, Synechococcus
(WH7803). Limnol. Oceanogr. 29, 370^374.
Fuhrman, J.A. and Azam, F. (1982) Thymidine incorporation
as a measure of heterotrophic bacterioplankton production in
marine surface waters: evaluation and ¢eld results. Mar. Biol.
66, 109^120.
Haas, L.W. and Webb, K.L. (1979) Nutritional mode of several nonpigmented micro£agellates from the York River estuary, Virginia. J. Exp. Mar. Biol. Ecol. 39, 125^134.
Fenchel, T. (1987) Ecology of Protozoa. Science Tech. Publishers, Madison, WI/Springer Verlag, Berlin.
Berman, T., Hoppe H.G. and Gocke, K. (1994) Response of
aquatic bacterial populations to substrate enrichment. Mar.
Ecol. Prog. Ser. 104, 173^184.
Kirchman, D.L. and Rich, J.H. (1997) Regulation of bacterial
growth by dissolved organic carbon and temperature in the
Equatorial Paci¢c Ocean. Mar. Ecol. 33, 11^20.
Sleigh, M.A. and Zubkov, M.V. (1998) Methods of estimating
bacterivory by protozoa. Eur. J. Protistol., in press.
Taylor, G.T. and Sullivan, C.W. (1984) The use of 14 C-labeled
bacteria as a tracer of ingestion and metabolism of bacterial
biomass by microbial grazers. J. Microbiol. Methods 3, 101^
124.
Sieracki, M.E., Haas, L.W., Caron, D.A. and Lessard, E.J.
(1987) E¡ect of ¢xation on particle retention by micro£agellates: underestimation of grazing rates. Mar. Ecol. Prog. Ser.
38, 251^258.
Owens, N.J.P., Burkill, P.H., Mantoura, R.F.C., Woodward,
E.M.S., Bellan, I.E., Aiken, J., Howland, R.J.M. and Llewellyn, C.A. (1993) Size-fractionated primary production and nitrogen assimilation in the northwestern Indian Ocean. DeepSea Res. II 40, 697^709.
Zubkov, M.V. and Sleigh, M.A. (1998) Heterotrophic nanoplankton biomass measured by a glucosaminidase assay.
FEMS Microb. Ecol. 25, 97^106.
Daro, M.H. A simpli¢ed 14 C method for grazing measurements on natural plankton populations. Helgolaënder Wiss.
Meeresunters. 31, 241^248.
Zubkov, M.V., Sleigh, M.A., Tarran, G.A., Burkill, P.H. and
Leakey, R.J.G. (1998) Picoplanktonic community structure on
an Atlantic transect from 50³N to 50³S. Deep-Sea Res. I, in
press.
Li, W.K.W., Jellett, J.F. and Dickie, P.M. (1995) DNA distribution in planktonic bacteria stained with TOTO or TOPRO. Limnol. Oceanogr. 40, 1485^1495.
Marie, D., Partensky, F., Jacquet, S. and Vaulot, D. (1997)
FEMSEC 945 20-8-98
102
[38]
[39]
[40]
[41]
[42]
M.V. Zubkov et al. / FEMS Microbiology Ecology 27 (1998) 85^102
Enumeration and cell cycle analysis of natural populations of
marine picoplankton by £ow cytometry using the nucleic acid
stain SYBR Green I. Appl. Environ. Microbiol. 63, 186^
193.
Landry, M.R., Kirstein, J. and Constantinou, J. (1995) A
re¢ned dilution technique for measuring the community grazing impact of microzooplankton, with experimental tests in
the central equatorial Paci¢c. Mar. Ecol. Prog. Ser. 120, 53^
63.
Servais, P., Billen, G. and Vives Rego, J. (1985) Rate of bacterial mortality in aquatic environments. Appl. Environ. Microbiol. 49, 1448^1454.
î.
Turk, V., Rehnstam, A.-S., Lundberg, E. and Hagstroëm, A
(1992) Release of bacterial DNA by marine nano£agellates, an
intermediate step in phosphorus regeneration. Appl. Environ.
Microbiol. 58, 3744^3750.
del Giorgio, P.A., Gasol, J.M., Vaqueè, D., Mura, P., Agusti,
S. and Daurte, C.M. (1996) Bacterioplankton community
structure: protists control net production and the proportion
of active bacteria in a coastal marine community. Limnol.
Oceanogr. 41, 1169^1179.
î . (1995) Total counts of marine
Zweifel, U.L. and Hagstroëm, A
[43]
[44]
[45]
[46]
[47]
bacteria include a large fraction of non-nucleoid-containing
bacteria (ghosts). Appl. Environ. Microbiol. 61, 2180^2185.
Leakey, R.J.G., Archer, S.D. and Grey, J. (1996) Microbial
dynamics in coastal waters of East Antarctica : bacterial production and nano£agellate bacterivory. Mar. Ecol. Prog. Ser.
142, 3^17.
î ., Azam, F., Andersson, A., Wikner, J. and RasHagstroëm, A
soulzadegan, F. (1988) Microbial loop in oligotrophic pelagic
marine ecosystem : possible roles of cyanobacteria and nano£agellates in the organic £uxes. Mar. Ecol. Prog. Ser. 49, 171^
178.
MacManus, G.B. and Fuhrman, J.A. (1988) Control of marine bacterioplankton populations: measurement and signi¢cance of grazing. Hydrobiologia 159, 51^62.
Sanders, R.W., Caron, D.A. and Berninger, U.-G. (1992) Relationship between bacteria and heterotrophic nanoplankton
in marine and fresh waters : an inter-ecosystem comparison.
Mar. Ecol. Prog. Ser. 86, 1^14.
Sherr, B.F., Sherr, E.B. and Pedroès-Alioè, C. (1989) Simultaneous measurement of bacterioplankton production and protozoan bacterivory in estuarine water. Mar. Ecol. Prog. Ser.
54, 209^219.
FEMSEC 945 20-8-98