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K . Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147
21
C mol leucine . This factor is an average conversion factor determined in several
experiments during the cruise from parallel measurements of biomass increase and
3
H-leucine incorporation in 0.8-mm filtered bottle incubations Gasol et al. submitted. For some of the substrate-enriched fractions we quantified bacterial abundance in
aggregates at the end of the experiments. Aggregated bacteria were directly counted in normal DAPI preparations when samples contained only smaller aggregates. When large
aggregates with more than several hundred bacteria appeared, these could not be properly quantified and samples were sonicated Branson Sonifier 250, ten bursts of 3 s
to disperse aggregated bacteria. From a first DAPI preparation only the number of freely dispersed single cells was obtained. The total number of bacteria was counted after a
second DAPI preparation of the sonicated sample. The difference of the two counts gives an estimate of the bacterial abundance in aggregates. In addition we counted and
sized maximal dimension bacterial aggregates at the end of the experiments from the DAPI preparations. To visualize whether bacterial aggregates were embedded in a
matrix of extracellular polysaccharides, selected samples were stained with alcian blue according to the procedure described by Logan et al. 1994.
Statistical analysis including t-test and analysis of variance ANOVA were performed using the software package
STATISTICA
.
3. Results
Yeast extract proved to be an ideal substrate for stimulating bacterial growth and increasing bacterial abundance, probably because besides organic carbon also inorganic
macro- and micronutrients are supplied. Experiment 3 compares the effects of the relatively high substrate pulse of yeast extract addition ¯ 50 mM organic C to the
addition of glucose, inorganic nitrogen, or a mixture of amino acids, all supplied at low concentrations similar to previously published substrate addition experiments Kirch-
man, 1990; Kirchman and Rich, 1997. The strong effect of yeast extract on bacterial productivity and abundance is shown in Fig. 2. The addition of glucose or inorganic
nitrogen had no effect on the development of bacterial abundance or leucine uptake compared to the unamended controls one-way repeated-measures ANOVA of the effects
of nutrient addition on bacterial abundance or leucine uptake, both p . 0.05. The addition of amino acids resulted in a moderate increase in bacterial abundance p ,
0.05 but not of leucine uptake p . 0.05 compared to the controls.
For the coupling between bacteria and protozoans after nutrient enrichment we examined altogether seven size-fractionation and nutrient addition experiments: at three
stations Exp. 1, 2, and 4 incubations with water from two depths 5 m, DCM, and at one station nutrient enrichment of unfiltered water from 5 m depth. Although the stations
were from different oceanic areas, the first two experiments from slightly more productive Northern subtropical areas and the second two experiments from the very
unproductive Southern subtropical Atlantic gyre, they all represented warm, oligotrophic
21
situations. The chlorophyll a concentrations were in the range 0.03–0.23 mg l at the
21
surface and in the range 0.17–0.36 mg l in the depth of the DCM Table 1. Bacterial
5 21
numbers were also within a narrow range, 3.8–8.1 3 10 ml
at the surface and
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. Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147 133
Fig. 2. Bacterial response to the addition of different nutrients in unfiltered incubations of experiment 3 5 m
21
depth. CONTR: Control bottles without nutrient additions, YE: yeast extract 1.5 mg l , GLC: glucose 7.4
mM, AA: mixture of ten amino acids, 100 mM; N: NH Cl 3.3 mM. A Leucine incorporation, mean and
4
range of two replicate treatments, each measured with triplicate subsamples not for AA because of the dilution with cold leucine; B development of bacterial cell numbers, mean and range of two replicate treatments.
5 21
2.2–5.1 3 10 ml at the DCM. Concentrations of Prochlorococcus and Synechococcus
were higher in surface than in the depth of the DCM Table 1, indicating that the deep chlorophyll maximum was probably made up by eukaryotic picoplankton Gasol et al.,
in preparation and by increased cell-specific pigment content. More differences were visible in bacterial productivity leucine incorporation and abundance of HNF. Here
experiment 2 was obviously enriched compared to the other stations. Although the station was not situated inside the North African upwelling area, the special hydrography
of the area as mentioned above suggest that it probably received relatively aged recently upwelled water with higher nutrient concentrations.
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K . Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147
Table 1
a
Physical and biological parameters for the stations at which microcosm experiments were performed Exp.
Depth Temp.
Bacteria HNF
T Bact Prochlorophytes
Synechococcus Chl a
d 5
21 21
5 21
3 21
21
m 8C
10 ml ml
days 10 ml
10 ml mg l
1 5
24.4 8.14
351 77.4
1.24 6.18
0.09 85
19.6 5.13
192 81.9
0.46 0.68
0.25 2
5 28.9
6.92 805
1.1 2.33
9.29 0.23
50 16.8
2.26 166
5.6 0.38
0.45 0.36
3 5
25.4 4.42
446 4.1
0.84 4.67
0.03 4
5 25.0
3.84 532
5.0 1.58
9.83 0.04
180 22.5
2.19 101
9.7 0.36
0.26 0.17
a
The second depth for experiments 1, 2 and 4 is the depth of the deep chlorophyll maximum DCM. Bacterial doubling time T is based on leucine incorporation data and the empirically determined conversion
d
factor of 0.62 kg C per mol of leucine. Concentrations of prochlorophytes and Synechococcus were determined by flow-cytometry as described in Gasol et al., submitted.
A general pattern in bacterial development, similar to all experiments, was observed. The bacterial population development is illustrated here only for the size-fractionation
experiment 1, surface and DCM Fig. 3, but the experiments 2 and 4 did not deviate essentially from this pattern. The addition of yeast extract always resulted in a rapid
21
increase of bacterial abundance, with growth rates in the range 0.07–0.13 h .
Maximum bacterial numbers were proportional to the amount of added yeast extract, which differed slightly between the experiments, and reached values between 3.4 and
6 21
9.6 3 10 ml Table 2. This level was approximately one order of magnitude higher
Fig. 3. Example of a fractionation and enrichment experiment exp. 1. Development of bacteria and HNF in
21
the fractions , 0.8 and , 5 mm, and with 1 N and without 2N nutrient addition 3 mg yeast extract l .
A Surface 5 m; B deep chlorophyll maximum 85 m. Mean and range of two replicate treatments.
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. Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147 135
Table 2
a
Summary of the enrichment experiments Exp.
Enrichment Depth
Maximum HNF
yeast extract m
bacterial numbers response time
21 6
21
mg l 10 ml
days 1
4.5 5
9.6361.00 3.0–3.8
4.5 85
8.9460.35 5.1–5.8
2 3.0
5 7.0060.59
3.0–4.8 3.0
50 5.2660.15
4.8–6.1 3
1.5 5
3.3660.24 5.0–6.0
4 2
5 3.8060.19
4.1–4.8 2
180 3.7860.42
4.8–5.5
a
Maximum bacterial numbers mean6S.D. of the fractions , 0.8 and , 5 mm after substrate addition. HNF response time is defined as the time period after which bacteria in the , 5-mm fractions were reduced to
about the initial levels due to HNF grazing.
than in the natural situation at the beginning. Within 24 h bacteria seemed already to have reached stationary phase after which bacterial numbers stayed more or less constant
until the end of the experiments in the , 0.8-mm fractions or until nanoflagellates developed in the , 5-mm fractions. HNF were comprised mainly by naked, colourless
forms, 3–5 mm in diameter, which increased within 1 day from nearly undetectable to
4 21
levels . 5 3 10 ml . For treatments in which we had fairly reliable estimates on the
initial HNF abundance before the peak, we estimated the population doubling time to be in the range 5–8 h. The HNF population peaks coincided with a rapid decline of the
5 21
bacteria to slightly above the initial levels 5–9 3 10 ml
. Peaks of HNF were short-lasting and they decreased again to low levels after the collapse of the bacterial
community. We defined the HNF response time to the bacterial enrichment as the time period which it took for the grazers to reduce bacteria to about the initial levels. This
corresponded generally to the peak in HNF abundance. The values were in the range of 3–6 days Table 2 with lower values in the warmer surface waters than in the DCM.
The variability in response time Table 2 and in HNF peak abundance Fig. 3 is due to the fact that HNF did not always increase exactly simultaneously in replicate treatments.
Bacterial abundance in the unenriched fractions did not remain constant although this is not visible from Fig. 3 in which bacteria of enriched and unenriched treatments are
plotted at the same scale. A 2–3-fold increase in bacterial abundance occurred in the unenriched treatments in all size fractions during the course of the experiments. Together
3
with an increase in mean cell volume from 0.05 to 0.07–0.09 mm this resulted in a considerable increase of bacterial biomass. For experiment 2, in which this stimulation
of bacterial growth was especially pronounced, this pattern is illustrated in Fig. 4. The bacterial growth was also expressed in an immediate and drastic increase in leucine
21
incorporation. The leucine incorporation reached about 2–4 nM h after 24 h of
incubation and remained at this high level until the end of the experiments. HNF
21
remained at such a low level , 300 ml in the unenriched fractions that a precise
enumeration with the amount of water usually filtered was not possible. The develop- ment of the bacterial communities in the different treatments did not show any signs of a
strong grazing impact, such as a change in size distribution or decrease in abundance.
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K . Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147
3
Fig. 4. Development of bacterial biomass closed symbols and H-Leucine incorporation open symbols in different size fractions of experiment 2 water from 5 m depth without nutrient addition. Means and ranges of
two replicate treatments.
There was no significant difference in development of bacterial biomass or leucine between the fractions , 0.8, , 5 and , 50 mm one-way repeated-measures ANOVA of
the effects of size-fractionation on bacterial biomass or leucine uptake, both p . 0.05. In contrast, the grazing impact of HNF on bacteria and the corresponding changes in
the morphological structure of the bacterial community could be studied in the enriched fractions. One very obvious change occurred in the size distribution of the bacterial
assemblage. The different influence of substrate and grazing can be seen when comparing the bacterial size structure in the , 0.8-mm fraction without grazers and in
the , 5-mm fraction strong grazing towards the end Fig. 5. The initial dominance of small cocci shifted after substrate addition towards larger-sized rods by which the mean
cell volume increased more than twofold. In treatments without grazers this cell size distribution remained more or less constant until the end of the incubations. In contrast,
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. Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147 137
Fig. 5. Bacterial size distribution and mean cell volume at times 0, 72, and 115 h during the incubation of , 0.8 and , 5-mm filtered and substrate-enriched water example from exp. 2, water from 5 m depth.
the size distribution shifted again to small coccoid cell volumes in the , 5-mm fractions after the peak in HNF development and the bacterial decline.
The second group of bacterial morphotypes, besides the small cocci, which still remained after the grazing peak, were normal rod-shaped bacteria attached in cell
clusters and aggregates. These bacterial aggregates, which did not appear in the non-enriched treatments, were enumerated and sized in DAPI preparations at the end of
the experiments, after HNF had reduced bacterial densities in the , 5-mm fractions to roughly the initial levels corresponds to HNF response time. The average size
distribution maximal linear dimension of these aggregates is shown in Fig. 6. Small
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K . Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147
Fig. 6. Average size distribution longest dimension of bacterial aggregates, pooled data from different experiments n 5 7.
aggregates with about 5–20 mm in size and harboring approximately 20–100 bacteria aggregate dominated about 70 of total aggregates. Additionally, large aggregates in
the range 50–100 mm occurred, with several hundred bacteria per aggregate. The aggregates could be stained with the dye Alcian blue, indicating that the bacteria are
embedded in a polysaccharide matrix Fig. 7 and thus looked similar to transparent exopolymer particles TEP described before Alldredge et al., 1993.
Bacterial aggregates were most obvious in the , 5-mm fractions after HNF had developed but that was due to the fact that nearly all freely suspended bacteria were
eliminated and not to the fact that aggregates developed only in these treatments. Bacterial aggregates were also present in the substrate-enriched , 0.8-mm fractions but
here their relative importance was much less due to the high number of freely suspended single cells. At the end of the experiments the , 0.8-mm fractions were dominated to
80–95 by freely suspended bacteria whereas in the , 5-mm fractions bacteria in aggregates made up 22–72 mean 51 of the total bacterial abundance. However,
when looking at the total numbers it is obvious that aggregated bacteria appeared in about the same amounts in , 5-mm and , 0.8-mm fractions but that freely suspended
bacteria were strongly reduced in the , 5-mm fractions Fig. 8.
4. Discussion
