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FEMS Microbiology Ecology 32 (2000) 143^155
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The microbial food web along salinity gradients
Carlos Pedrös-Aliö *, Juan I. Calderön-Paz, Marlie H. MacLean, Glo©ria Medina,
Ce©lia Marrasë, Josep M. Gasol, Nüria Guixa-Boixereu
Departament de Biologia Marina i Oceanogra¢a, Institut de Cie©ncies del Mar, CSIC, Passeig Joan de Borbö s/n, 08039 Barcelona, Spain
Received 29 January 2000; accepted 15 March 2000
Abstract
The microbial food web was studied along a gradient of salinity in two solar salterns used for the commercial production of salt. The
different ponds in the salterns provide a wide range of ecosystems with food webs of different complexities. Abundance of prokaryotes, cell
volume, prokaryotic heterotrophic production, chlorophyll a, abundance of heterotrophic flagellates, ciliates and phytoplankton were
determined in several ponds in each saltern. Increases in salinity resulted in a progressive reduction in the abundance and number of
different groups of eukaryotic microorganisms present, but an increase in biomass of prokaryotes. Maximal activity of both phyto- and
bacterioplankton was found at a salinity of around 100x, where there was also a maximum in chlorophyll a concentration. Growth rates
of heterotrophic prokaryotes decreased with increasing salinity. Bacterivory disappeared above 250x salinity, whereas other loss factors
such as viral lysis appeared to be of minor importance throughout the gradient [Guixa-Boixereu et al. (1996) Aquat. Microb. Ecol. 11, 215^
227]. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Solar saltern; Archaea; Bacterium ; Flagellate ; Phytoplankton; Bacterivory; Heterotrophic production
1. Introduction
Multi-pond solar salterns provide a range of environments with di¡erent salinity, from that of seawater up to
sodium chloride saturation and sometimes even beyond
(chapter 13 in [1,2]). As water evaporates and salinity increases, water is pumped or fed by gravity to the next
pond, such that salinity in each particular pond is kept
within narrow limits, essentially constant. Each pond can
thus be considered at equilibrium and the biota in any
given pond as a well-adapted and established community
for that particular salinity. The two extremes of salinity
provide one of the most common habitats (seawater) and
one of the most extreme habitats in the world (calcium
and magnesium chloride-saturated brines). In no other
* Corresponding author. Tel. : +34 (93) 221-6416;
Fax: +34 (93) 221-7340; E-mail: [email protected]
system is there such a complete gradient of environments
with di¡erent salinities available within walking distance.
Thus, the response of the food web structure, the £uxes of
elements, the diversity of the community and the adaptations of organisms can easily be traced by simultaneously
analyzing several di¡erent ponds along the gradient. Salterns are particularly interesting for microbial ecology,
since large organisms disappear early in the salinity gradient, and predominantly or exclusively microbial communities are found at the highest salinity.
Given these advantages, it is surprising how few studies
of the microbial ecology of saltern ponds have been carried out. Saltern ponds have been used mostly for biogeochemical studies [1], as models for ancient evaporitic environments [3], and as a source of halophilic and
halotolerant microorganisms [4^6]. Ecological studies
have been limited to descriptions of the peculiar organisms
found at di¡erent salinities, with attention to their respective salinity ranges [7], but with little concern for quantitation of their biomass and activities. In particular, there is
an extensive literature describing the most conspicuous
members of the microbial mats found at the bottom of
many ponds, but again, with little quanti¢cation of biomass and activities [8^11].
As a result, the biota of salterns and the existence of a
0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 2 5 - 8
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trend of a decreasing number of species as salinity increases are well-characterized, but most functional aspects
remain unknown. Thus, for example, there is debate about
whether Dunaliella and haloarchaea grow fast, slow or not
at all at the ponds where they show maximal abundance
[1,2,12], or about what is the salinity of maximal primary
production. In particular, some trophic relationships such
as herbivory and bacterivory have never been measured in
the ¢eld. Thus, we were interested in determining a series
of microbial food web parameters along the salinity gradient, with special attention to protistan bacterivory, protistan biomass, as well as production and speci¢c growth
rates of prokaryotic organisms.
Study of the microbial food web in saltern ponds o¡ers
an additional advantage. A paradigm of planktonic food
web structure and function emerged during the 80s [13^16]
and many aspects have been tested experimentally in a
large number of marine and freshwater environments.
The time has come, therefore, when analysis of systems
which depart from the standard may fruitfully show the
inconsistencies of the current paradigm. Salterns also provide systems with progressively more simpli¢ed food webs.
What happens when grazers of prokaryotes disappear ?
How does a food web formed only by prokaryotes function ? Do lytic viruses become an important loss factor?
These and many other questions could be answered by
studying saltern ponds. In this paper, we describe the microbial food webs in two di¡erent salterns. In a separate
paper [17], we compare the results from the salterns to the
current paradigm of microbial plankton.
2. Materials and methods
2.1. Salterns
We studied two di¡erent solar salterns on the Mediterranean coast of the Iberian Peninsula: `La Trinitat' in the
Ebro River Delta (Tarragona, Spain, 40³35PN, 0³41PE)
and `Bras del Port' in Santa Pola (Alicante, Spain,
38³12PN, 0³36PW). The Bras del Port salterns have been
used for microbiological studies in the past, especially as a
source of halophilic bacteria and archaea [2,4,18]. On the
other hand, the microbial plankton of La Trinitat (Fig. 1)
has not been studied before. The two salterns are operated
for commercial salt production in di¡erent ways. Bras del
Port is active year-round. Thus, each pond tends to provide a very stable environment. La Trinitat does not operate during the fall and winter and, therefore, ponds do not
reach their de¢nitive salinity until late June or July. As a
result, there is a seasonal succession in each pond, and the
crystallizers are drained and salt is harvested in August
every year. The most complete sampling was carried out
in late July in both salterns (July 1993 for Bras del Port
and July 1994 for La Trinitat) to ensure that ponds had
reached their de¢nitive salinity. Additional samplings were
carried out in La Trinitat during the spring (May 1992 and
May 1993) to get some impression of seasonal changes.
Sampling was carried out with a bucket ¢xed to the end
of a pole. The corners of the ponds were avoided since
organisms tend to accumulate in the downwind corners.
Between three and four ponds could be sampled and proc-
Fig. 1. La Trinitat salterns, Delta de l'Ebre, Tarragona (Spain). Note the main seawater intake (in Badia dels Alfacs, to the right of the pier) and the
spiral layout of the increasingly saline ponds: `depösitos', `calentadores' (1^10) and ¢nally, `cristalizadores' (crystallizers).
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essed in a day. When nine or 10 ponds were sampled, the
whole operation took 2 or 3 days. All samples were ¢xed
and incubations carried out in the ¢eld.
2.2. Physico-chemical analyses
Temperature was measured with a mercury thermometer. Opaque BOD bottles were ¢lled and ¢xed for dissolved oxygen determinations by a modi¢cation of the
Winkler titration [19]. Samples for inorganic nutrients
were collected in acid-rinsed tubes and frozen until analysis. Ammonia, nitrate, nitrite and phosphate were analyzed by conventional methods [20] with a Technicon
autoanalyzer. Samples had to be diluted to approximate
sea water salinity before measurements were carried out.
Salinity was determined in the ¢eld with a hand-held refractometer. For salinity higher than 130x, water from
the ponds had to be diluted with distilled water to fall
within the scale of the refractometer.
2.3. Abundance of microorganisms
Chlorophyll a was determined by £uorescence in acetone extracts [21]. Water samples were ¢ltered on GF/F
glass ¢ber ¢lters and the ¢lters placed in tubes with acetone. The tubes were kept cool until processed. Glass
pearls were added to each tube and these were violently
agitated in a high-speed cell disrupter (Vibrogen-Zellmu«hle, Germany) to break up the cells. After half an
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hour in the dark, the tubes were spun in a benchtop centrifuge and the £uorescence of the supernatant read with a
Turner Designs £uorometer.
Duplicate 30-ml samples were ¢xed with formaldehyde
for counts of prokaryotes and £agellates by epi£uorescence microscopy. Samples used for the determination of
prokaryotic abundance had to be diluted from 10 to
100 times and were then ¢ltered through 0.2-Wm pore diameter black polycarbonate ¢lters and stained with DAPI
(0.1 Wg ml31 ¢nal concentration) for 5 min before drying
the ¢lters by suction [22]. Filters were then mounted on
microscope slides with non-£uorescent oil (R.P. Cargille
Lab.). Filters were counted by epi£uorescence microscopy
with a Nikon Diaphot microscope. About 200^400 cells
were counted per sample. Cell volumes were determined
with an image analysis system measuring at least 200 cells
per sample. A Hamamatsu C2400-08 video camera was
used to examine microscopic preparations. Images were
captured in a PC with the software MIP (from Microm
Espan¬a SA). The video images were downloaded to a
Macintosh computer and analyzed with the shareware
program NIH Image. Objects occupying less than seven
pixels (equivalent to an sphere with diameter less than
0.2 Wm) were discarded. The remaining bacteria-like objects were measured and the volume calculated from area
and perimeter measurements with the formula of Fry [23].
The system was calibrated with £uorescent latex beads and
with natural bacterioplankton samples measured simultaneously by phase contrast microscopy and by epi£uores-
Fig. 2. Physico-chemical characteristics of the di¡erent ponds in the Bras del Port (A, C) and La Trinitat (B, D) salterns during samplings carried out
in July 1993 and July 1994, respectively. (A, B) Temperature and dissolved oxygen; (C, D) Concentration of inorganic nutrients (ammonia, nitrate, nitrite and phosphate).
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Fig. 3. Abundance and biomass estimates of autotrophic microbial plankton in Bras del Port and La Trinitat salterns. (A) Biovolume of phytoplankton
and chlorophyll a concentrations in Bras del Port, July 1993; (B) Biovolume of phytoplankton and chlorophyll a concentrations in La Trinitat, July
1994 ; (C) Biovolume of the di¡erent phytoplankton groups detected in Bras del Port; (D) Biovolume of the di¡erent phytoplankton groups detected in
La Trinitat; (E) Diversity of ciliates and phototrophic microbes as measured by S (total number of taxons), and two diversity indices, Maregalef's and
Berger-Parker's for Bras del Port; (F) Same as (E) for La Trinitat. Note the two peaks of phytoplankton biomass (in chlorophyll a or biovolume) at
V100x and V250x salinity.
cence [24]. Biomass was calculated by using the carbon
to volume relationship derived by Norland [25] from the
data of Simon and Azam [26]: pg C cell31 = 0.12U(Wm3
cell31 )0:7 .
For the square archaea, we measured length and width
of a representative number of cells. We assumed cells to be
a parallel sided with the two measured dimensions plus
height taken from the electron micrographs of Stoeckenius
[27].
The abundance of nano and microplankton was determined by the Utermo«hl technique. Samples were ¢xed
with Lugol's solution and kept in the dark until counted.
Volumes of 100 ml were allowed to settle for 24^48 h and
examined in a Zeiss inverted microscope. The whole bottom chamber was examined at 20U and 40U magni¢cation for the larger and less abundant microorganisms.
Then, 200U and 400U magni¢cations were used to count
the remaining microorganisms in transects chosen at random. Cells were assigned to morphotypes and these were
assigned to one of the large taxonomic groups shown in
Fig. 3. Linear dimensions of at least 20 cells per taxon
were determined and the volume calculated assuming closest geometrical shapes.
2.4. Microbial activities
Primary production and respiration in the plankton
were measured by oxygen changes in clear and dark
BOD bottles incubated in the ponds for about 4 h around
noon. Four clear and four dark bottles were incubated in
each pond. Some ponds were supersaturated with oxygen
while others had very low oxygen concentrations due to
the low solubility of oxygen at extremely high salinity and
temperatures. Thus, we decreased the oxygen concentration in the samples for the clear bottles by stripping with
oxygen-free nitrogen gas in a 1 liter graduated cylinder. A
portion of this water was ¢xed in a 150-ml BOD bottle for
Winkler titration and the rest was distributed to the incubation bottles. In addition, we measured oxygen with an
electrode before starting the incubations and at the end by
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sacri¢cing one of the four replicates. This aided in determining the best incubation times for the incubations carried out the following day. The other three replicates were
¢xed for Winkler titration in the laboratory.
We carried out [3 H]leucine incorporation assays in all
samplings according to Kirchman [28] with slight modi¢cations. In one case (La Trinitat 1994), we also measured
[3 H]thymidine incorporation according to Bell [29]. And in
two cases (La Trinitat 1992 and 1993), we determined the
frequency of dividing plus divided cells (FDDC; [30]). The
thymidine incorporation data have already been presented
in Guixa-Boixereu et al. (Fig. 3A in [35]) and will not be
shown here. The two measurements agreed fairly well,
producing doubling times that were not signi¢cantly di¡erent. The ratio Leu/TdR was 21.3 on average (S.E.M. 3.7).
Only in the 182.5% salinity pond was there a signi¢cant
departure from this ratio (47.3).
In the radioactivity incubations, 5-ml samples were incubated with 40 nM leucine (1:9 hot:cold v/v, around 150
Ci mmol31 ) in plastic scintillation vials. This concentration was shown to be saturating in experiments carried out
in the May 1993 visit to La Trinitat. Two replicates plus a
formaldehyde-killed control (4% ¢nal concentration) were
incubated for each pond. Incubations were carried out in
the dark at ambient temperature for 45 min. Samples were
kept in a cooler with ice until returned to the laboratory.
There, samples were ¢ltered and counted in an LKBRackbeta liquid scintillation counter.
Oren [12,31] introduced a way to determine the activity
of bacteria and archaea separately in the same samples.
The technique uses bile salts to lyse archaea. Thus, activity
in samples treated with bile salt corresponds to bacteria,
while the activity in untreated samples corresponds to bacteria plus archaea. However, this technique is not absolutely foolproof since Halococcus and some Halobacterium
species are not lysed by sodium taurocholate (the detergent commonly used; [12,31]), although Halococcus is believed to play a small role in natural systems [2,12,31]. A
double check can be done by carrying out parallel incubations with an inhibitor of bacteria. On two occasions, we
used this methodology to assign leucine incorporation to
bacteria or to archaea. Erythromycin (at a ¢nal concentration of 20 Wg ml31 ) was used to inhibit bacterial leucine
incorporation (i.e. protein synthesis). Na-taurocholate (at
a ¢nal concentration of 50 Wg ml31 ) was used to lyse archaea and thus avoid leucine incorporation by these organisms [12,31]. All inhibitors were added before the start
of incubations.
Data for leucine incorporation were converted to estimates of prokaryotic production by using the conversion
factor 3.1 kg C mol31 incorporated [26]. Speci¢c growth
rates (W) and doubling times (Dt ) were calculated from
these production estimates and those of total prokaryotic
biomass. We determined empirical conversion factors in a
recent visit to the Bras del Port salterns (May 1999). Since
one could argue that conversion factors could have been
147
di¡erent in 1993, we have chosen to present the growth
rates calculated with both the standard conversion factor
[26] and with the conversion factors determined in 1999.
Despite di¡erences in absolute values, the trends and
ranges of values are essentially the same, thus giving con¢dence to our conclusions.
The term bacterivory will be used for convenience when
designing predation on both bacteria and archaea. Bacterivory was determined with £uorescently labeled bacteria
(FLB; [32]), using the FLB disappearance method [33].
FLB were prepared from a heterotrophic bacterium isolated from the Mediterranean coast (1U0.8 Wm, biovolume : 0.42 Wm3 ). This cell size is a compromise between
the average cell volume at the lowest salinity ponds (0.084
Wm3 ) and at the highest salinity where bacterivory was
measured (0.916 Wm3 ). Bacterivory experiments were carried out in all ponds except for the crystallizers (with salinity s 300x). At these high salinities, no predators were
present, since neither £agellates nor ciliates can live in the
crystallizers. Samples of 1 liter were incubated in acidwashed 2.5-l polycarbonate bottles in the dark and ambient temperature for 48 h. Two replicates and a formaldehyde-killed control (1% ¢nal concentration) were incubated per pond. Bacteria and FLB were counted at
times 0 and 48 h in the experimental bottles and in the
controls, and bacterivory was calculated using the formula
of Salat and Marrasë [34]. Bacterivory results from La
Trinitat appeared in a previous paper [35] where they
were compared to viral lysis. Here they are presented
again for comparative purposes with the results from the
Bras del Port salterns.
3. Results
The data sets for Bras del Port and La Trinitat collected
in late July are presented together. These correspond to
fully developed stable conditions in the di¡erent ponds
and are considered representative of the salterns. The
data collected in La Trinitat during the spring are referred
to only sporadically to make speci¢c points.
3.1. Physico-chemical conditions
Fig. 2A,B presents temperature and oxygen concentration in the salterns. As salinity increased, temperature also
increased due to the higher calori¢c capacity of the brines,
while oxygen decreased due to the lower solubility of oxygen in the brines. Supersaturation was common in many
ponds where primary production quickly overrode the solubility of oxygen for the given temperature and salinity.
Inorganic nutrient levels were similar to those reported
for other salterns (for example the Western Salt salterns ;
[36]). Nitrate was found in higher concentrations at the
highest salinities (Fig. 2C,D). Ammonia was, by far, the
most abundant inorganic nutrient, perhaps due to excre-
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Fig. 4. Abundance and biomass estimates of heterotrophic microbial plankton and microbial activities in Bras del Port and La Trinitat salterns. (A)
Heterotrophic prokaryotes (PN) and biovolume of ciliates in Bras del Port, July 1993; (B) Heterotrophic prokaryotes (PN), heterotrophic nano£agellates (HNF) and biovolume of ciliates in La Trinitat in July 1994; (C) Total prokaryotic leucine incorporation (Leu, total), leucine incorporation in the
presence of the protein synthesis inhibitor erythromycin, speci¢c for bacteria (Leu+E), and leucine incorporation in the presence of the lysing agent
taurocholate, speci¢c for archaea (Leu+T) in Bras del Port; (C) Same as (D) for La Trinitat in July 1994 ; (E) Estimates of primary production and respiration in Bras del Port; (F) Total prokaryotic leucine incorporation (Leu), frequency of dividing (FDC), and dividing plus divided (FDDC) cells in
La Trinitat in May 1993. Error bars indicating standard error of the mean are shown for activity measurements. When bars are not visible, error is
smaller than symbol.
tion by protists and invertebrates. Phosphate concentrations showed opposite trends with salinity in Bras del
Port and La Trinitat, with a slight increase in the former
and a slight decrease in the latter. Nutrient concentrations
along the salinity gradient have been measured in several
salterns. There are large di¡erences in nutrient concentration among di¡erent salterns, to the point that salterns can
be classi¢ed as oligo-, meso- or eutrophic just as any other
aquatic environments [1]. These di¡erences are re£ected
both in planktonic chlorophyll a content and in primary
production, as well as in the predominance of autotrophic
production in the plankton or in the benthos. In this regard, both La Trinitat and Bras del Port salterns could be
assigned to the eutrophic saltern types.
3.2. The communities
In almost all cases, chlorophyll a concentration pre-
sented a similar pattern (Fig. 3A,B). Usually, chlorophyll
a increased from seawater up to a salinity around 100x
where it reached the highest concentration, then it decreased. In some cases, for example in Bras del Port, there
was a second smaller maximum around 250x. The ¢rst
maximum was due to diatoms and especially to ¢lamentous cyanobacteria (Fig. 3C,D). In e¡ect, the water in
these ponds had an intense blue^green color. These ponds
showed the highest concentrations of feeding birds, including £amingos, Mediterranean gulls and waders in the
shores. Many of the diatoms could be identi¢ed as benthic
and had probably been dislodged from the top sediment
layers by the biotic and/or wind-induced turbulence. The
water turned to brownish colors in successive ponds. The
second peak in Bras del Port was due to Dunaliella salina
(Fig. 3C). The maximal abundance of this alga sometimes coincided with the chlorophyll a maximum, but at
other times, the numbers kept increasing up to the crys-
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tallizers, despite the fact that pigment concentrations decreased.
A total of 50 (in La Trinitat) or 79 (in Bras del Port)
microbial taxons were identi¢ed by light microscopy. We
calculated a diversity index sensitive to richness (Margalef's) and another one sensitive to eveness (Berger-Parker's). The decrease in diversity with increasing salinity can
be appreciated with any of the indices chosen (Fig. 3E,F),
as well as in the total number of taxons (S).
The total number of heterotrophic prokaryotes (PN;
bacteria plus archaea) increased by an order of magnitude
from the lowest salinity up to around 200x and thereafter remained approximately constant (Fig. 4A,B). In
July, the crystallizers had an intense purplish color, due
to the halophilic archaea [37]. Usually, the cell number at
the lowest salinity pond was already very high, around
1010 cells l31 , and reached 1011 cells l31 at 200^250x
salinity. In the crystallizers, not only the cell number
was around 1011 cells l31 , but the cells were very large
rods and square-shaped archaea (mean cellular size of
0.72 Wm3 ). Consequently, biomass in these ponds was extremely large. The only case that departed from this pattern was the May 1992 sampling in La Trinitat (data not
shown), where the initial cell abundance was only 3U109
cells l31 but very rapidly increased to 1011 cells l31 at
100x salinity. As stated earlier, the spring situation can
be quite variable depending on the particular weather conditions, while the summer situation is very stable.
Heterotrophic nano£agellates (HNF) and ciliates decreased with increasing salinity, disappearing around
250x salinity (Fig. 4A,B). There were abundant Artemia
salina individuals around this salinity that coincided with
abundant D. salina populations.
3.3. Microbial activities
Planktonic primary production and respiration measurements are only available for Bras del Port salterns
(Fig. 4E). There were two peaks of primary production
coinciding with the two chlorophyll a peaks. Production
was about twice as high in the Dunaliella peak (with about
half the amount of chlorophyll a, but with higher biomass)
as in the cyanobacteria^diatom peak. This would be easier
to understand if the diatoms were inactive, having been
taken away from their natural benthic habitat. Due to the
uncertainties of the oxygen determinations in these highly
saline waters [19], however, these values can only be taken
as relative and approximate. Respiration was very low
compared to primary production. However, the values
presented in Fig. 4E are hourly values based on incubations that bracketed noon. Therefore, daily values of respiration could be several times higher while primary production values could be estimated to be at most twice the
measured values.
Activity of heterotrophic prokaryotes (HPP) was measured in two di¡erent ways. Leucine incorporation was
149
Fig. 5. Doubling times (Dt ) of the prokaryotic assemblage in each pond
throughout the salinity gradients of La Trinitat and Bras del Port salterns in di¡erent years. The values of heterotrophic prokaryotic production used to calculate doubling times were obtained with the standard
conversion factor (open symbols) or with empirical conversion factors
obtained in a di¡erent visit to the salterns (¢lled symbols, see text).
measured in Bras del Port and La Trinitat in 1993 and
1994 (Fig. 4C,D), and FDDC was determined in La Trinitat in 1992 and 1993 (Fig. 4F). In general, activity increased from the lowest salinity up to 150x. Next, activities decreased with increasing salinity up to 250^300x.
Finally, activity increased again at the highest salinity
ponds, i.e. the crystallizers. The activity maximum at
100^150x seems a logical consequence of the maxima
in both chlorophyll a and primary production at these
salinities. Between the two maxima, lower values could
be expected as neither heterotrophic bacteria nor heterotrophic archaea encountered optimal salinity conditions
for growth.
We used speci¢c protein synthesis inhibitors for prokaryotes in the summer samplings to assign leucine incorporation to either bacteria or archaea (Fig. 4C,D). Similar
patterns were observed in La Trinitat and Bras del Port.
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Taurocholate completely inhibited leucine incorporation in
the highest salinity ponds, while relatively little or no inhibition was found at the lowest salinities. The pattern of
leucine incorporation with erythromycin was the opposite.
Nevertheless, at the highest salinities, erythromycin inhibited about half of the leucine incorporation. There have
been recent reports that at least one halophilic bacterium
was also present in the crystallizers (J. Antön and R. Rossellö-Mora, personal communication). However, given the
complete inhibition by taurocholate in these ponds, it is
di¤cult to decide whether there was partial inhibition of
the archaea by erythromycin or whether the bacterium
was sensitive to taurocholate.
Doubling times (Dt ) of the prokaryotic assemblages
were calculated in two di¡erent ways: ¢rst, a standard
conversion factor from the literature [26] was used to calculate PHP from leucine incorporation and, second, conversion factors determined in a recent visit to Bras del Port
in May 1999 were used for the same purpose. The two
estimates of Dt followed a similar trend with salinity
(Fig. 5). The same pattern was found both in Bras del
Port and in La Trinitat during May 93 and July 94. Dt
values were relatively short at the lower salinities and progressively increased along the salinity gradient. In fact, at
salinities 6 100x, Dt was very short (around 0.3 days,
W = 2.2 days31 ). At higher salinities, Dt was moderate (1^
20 days, W = 0.1^0.8 days31 ) in the summer samplings of
La Trinitat and Bras del Port, to very long (70 days,
W = 0.01 days31 ) in the spring sampling of La Trinitat.
As absolute values, these Dt , as those obtained by Oren
[12], could not be considered especially long (even in the
crystallizers). However, when these Dt are considered in
their high temperature context by using the predictive
equations of White et al. [38], they appear to be considerably lower than expected. Interestingly, only values of Dt
from salinities lower than 150x were similar to their
predictions according to White et al. [38].
Bacterivory was estimated in the summer visits to La
Trinitat and Bras del Port (Fig. 6). In both cases, no
bacterivory could be detected at salinities higher than
200x, in accordance with the low concentrations (or
even the absence) of HNF and ciliates (Fig. 4A,B). Bacterivory, on the other hand, was very intense at lower
salinities. In La Trinitat, it was maximal between 60 and
150x salinity and decreased both at higher and lower
salinities. In Bras del Port, bacterivory was uniformly
high throughout the lower range of salinities up to
250x. We calculated the percent of bacterial biomass
and production culled down by bacterivores every day.
In La Trinitat, 100% of heterotrophic prokaryotic production and 200% of the biomass were eaten per day. In Bras
del Port, the percentages were much lower, between 20
and 100% of the biomass and less than 40% of production.
4. Discussion
The sequential changes in the physico-chemical conditions and phytoplankton groups along the salinity gradient of salterns have been repeatedly described [1,2,39].
The disappearance of diatoms as salinity reaches 200x,
the predominance of cyanobacteria at around 100x, and
the peak of D. salina at 250x found in the salterns we
studied are all common features of this general pattern of
species substitution along the gradient. The changes in
prokaryotic heterotrophs have also been recorded through
the isolation of pure cultures from di¡erent ponds [4].
Thus, halotolerant bacteria predominate up to about
100x salinity; moderate halophilic bacteria are the
most abundant isolates in the 100^200x salinity range;
next, extreme halophilic bacteria plus halophilic archaea
coexist up to about 300x salinity ; and, ¢nally, only extreme halophilic prokaryotes have been isolated from the
higher salinity ponds including the crystallizers [2,12,31].
Recent molecular evidence suggests that these crystallizer
ponds may be co-cultures of a single phylotype of a new
halophilic archaea [40] and a couple of extremely halophilic bacteria (J. Antön and R. Rossellö-Mora, personal
communication). The progressive increase in salinity
makes each pond a more extreme environment than the
Fig. 6. Bacterivory in Bras del Port (A) and La Trinitat (B) salterns. %PN: bacterivory as percentage of prokaryotic biomass. %PHP : bacterivory as
percentage of prokaryotic heterotrophic production. Note the disappearance of bacterivory from 250x salinity on. The horizontal arrow indicates
100% of production or biomass.
FEMSEC 1126 10-5-00
C. Pedrös-Aliö et al. / FEMS Microbiology Ecology 32 (2000) 143^155
former and this, according to ecological theory, should
cause a decrease in the number of species present [41].
We were interested in seeing how this decrease in the
number of di¡erent organisms would a¡ect the structure
of the microbial food web. We expected that as more
complex organisms disappeared, less and less trophic steps
would be found. Eventually, all phagotrophic organisms
would disappear and only osmotrophic (i.e. prokaryotic)
relationships would be present. The question, therefore,
was whether the factors controlling prokaryotic abundance and production were the same in these extreme environments as in the more `common' systems found at
lower salinities.
Extremely few data are available on biomass and activity of bacterioplankton in salterns. The data of Rodr|¨guezValera et al. [4,18] were obtained by plate counts and are
more relevant for diversity studies than for biomass determinations. The data of Javor [42] only considered ponds
with salinities above 300x and, moreover, the total numbers found were strangely low when compared to more
recent estimates. Thus, we are only left with the counts
of Oren at the Eilat saltern [12,31], carried out by phase
contrast microscopy in counting chambers. In that study,
total prokaryotic counts increased sharply from sea water
salinity up to about 150x salinity. From a salinity of
150x upward, the total cell number did not seem to
change very much, oscillating around 5U1010 cells l31 .
These cell concentrations are towards the high end of
the range found in natural planktonic systems [43]. The
numbers obtained in the present work con¢rm this pattern.
Data on activity of bacterioplankton of solar salterns
are very scarce as well. The only reliable studies (i.e. using
relatively accurate techniques such as thymidine or leucine
incorporation) are those by Oren in an Eilat saltern. Oren
[12,31] estimated doubling times to be in the range of 1^23
days along the salinity gradient and, therefore, concluded
that, even in the most concentrated brines, doubling times
were not especially long. Both `bacteria' and `archaea' live
throughout the salinity gradient and eventually coexist in
several intermediate ponds. Oren [12,31] introduced an
ingenious way to determine separately the total number
and activity of bacteria and archaea in the same samples.
The technique uses bile salts to lyse archaea, and selective
inhibitors of bacterial protein synthesis (and thus leucine
incorporation) and/or DNA replication (and thus thymidine incorporation). In this manner, Oren [12,31] was able
to assign leucine and thymidine incorporation to each one
of these two groups of prokaryotes, and concluded that at
salinities of 250x and above, all thymidine incorporation
could be attributed to halophilic archaea. We used this
technique to assign leucine incorporation to bacteria or
to archaea. At the lower salinities, all incorporation was
clearly due to bacteria. At the highest salinities, the
archaeal inhibitor completely inhibited leucine incorporation, suggesting that the activity in the salterns was en-
151
tirely due to archaea. The bacterial inhibitor, however,
also caused an inhibition between 50 and 75% of the activity. This was found in both salterns studied. Thus, either erythromycin partially inhibits the archaea, or part of
the activity is due to bacteria sensitive to taurocholate.
More information is needed about the extreme halophilic
bacterium recently discovered in these ponds (J. Antön
and R. Rossellö-Mora, personal communication) to be
able to interpret our results.
4.1. Biotopes and communities
Although the gradient of salinity is practically continuous, it has been broken down into di¡erent regions to
describe the biota by Por [44], Ort|¨-Cabo et al. [3] and
Rodr|¨guez-Valera [2]. For the sake of clarity, we will describe only three di¡erent communities of major microbial
interest found at di¡erent points (Table 1 and Fig. 7). The
community in the ponds with salinities lower than 100x
will not be considered since it is essentially similar to the
coastal marine community. The next saltern community is
found between 100 and 150x salinity. It has a very large
phytoplankton biomass formed mostly by cyanobacteria,
green algae and diatoms, and primary production is high.
Abundance of heterotrophic prokaryotes (PN) is around
5U1010 cells l31 and both ciliates and HNF are present in
signi¢cant numbers. Prokaryotic heterotrophic production
(PHP) is very high, resulting in doubling times around
30 h, and is essentially all due to bacteria. Bacterivory is
intense, resulting in most of the PHP being removed daily
(Table 1 and Fig. 7).
The community found around 200^250x salinity may
or may not show the maximum of chlorophyll a almost
exclusively due to D. salina. A peak in primary production
may also be present. In these ponds, A. salina plays the
role of main predator on D. salina. Artemia is commonly
thought to severely reduce the populations of Dunaliella
when it is present in large numbers and, in turn, may be
cropped for commercial purposes or fed upon by birds.
The PN reach very high abundance, around 1011 cells l31 ,
but the activity is lower than at lower salinities, resulting
in doubling times between 2 and 6 days. The activity seems
to be approximately equally divided between bacteria and
archaea. Both ciliates and HNF are found in very low
abundance. Thus, bacterivory is below detection limits
(Table 1).
Finally, the community found above 300x salinity has
almost no chlorophyll a. If chlorophyll a is present, the
algae responsible (always D. salina) sometimes seem to be
in a non-functional state. PN are very abundant, also
around 1011 cells l31 , but the activity is generally low,
resulting in doubling times between 2 and 50 days (Table
1). The activity is mostly due to archaea. There are only
one archaeal and one or two bacterial dominant phylotypes in the plankton and there is no bacterivory.
Assuming a standard factor of 0.37 mg C ¢xed per mg
FEMSEC 1126 10-5-00
152
C. Pedrös-Aliö et al. / FEMS Microbiology Ecology 32 (2000) 143^155
O2 produced [45], it was possible to obtain rough estimates of carbon planktonic primary production for Bras
del Port samples. Assuming that prokaryotes respire 50%
of the carbon taken up, the prokaryotic carbon demand
was always (up to 280x salinity) higher than 100% primary production, while usual values in other, `normal'
systems are about 30^60%. Values similar to those found
in our salterns have been found only in typically heterotrophic systems such as benthos or estuaries, where most
PHP is sustained from allochthonous organic sources [46].
The organic carbon needed to sustain this PHP probably
originated by primary production in previous ponds. In
addition, birds and other animals present at intermediate
salinities (insect larvae, some other invertebrates and large
£ocks of £amingos, gulls and waders) can also play a role
in providing sources of organic matter through excretion.
Finally, along the salinity gradient up to 300x, there is a
rich benthic community, whose primary production could
be at least partially transferred to the planktonic food
web. The production of the benthic mats, however, has
never been evaluated in salterns.
In this regard, crystallizer ponds o¡er an especially interesting situation. The two ¢rst planktonic communities
(around 150x and 250x) have primary producers, but
that of the crystallizers does not. This is especially remarkable because of the fact that the largest prokaryotic biomass can be found in the latter community. Since there are
no benthic communities or macroorganisms at these salinities, the organic sources needed to sustain these prokaryotic communities must come from previous ponds, especially the D. salina ponds. In some cases, particularly early
in the season, Dunaliella cells might be active in the crystallizers.
4.2. Prokaryotic loss factors
We have studied the role of viruses in salterns in a
separate paper [35]. Brie£y, no incidence of viruses could
be detected in La Trinitat below 150x salinity, and the
impact of viruses was extremely moderate above this salinity. At most, 10% of the heterotrophic production was
removed by viral lysis per day. The non-detectable impact
of viruses at low salinities is probably related to the higher
diversity of the organisms present. If di¡erent populations
of bacteria were growing simultaneously, probably, any
particular naked virion would be inactivated by the strong
sun radiation in the shallow ponds before it had a chance
to hit a suitable prey cell. As diversity decreased along the
salinity gradient, however, chances of hitting sensitive
hosts increase and viruses might begin to play a role.
The reasons why there was not a higher impact of viruses
in the crystallizers, where probably a single species of archaea is present [40], are most likely related to the peculiar
dynamics of phage^host systems of archaea (see [35]).
The community found between 100 and 150x had signi¢cant bacterivory, while the other two communities did
not. This was re£ected in the faster speci¢c growth rates of
the PN in the ¢rst community than in the other two. It
seems that bacterivory keeps the PN numbers lower than
the potential abundance (or carrying capacity), which is
probably the one reached in ponds with higher salinities.
Thus, the bacteria in the ¢rst community are probably
Table 1
Average values (or ranges) of di¡erent microbiological variables in the four domains of the two solar salterns studied
Saltern
La Trinitat
Bras del Port
Salinity domain (x)
6 100
100^150
200^250
s 300
6 100
100^150
200^250
s 300
Chlorophyll a (Wg l31 )
Cyanobacteria (% of biovolume)
Diatoms (%)
Green algae (%)
Dunaliella (%)
Prokaryotes PN (cells l31 )
Viruses (VLP l31 )
HNF (cells l31 )
Ciliates (mm3 l31 )
Primary production (mg C l31 day31 )
PHP (mg C l31 day31 )a
Bacteria (%)
Archaea (%)
Bacterivory (cells l31 day31 )b
(% of PHP)
Viral lysis (cells l31 day31 )b
(% of PHP)
4.2
0^9
46^90
51^0
0
9.8U109
4.4U1010
5.6U107
6.4
nd
0.6
100
0
8.9U108
65
0
0
19.5
20^100
80^69
0
0
1.1U1010
5.6U1010
3.7U108
3.4
nd
1.1
100
0
1.3U109
91
1.1U106
2
12.8
100^4
0
0
0^96
4.4U1010
4.4U1011
8.5U107
3.2
nd
0.7
30
70
3.2U108
19
1.6U107
11
1.9
0.9^0
0
0
99^100
1.3U1011
1.3U1012
0
0
nd
0.8
v2
998
0
0
1.6U107
7
4.8
1
68^99
0^32
0
1.2U1010
4U1010
nd
0.13
bd
0.06
91^62
4^9
1.7U107
43
nd
nd
4.1
5^88
0^1
95^10
0^6
4.6U1010
5U1010
nd
0.10
0.7
0.08
83
48
9.7U106
40^1
nd
nd
3.6
9^4
0
17^0
74^96
9.1U1010
7U1011
nd
0.02
0.07^1.27
0.09
25
91
0
0
nd
nd
3.5
1^0
0
0
99^100
5.5U1010
1012
nd
6 0.01
bd
0.10
v0
9100
0
0
nd
nd
Symbols: nd, not determined; bd, below detection limits on the single date measured.
a
The percent distribution between bacteria and archaea does not always add up to 100% because the contributions were calculated from the percent of
leucine incorporation inhibited by erythromycin and taurocholate, respectively (see text).
b
Data from [35].
FEMSEC 1126 10-5-00
C. Pedrös-Aliö et al. / FEMS Microbiology Ecology 32 (2000) 143^155
153
Fig. 7. Simpli¢ed scheme of the microbial food web at three points along the salinity gradient.
growing at their maximal speci¢c growth rates. The picture is completely opposite in the other two communities:
in the absence of bacterivory, biomass is enormous but
speci¢c growth rates are moderate to slow (Fig. 5). In
fact, fastest doubling times for halophilic archaea in the
laboratory are around 0.25 days, more than eight times
faster than those recorded in the salterns. The three communities, therefore, provide a very clear example of the
way in which speci¢c growth rates and biomass of PN
are controlled in the plankton: predation coincides with
high speci¢c growth rates and lower total biomass. The
absence of predation is found in the opposite situation:
slow growth and large biomass (see [17,47]).
Considering the whole food web in the three communities, their structure and £uxes su¡er dramatic changes
along the salinity gradient (Figs. 3, 4, 6 and 7). The
100^150x salinity community has an intricate web, including benthos, a large variety of phytoplankton taxons
and macroorganisms. As we proceed to the 250x salinity
community, the web has been dramatically simpli¢ed.
There is only one primary producer D. salina and only
one predator A. salina, forming a single, linear, and short
`classical' food chain. In this community, bacterivores disappear and viruses play a small role. Finally, there are
essentially no primary producers and no predators in the
crystallizers. Only two or three prokaryotes and their viruses grow slowly and maintain a tremendous amount of
biomass. Thus, the di¡erent ponds along the salterns provide a whole range of ecosystems with food webs of widely
di¡erent complexities.
FEMSEC 1126 10-5-00
154
C. Pedrös-Aliö et al. / FEMS Microbiology Ecology 32 (2000) 143^155
Acknowledgements
This study was supported by DGICYT Grants PB91075 and PB95-0222-C02-01. Dr. M.H. MacLean was supported by fellowships from the Royal Society and the
Spanish Ministerio de Educaciön y Ciencia. We thank
Mr. Juan Duch from INFOSA SA and Mr. Miguel Cuervo-Arango for permission to work at La Trinitat and Bras
del Port salterns, respectively. Laura Ar|¨n determined
chlorophyll a values in the 1992 and 1993 visits to La
Trinitat and R. Guerrero greatly facilitated logistics during the same visits. F. Rodr|¨guez-Valera provided invaluable help during the work at the Bras del Port salterns.
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