The microbial food web along salinity gradients

The microbial food web along salinity gradients

Carlos Pedros-Alio *, Juan I. Calderon-Paz, Marlie H. MacLean, Glo©ria Medina,Ce©lia Marrase, Josep M. Gasol, Nuria Guixa-Boixereu

Departament de Biologia Marina i Oceanogra¢a, Institut de Cie©ncies del Mar, CSIC, Passeig Joan de Borbo 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. Thedifferent ponds in the salterns provide a wide range of ecosystems with food webs of different complexities. Abundance of prokaryotes, cellvolume, prokaryotic heterotrophic production, chlorophyll a, abundance of heterotrophic flagellates, ciliates and phytoplankton weredetermined in several ponds in each saltern. Increases in salinity resulted in a progressive reduction in the abundance and number ofdifferent groups of eukaryotic microorganisms present, but an increase in biomass of prokaryotes. Maximal activity of both phyto- andbacterioplankton was found at a salinity of around 100x, where there was also a maximum in chlorophyll a concentration. Growth ratesof heterotrophic prokaryotes decreased with increasing salinity. Bacterivory disappeared above 250x salinity, whereas other loss factorssuch 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 environ-ments with di¡erent salinity, from that of seawater up tosodium chloride saturation and sometimes even beyond(chapter 13 in [1,2]). As water evaporates and salinity in-creases, water is pumped or fed by gravity to the nextpond, such that salinity in each particular pond is keptwithin narrow limits, essentially constant. Each pond canthus be considered at equilibrium and the biota in anygiven pond as a well-adapted and established communityfor that particular salinity. The two extremes of salinityprovide one of the most common habitats (seawater) andone of the most extreme habitats in the world (calciumand magnesium chloride-saturated brines). In no other

system is there such a complete gradient of environmentswith di¡erent salinities available within walking distance.Thus, the response of the food web structure, the £uxes ofelements, the diversity of the community and the adapta-tions of organisms can easily be traced by simultaneouslyanalyzing several di¡erent ponds along the gradient. Salt-erns are particularly interesting for microbial ecology,since large organisms disappear early in the salinity gra-dient, and predominantly or exclusively microbial com-munities are found at the highest salinity.

Given these advantages, it is surprising how few studiesof the microbial ecology of saltern ponds have been car-ried out. Saltern ponds have been used mostly for biogeo-chemical studies [1], as models for ancient evaporitic envi-ronments [3], and as a source of halophilic andhalotolerant microorganisms [4^6]. Ecological studieshave been limited to descriptions of the peculiar organismsfound at di¡erent salinities, with attention to their respec-tive salinity ranges [7], but with little concern for quanti-tation of their biomass and activities. In particular, there isan extensive literature describing the most conspicuousmembers of the microbial mats found at the bottom ofmany ponds, but again, with little quanti¢cation of bio-mass 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

* Corresponding author. Tel. : +34 (93) 221-6416;Fax: +34 (93) 221-7340; E-mail : [email protected]

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trend of a decreasing number of species as salinity in-creases are well-characterized, but most functional aspectsremain unknown. Thus, for example, there is debate aboutwhether Dunaliella and haloarchaea grow fast, slow or notat all at the ponds where they show maximal abundance[1,2,12], or about what is the salinity of maximal primaryproduction. In particular, some trophic relationships suchas herbivory and bacterivory have never been measured inthe ¢eld. Thus, we were interested in determining a seriesof microbial food web parameters along the salinity gra-dient, with special attention to protistan bacterivory, pro-tistan biomass, as well as production and speci¢c growthrates of prokaryotic organisms.

Study of the microbial food web in saltern ponds o¡ersan additional advantage. A paradigm of planktonic foodweb structure and function emerged during the 80s [13^16]and many aspects have been tested experimentally in alarge number of marine and freshwater environments.The time has come, therefore, when analysis of systemswhich depart from the standard may fruitfully show theinconsistencies of the current paradigm. Salterns also pro-vide systems with progressively more simpli¢ed food webs.What happens when grazers of prokaryotes disappear?How does a food web formed only by prokaryotes func-tion? Do lytic viruses become an important loss factor?These and many other questions could be answered bystudying saltern ponds. In this paper, we describe the mi-crobial food webs in two di¡erent salterns. In a separatepaper [17], we compare the results from the salterns to thecurrent paradigm of microbial plankton.

2. Materials and methods

2.1. Salterns

We studied two di¡erent solar salterns on the Mediter-ranean coast of the Iberian Peninsula: `La Trinitat' in theEbro River Delta (Tarragona, Spain, 40³35PN, 0³41PE)and `Bras del Port' in Santa Pola (Alicante, Spain,38³12PN, 0³36PW). The Bras del Port salterns have beenused for microbiological studies in the past, especially as asource of halophilic bacteria and archaea [2,4,18]. On theother hand, the microbial plankton of La Trinitat (Fig. 1)has not been studied before. The two salterns are operatedfor commercial salt production in di¡erent ways. Bras delPort is active year-round. Thus, each pond tends to pro-vide a very stable environment. La Trinitat does not oper-ate during the fall and winter and, therefore, ponds do notreach their de¢nitive salinity until late June or July. As aresult, there is a seasonal succession in each pond, and thecrystallizers are drained and salt is harvested in Augustevery year. The most complete sampling was carried outin late July in both salterns (July 1993 for Bras del Portand July 1994 for La Trinitat) to ensure that ponds hadreached their de¢nitive salinity. Additional samplings werecarried out in La Trinitat during the spring (May 1992 andMay 1993) to get some impression of seasonal changes.

Sampling was carried out with a bucket ¢xed to the endof a pole. The corners of the ponds were avoided sinceorganisms 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 thespiral layout of the increasingly saline ponds: `depositos', `calentadores' (1^10) and ¢nally, `cristalizadores' (crystallizers).

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essed in a day. When nine or 10 ponds were sampled, thewhole operation took 2 or 3 days. All samples were ¢xedand incubations carried out in the ¢eld.

2.2. Physico-chemical analyses

Temperature was measured with a mercury thermome-ter. Opaque BOD bottles were ¢lled and ¢xed for dis-solved oxygen determinations by a modi¢cation of theWinkler titration [19]. Samples for inorganic nutrientswere collected in acid-rinsed tubes and frozen until analy-sis. Ammonia, nitrate, nitrite and phosphate were ana-lyzed by conventional methods [20] with a Techniconautoanalyzer. Samples had to be diluted to approximatesea water salinity before measurements were carried out.Salinity was determined in the ¢eld with a hand-held re-fractometer. For salinity higher than 130x, water fromthe ponds had to be diluted with distilled water to fallwithin the scale of the refractometer.

2.3. Abundance of microorganisms

Chlorophyll a was determined by £uorescence in ace-tone extracts [21]. Water samples were ¢ltered on GF/Fglass ¢ber ¢lters and the ¢lters placed in tubes with ace-tone. The tubes were kept cool until processed. Glasspearls were added to each tube and these were violentlyagitated in a high-speed cell disrupter (Vibrogen-Zell-mu«hle, Germany) to break up the cells. After half an

hour in the dark, the tubes were spun in a benchtop cen-trifuge and the £uorescence of the supernatant read with aTurner Designs £uorometer.

Duplicate 30-ml samples were ¢xed with formaldehydefor counts of prokaryotes and £agellates by epi£uores-cence microscopy. Samples used for the determination ofprokaryotic abundance had to be diluted from 10 to100 times and were then ¢ltered through 0.2-Wm pore di-ameter black polycarbonate ¢lters and stained with DAPI(0.1 Wg ml31 ¢nal concentration) for 5 min before dryingthe ¢lters by suction [22]. Filters were then mounted onmicroscope slides with non-£uorescent oil (R.P. CargilleLab.). Filters were counted by epi£uorescence microscopywith a Nikon Diaphot microscope. About 200^400 cellswere counted per sample. Cell volumes were determinedwith an image analysis system measuring at least 200 cellsper sample. A Hamamatsu C2400-08 video camera wasused to examine microscopic preparations. Images werecaptured in a PC with the software MIP (from MicromEspan¬a SA). The video images were downloaded to aMacintosh computer and analyzed with the sharewareprogram NIH Image. Objects occupying less than sevenpixels (equivalent to an sphere with diameter less than0.2 Wm) were discarded. The remaining bacteria-like ob-jects were measured and the volume calculated from areaand perimeter measurements with the formula of Fry [23].The system was calibrated with £uorescent latex beads andwith natural bacterioplankton samples measured simulta-neously 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 outin July 1993 and July 1994, respectively. (A, B) Temperature and dissolved oxygen; (C, D) Concentration of inorganic nutrients (ammonia, nitrate, ni-trite and phosphate).

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cence [24]. Biomass was calculated by using the carbonto volume relationship derived by Norland [25] from thedata 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 bea parallel sided with the two measured dimensions plusheight taken from the electron micrographs of Stoeckenius[27].

The abundance of nano and microplankton was deter-mined by the Utermo«hl technique. Samples were ¢xedwith Lugol's solution and kept in the dark until counted.Volumes of 100 ml were allowed to settle for 24^48 h andexamined in a Zeiss inverted microscope. The whole bot-tom chamber was examined at 20U and 40U magni¢ca-tion for the larger and less abundant microorganisms.Then, 200U and 400U magni¢cations were used to countthe remaining microorganisms in transects chosen at ran-dom. Cells were assigned to morphotypes and these wereassigned to one of the large taxonomic groups shown in

Fig. 3. Linear dimensions of at least 20 cells per taxonwere determined and the volume calculated assuming clos-est geometrical shapes.

2.4. Microbial activities

Primary production and respiration in the planktonwere measured by oxygen changes in clear and darkBOD bottles incubated in the ponds for about 4 h aroundnoon. Four clear and four dark bottles were incubated ineach pond. Some ponds were supersaturated with oxygenwhile others had very low oxygen concentrations due tothe low solubility of oxygen at extremely high salinity andtemperatures. Thus, we decreased the oxygen concentra-tion in the samples for the clear bottles by stripping withoxygen-free nitrogen gas in a 1 liter graduated cylinder. Aportion of this water was ¢xed in a 150-ml BOD bottle forWinkler titration and the rest was distributed to the incu-bation bottles. In addition, we measured oxygen with anelectrode before starting the incubations and at the end by

Fig. 3. Abundance and biomass estimates of autotrophic microbial plankton in Bras del Port and La Trinitat salterns. (A) Biovolume of phytoplanktonand chlorophyll a concentrations in Bras del Port, July 1993; (B) Biovolume of phytoplankton and chlorophyll a concentrations in La Trinitat, July1994; (C) Biovolume of the di¡erent phytoplankton groups detected in Bras del Port; (D) Biovolume of the di¡erent phytoplankton groups detected inLa Trinitat ; (E) Diversity of ciliates and phototrophic microbes as measured by S (total number of taxons), and two diversity indices, Maregalef's andBerger-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) atV100x and V250x salinity.

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sacri¢cing one of the four replicates. This aided in deter-mining the best incubation times for the incubations car-ried out the following day. The other three replicates were¢xed for Winkler titration in the laboratory.

We carried out [3H]leucine incorporation assays in allsamplings according to Kirchman [28] with slight modi¢-cations. In one case (La Trinitat 1994), we also measured[3H]thymidine incorporation according to Bell [29]. And intwo cases (La Trinitat 1992 and 1993), we determined thefrequency of dividing plus divided cells (FDDC; [30]). Thethymidine incorporation data have already been presentedin Guixa-Boixereu et al. (Fig. 3A in [35]) and will not beshown here. The two measurements agreed fairly well,producing doubling times that were not signi¢cantly di¡er-ent. 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¢cantdeparture from this ratio (47.3).

In the radioactivity incubations, 5-ml samples were in-cubated with 40 nM leucine (1:9 hot:cold v/v, around 150Ci mmol31) in plastic scintillation vials. This concentra-tion was shown to be saturating in experiments carried outin the May 1993 visit to La Trinitat. Two replicates plus aformaldehyde-killed control (4% ¢nal concentration) wereincubated for each pond. Incubations were carried out inthe dark at ambient temperature for 45 min. Samples werekept in a cooler with ice until returned to the laboratory.There, samples were ¢ltered and counted in an LKB-Rackbeta liquid scintillation counter.

Oren [12,31] introduced a way to determine the activityof bacteria and archaea separately in the same samples.The technique uses bile salts to lyse archaea. Thus, activityin samples treated with bile salt corresponds to bacteria,while the activity in untreated samples corresponds to bac-teria plus archaea. However, this technique is not abso-lutely foolproof since Halococcus and some Halobacteriumspecies are not lysed by sodium taurocholate (the deter-gent commonly used; [12,31]), although Halococcus is be-lieved to play a small role in natural systems [2,12,31]. Adouble check can be done by carrying out parallel incuba-tions with an inhibitor of bacteria. On two occasions, weused this methodology to assign leucine incorporation tobacteria or to archaea. Erythromycin (at a ¢nal concen-tration of 20 Wg ml31) was used to inhibit bacterial leucineincorporation (i.e. protein synthesis). Na-taurocholate (ata ¢nal concentration of 50 Wg ml31) was used to lyse ar-chaea and thus avoid leucine incorporation by these or-ganisms [12,31]. All inhibitors were added before the startof incubations.

Data for leucine incorporation were converted to esti-mates of prokaryotic production by using the conversionfactor 3.1 kg C mol31 incorporated [26]. Speci¢c growthrates (W) and doubling times (Dt) were calculated fromthese production estimates and those of total prokaryoticbiomass. We determined empirical conversion factors in arecent visit to the Bras del Port salterns (May 1999). Sinceone could argue that conversion factors could have been

di¡erent in 1993, we have chosen to present the growthrates calculated with both the standard conversion factor[26] and with the conversion factors determined in 1999.Despite di¡erences in absolute values, the trends andranges of values are essentially the same, thus giving con-¢dence to our conclusions.

The term bacterivory will be used for convenience whendesigning predation on both bacteria and archaea. Bacter-ivory was determined with £uorescently labeled bacteria(FLB; [32]), using the FLB disappearance method [33].FLB were prepared from a heterotrophic bacterium iso-lated from the Mediterranean coast (1U0.8 Wm, bio-volume: 0.42 Wm3). This cell size is a compromise betweenthe average cell volume at the lowest salinity ponds (0.084Wm3) and at the highest salinity where bacterivory wasmeasured (0.916 Wm3). Bacterivory experiments were car-ried out in all ponds except for the crystallizers (with sal-inity s 300x). At these high salinities, no predators werepresent, since neither £agellates nor ciliates can live in thecrystallizers. Samples of 1 liter were incubated in acid-washed 2.5-l polycarbonate bottles in the dark and ambi-ent temperature for 48 h. Two replicates and a formalde-hyde-killed control (1% ¢nal concentration) were incu-bated per pond. Bacteria and FLB were counted attimes 0 and 48 h in the experimental bottles and in thecontrols, and bacterivory was calculated using the formulaof Salat and Marrase [34]. Bacterivory results from LaTrinitat appeared in a previous paper [35] where theywere compared to viral lysis. Here they are presentedagain for comparative purposes with the results from theBras del Port salterns.

3. Results

The data sets for Bras del Port and La Trinitat collectedin late July are presented together. These correspond tofully developed stable conditions in the di¡erent pondsand are considered representative of the salterns. Thedata collected in La Trinitat during the spring are referredto only sporadically to make speci¢c points.

3.1. Physico-chemical conditions

Fig. 2A,B presents temperature and oxygen concentra-tion in the salterns. As salinity increased, temperature alsoincreased due to the higher calori¢c capacity of the brines,while oxygen decreased due to the lower solubility of oxy-gen in the brines. Supersaturation was common in manyponds where primary production quickly overrode the sol-ubility of oxygen for the given temperature and salinity.

Inorganic nutrient levels were similar to those reportedfor other salterns (for example the Western Salt salterns;[36]). Nitrate was found in higher concentrations at thehighest salinities (Fig. 2C,D). Ammonia was, by far, themost abundant inorganic nutrient, perhaps due to excre-

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tion by protists and invertebrates. Phosphate concentra-tions showed opposite trends with salinity in Bras delPort and La Trinitat, with a slight increase in the formerand a slight decrease in the latter. Nutrient concentrationsalong the salinity gradient have been measured in severalsalterns. There are large di¡erences in nutrient concentra-tion among di¡erent salterns, to the point that salterns canbe classi¢ed as oligo-, meso- or eutrophic just as any otheraquatic environments [1]. These di¡erences are re£ectedboth in planktonic chlorophyll a content and in primaryproduction, as well as in the predominance of autotrophicproduction in the plankton or in the benthos. In this re-gard, both La Trinitat and Bras del Port salterns could beassigned 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, chlorophylla increased from seawater up to a salinity around 100xwhere it reached the highest concentration, then it de-creased. In some cases, for example in Bras del Port, therewas a second smaller maximum around 250x. The ¢rstmaximum was due to diatoms and especially to ¢lamen-tous cyanobacteria (Fig. 3C,D). In e¡ect, the water inthese ponds had an intense blue^green color. These pondsshowed the highest concentrations of feeding birds, includ-ing £amingos, Mediterranean gulls and waders in theshores. Many of the diatoms could be identi¢ed as benthicand had probably been dislodged from the top sedimentlayers by the biotic and/or wind-induced turbulence. Thewater turned to brownish colors in successive ponds. Thesecond peak in Bras del Port was due to Dunaliella salina(Fig. 3C). The maximal abundance of this alga some-times coincided with the chlorophyll a maximum, but atother times, the numbers kept increasing up to the crys-

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£agel-lates (HNF) and biovolume of ciliates in La Trinitat in July 1994; (C) Total prokaryotic leucine incorporation (Leu, total), leucine incorporation in thepresence of the protein synthesis inhibitor erythromycin, speci¢c for bacteria (Leu+E), and leucine incorporation in the presence of the lysing agenttaurocholate, 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 res-piration in Bras del Port; (F) Total prokaryotic leucine incorporation (Leu), frequency of dividing (FDC), and dividing plus divided (FDDC) cells inLa Trinitat in May 1993. Error bars indicating standard error of the mean are shown for activity measurements. When bars are not visible, error issmaller than symbol.

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tallizers, despite the fact that pigment concentrations de-creased.

A total of 50 (in La Trinitat) or 79 (in Bras del Port)microbial taxons were identi¢ed by light microscopy. Wecalculated a diversity index sensitive to richness (Marga-lef's) and another one sensitive to eveness (Berger-Park-er's). The decrease in diversity with increasing salinity canbe 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 magnitudefrom the lowest salinity up to around 200x and there-after remained approximately constant (Fig. 4A,B). InJuly, the crystallizers had an intense purplish color, dueto the halophilic archaea [37]. Usually, the cell number atthe lowest salinity pond was already very high, around1010 cells l31, and reached 1011 cells l31 at 200^250xsalinity. In the crystallizers, not only the cell numberwas around 1011 cells l31, but the cells were very largerods and square-shaped archaea (mean cellular size of0.72 Wm3). Consequently, biomass in these ponds was ex-tremely large. The only case that departed from this pat-tern was the May 1992 sampling in La Trinitat (data notshown), where the initial cell abundance was only 3U109

cells l31 but very rapidly increased to 1011 cells l31 at100x salinity. As stated earlier, the spring situation canbe quite variable depending on the particular weather con-ditions, while the summer situation is very stable.

Heterotrophic nano£agellates (HNF) and ciliates de-creased with increasing salinity, disappearing around250x salinity (Fig. 4A,B). There were abundant Artemiasalina individuals around this salinity that coincided withabundant D. salina populations.

3.3. Microbial activities

Planktonic primary production and respiration mea-surements are only available for Bras del Port salterns(Fig. 4E). There were two peaks of primary productioncoinciding with the two chlorophyll a peaks. Productionwas about twice as high in the Dunaliella peak (with abouthalf the amount of chlorophyll a, but with higher biomass)as in the cyanobacteria^diatom peak. This would be easierto understand if the diatoms were inactive, having beentaken away from their natural benthic habitat. Due to theuncertainties of the oxygen determinations in these highlysaline waters [19], however, these values can only be takenas relative and approximate. Respiration was very lowcompared to primary production. However, the valuespresented in Fig. 4E are hourly values based on incuba-tions that bracketed noon. Therefore, daily values of res-piration could be several times higher while primary pro-duction values could be estimated to be at most twice themeasured values.

Activity of heterotrophic prokaryotes (HPP) was mea-sured in two di¡erent ways. Leucine incorporation was

measured in Bras del Port and La Trinitat in 1993 and1994 (Fig. 4C,D), and FDDC was determined in La Tri-nitat in 1992 and 1993 (Fig. 4F). In general, activity in-creased from the lowest salinity up to 150x. Next, activ-ities decreased with increasing salinity up to 250^300x.Finally, activity increased again at the highest salinityponds, i.e. the crystallizers. The activity maximum at100^150x seems a logical consequence of the maximain both chlorophyll a and primary production at thesesalinities. Between the two maxima, lower values couldbe expected as neither heterotrophic bacteria nor hetero-trophic archaea encountered optimal salinity conditionsfor growth.

We used speci¢c protein synthesis inhibitors for pro-karyotes in the summer samplings to assign leucine incor-poration to either bacteria or archaea (Fig. 4C,D). Similarpatterns were observed in La Trinitat and Bras del Port.

Fig. 5. Doubling times (Dt) of the prokaryotic assemblage in each pondthroughout the salinity gradients of La Trinitat and Bras del Port salt-erns in di¡erent years. The values of heterotrophic prokaryotic produc-tion used to calculate doubling times were obtained with the standardconversion factor (open symbols) or with empirical conversion factorsobtained in a di¡erent visit to the salterns (¢lled symbols, see text).

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Taurocholate completely inhibited leucine incorporation inthe highest salinity ponds, while relatively little or no in-hibition was found at the lowest salinities. The pattern ofleucine incorporation with erythromycin was the opposite.Nevertheless, at the highest salinities, erythromycin inhib-ited about half of the leucine incorporation. There havebeen recent reports that at least one halophilic bacteriumwas also present in the crystallizers (J. Anton and R. Ros-sello-Mora, personal communication). However, given thecomplete inhibition by taurocholate in these ponds, it isdi¤cult to decide whether there was partial inhibition ofthe archaea by erythromycin or whether the bacteriumwas sensitive to taurocholate.

Doubling times (Dt) of the prokaryotic assemblageswere calculated in two di¡erent ways: ¢rst, a standardconversion factor from the literature [26] was used to cal-culate PHP from leucine incorporation and, second, con-version factors determined in a recent visit to Bras del Portin May 1999 were used for the same purpose. The twoestimates of Dt followed a similar trend with salinity(Fig. 5). The same pattern was found both in Bras delPort and in La Trinitat during May 93 and July 94. Dt

values were relatively short at the lower salinities and pro-gressively increased along the salinity gradient. In fact, atsalinities 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 ofLa 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 thecrystallizers). However, when these Dt are considered intheir high temperature context by using the predictiveequations of White et al. [38], they appear to be consid-erably lower than expected. Interestingly, only values of Dt

from salinities lower than 150x were similar to theirpredictions according to White et al. [38].

Bacterivory was estimated in the summer visits to LaTrinitat and Bras del Port (Fig. 6). In both cases, nobacterivory could be detected at salinities higher than

200x, in accordance with the low concentrations (oreven the absence) of HNF and ciliates (Fig. 4A,B). Bac-terivory, on the other hand, was very intense at lowersalinities. In La Trinitat, it was maximal between 60 and150x salinity and decreased both at higher and lowersalinities. In Bras del Port, bacterivory was uniformlyhigh throughout the lower range of salinities up to250x. We calculated the percent of bacterial biomassand production culled down by bacterivores every day.In La Trinitat, 100% of heterotrophic prokaryotic produc-tion and 200% of the biomass were eaten per day. In Brasdel Port, the percentages were much lower, between 20and 100% of the biomass and less than 40% of production.

4. Discussion

The sequential changes in the physico-chemical condi-tions and phytoplankton groups along the salinity gra-dient of salterns have been repeatedly described [1,2,39].The disappearance of diatoms as salinity reaches 200x,the predominance of cyanobacteria at around 100x, andthe peak of D. salina at 250x found in the salterns westudied are all common features of this general pattern ofspecies substitution along the gradient. The changes inprokaryotic heterotrophs have also been recorded throughthe isolation of pure cultures from di¡erent ponds [4].Thus, halotolerant bacteria predominate up to about100x salinity; moderate halophilic bacteria are themost abundant isolates in the 100^200x salinity range;next, extreme halophilic bacteria plus halophilic archaeacoexist up to about 300x salinity; and, ¢nally, only ex-treme halophilic prokaryotes have been isolated from thehigher salinity ponds including the crystallizers [2,12,31].Recent molecular evidence suggests that these crystallizerponds may be co-cultures of a single phylotype of a newhalophilic archaea [40] and a couple of extremely halo-philic bacteria (J. Anton and R. Rossello-Mora, personalcommunication). The progressive increase in salinitymakes 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 aspercentage of prokaryotic heterotrophic production. Note the disappearance of bacterivory from 250x salinity on. The horizontal arrow indicates100% of production or biomass.

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former and this, according to ecological theory, shouldcause a decrease in the number of species present [41].We were interested in seeing how this decrease in thenumber of di¡erent organisms would a¡ect the structureof the microbial food web. We expected that as morecomplex organisms disappeared, less and less trophic stepswould be found. Eventually, all phagotrophic organismswould disappear and only osmotrophic (i.e. prokaryotic)relationships would be present. The question, therefore,was whether the factors controlling prokaryotic abun-dance and production were the same in these extreme en-vironments as in the more `common' systems found atlower salinities.

Extremely few data are available on biomass and activ-ity of bacterioplankton in salterns. The data of Rodr|guez-Valera et al. [4,18] were obtained by plate counts and aremore relevant for diversity studies than for biomass deter-minations. The data of Javor [42] only considered pondswith salinities above 300x and, moreover, the total num-bers found were strangely low when compared to morerecent estimates. Thus, we are only left with the countsof Oren at the Eilat saltern [12,31], carried out by phasecontrast microscopy in counting chambers. In that study,total prokaryotic counts increased sharply from sea watersalinity up to about 150x salinity. From a salinity of150x upward, the total cell number did not seem tochange very much, oscillating around 5U1010 cells l31.These cell concentrations are towards the high end ofthe range found in natural planktonic systems [43]. Thenumbers obtained in the present work con¢rm this pat-tern.

Data on activity of bacterioplankton of solar salternsare very scarce as well. The only reliable studies (i.e. usingrelatively accurate techniques such as thymidine or leucineincorporation) are those by Oren in an Eilat saltern. Oren[12,31] estimated doubling times to be in the range of 1^23days along the salinity gradient and, therefore, concludedthat, even in the most concentrated brines, doubling timeswere not especially long. Both `bacteria' and `archaea' livethroughout the salinity gradient and eventually coexist inseveral intermediate ponds. Oren [12,31] introduced aningenious way to determine separately the total numberand activity of bacteria and archaea in the same samples.The technique uses bile salts to lyse archaea, and selectiveinhibitors of bacterial protein synthesis (and thus leucineincorporation) and/or DNA replication (and thus thymi-dine incorporation). In this manner, Oren [12,31] was ableto assign leucine and thymidine incorporation to each oneof these two groups of prokaryotes, and concluded that atsalinities of 250x and above, all thymidine incorporationcould be attributed to halophilic archaea. We used thistechnique to assign leucine incorporation to bacteria orto archaea. At the lower salinities, all incorporation wasclearly due to bacteria. At the highest salinities, thearchaeal inhibitor completely inhibited leucine incorpora-tion, suggesting that the activity in the salterns was en-

tirely due to archaea. The bacterial inhibitor, however,also caused an inhibition between 50 and 75% of the ac-tivity. This was found in both salterns studied. Thus, ei-ther erythromycin partially inhibits the archaea, or part ofthe activity is due to bacteria sensitive to taurocholate.More information is needed about the extreme halophilicbacterium recently discovered in these ponds (J. Antonand R. Rossello-Mora, personal communication) to beable to interpret our results.

4.1. Biotopes and communities

Although the gradient of salinity is practically continu-ous, it has been broken down into di¡erent regions todescribe the biota by Por [44], Ort|-Cabo et al. [3] andRodr|guez-Valera [2]. For the sake of clarity, we will de-scribe only three di¡erent communities of major microbialinterest found at di¡erent points (Table 1 and Fig. 7). Thecommunity in the ponds with salinities lower than 100xwill not be considered since it is essentially similar to thecoastal marine community. The next saltern community isfound between 100 and 150x salinity. It has a very largephytoplankton biomass formed mostly by cyanobacteria,green algae and diatoms, and primary production is high.Abundance of heterotrophic prokaryotes (PN) is around5U1010 cells l31 and both ciliates and HNF are present insigni¢cant numbers. Prokaryotic heterotrophic production(PHP) is very high, resulting in doubling times around30 h, and is essentially all due to bacteria. Bacterivory isintense, resulting in most of the PHP being removed daily(Table 1 and Fig. 7).

The community found around 200^250x salinity mayor may not show the maximum of chlorophyll a almostexclusively due to D. salina. A peak in primary productionmay also be present. In these ponds, A. salina plays therole of main predator on D. salina. Artemia is commonlythought to severely reduce the populations of Dunaliellawhen it is present in large numbers and, in turn, may becropped 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, resultingin doubling times between 2 and 6 days. The activity seemsto be approximately equally divided between bacteria andarchaea. Both ciliates and HNF are found in very lowabundance. Thus, bacterivory is below detection limits(Table 1).

Finally, the community found above 300x salinity hasalmost no chlorophyll a. If chlorophyll a is present, thealgae responsible (always D. salina) sometimes seem to bein a non-functional state. PN are very abundant, alsoaround 1011 cells l31, but the activity is generally low,resulting in doubling times between 2 and 50 days (Table1). The activity is mostly due to archaea. There are onlyone archaeal and one or two bacterial dominant phylo-types in the plankton and there is no bacterivory.

Assuming a standard factor of 0.37 mg C ¢xed per mg

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O2 produced [45], it was possible to obtain rough esti-mates of carbon planktonic primary production for Brasdel Port samples. Assuming that prokaryotes respire 50%of the carbon taken up, the prokaryotic carbon demandwas always (up to 280x salinity) higher than 100% pri-mary production, while usual values in other, `normal'systems are about 30^60%. Values similar to those foundin our salterns have been found only in typically hetero-trophic systems such as benthos or estuaries, where mostPHP is sustained from allochthonous organic sources [46].The organic carbon needed to sustain this PHP probablyoriginated by primary production in previous ponds. Inaddition, birds and other animals present at intermediatesalinities (insect larvae, some other invertebrates and large£ocks of £amingos, gulls and waders) can also play a rolein providing sources of organic matter through excretion.Finally, along the salinity gradient up to 300x, there is arich benthic community, whose primary production couldbe at least partially transferred to the planktonic foodweb. The production of the benthic mats, however, hasnever been evaluated in salterns.

In this regard, crystallizer ponds o¡er an especially in-teresting situation. The two ¢rst planktonic communities(around 150x and 250x) have primary producers, butthat of the crystallizers does not. This is especially remark-able because of the fact that the largest prokaryotic bio-mass can be found in the latter community. Since there areno benthic communities or macroorganisms at these salin-ities, the organic sources needed to sustain these prokary-otic communities must come from previous ponds, espe-cially the D. salina ponds. In some cases, particularly early

in the season, Dunaliella cells might be active in the crys-tallizers.

4.2. Prokaryotic loss factors

We have studied the role of viruses in salterns in aseparate paper [35]. Brie£y, no incidence of viruses couldbe detected in La Trinitat below 150x salinity, and theimpact of viruses was extremely moderate above this sal-inity. At most, 10% of the heterotrophic production wasremoved by viral lysis per day. The non-detectable impactof viruses at low salinities is probably related to the higherdiversity of the organisms present. If di¡erent populationsof bacteria were growing simultaneously, probably, anyparticular naked virion would be inactivated by the strongsun radiation in the shallow ponds before it had a chanceto hit a suitable prey cell. As diversity decreased along thesalinity gradient, however, chances of hitting sensitivehosts increase and viruses might begin to play a role.The reasons why there was not a higher impact of virusesin the crystallizers, where probably a single species of ar-chaea is present [40], are most likely related to the peculiardynamics of phage^host systems of archaea (see [35]).

The community found between 100 and 150x had sig-ni¢cant bacterivory, while the other two communities didnot. This was re£ected in the faster speci¢c growth rates ofthe PN in the ¢rst community than in the other two. Itseems that bacterivory keeps the PN numbers lower thanthe potential abundance (or carrying capacity), which isprobably the one reached in ponds with higher salinities.Thus, the bacteria in the ¢rst community are probably

Table 1Average 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) 4.2 19.5 12.8 1.9 4.8 4.1 3.6 3.5Cyanobacteria (% of biovolume) 0^9 20^100 100^4 0.9^0 1 5^88 9^4 1^0Diatoms (%) 46^90 80^69 0 0 68^99 0^1 0 0Green algae (%) 51^0 0 0 0 0^32 95^10 17^0 0Dunaliella (%) 0 0 0^96 99^100 0 0^6 74^96 99^100Prokaryotes PN (cells l31) 9.8U109 1.1U1010 4.4U1010 1.3U1011 1.2U1010 4.6U1010 9.1U1010 5.5U1010

Viruses (VLP l31) 4.4U1010 5.6U1010 4.4U1011 1.3U1012 4U1010 5U1010 7U1011 1012

HNF (cells l31) 5.6U107 3.7U108 8.5U107 0 nd nd nd ndCiliates (mm3 l31) 6.4 3.4 3.2 0 0.13 0.10 0.02 6 0.01Primary production (mg C l31 day31) nd nd nd nd bd 0.7 0.07^1.27 bdPHP (mg C l31 day31)a 0.6 1.1 0.7 0.8 0.06 0.08 0.09 0.10Bacteria (%) 100 100 30 v2 91^62 83 25 v0Archaea (%) 0 0 70 998 4^9 48 91 9100Bacterivory (cells l31 day31)b 8.9U108 1.3U109 3.2U108 0 1.7U107 9.7U106 0 0(% of PHP) 65 91 19 0 43 40^1 0 0Viral lysis (cells l31 day31)b 0 1.1U106 1.6U107 1.6U107 nd nd nd nd(% of PHP) 0 2 11 7 nd nd nd nd

Symbols: nd, not determined; bd, below detection limits on the single date measured.aThe percent distribution between bacteria and archaea does not always add up to 100% because the contributions were calculated from the percent ofleucine incorporation inhibited by erythromycin and taurocholate, respectively (see text).bData from [35].

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growing at their maximal speci¢c growth rates. The pic-ture is completely opposite in the other two communities:in the absence of bacterivory, biomass is enormous butspeci¢c growth rates are moderate to slow (Fig. 5). Infact, fastest doubling times for halophilic archaea in thelaboratory are around 0.25 days, more than eight timesfaster than those recorded in the salterns. The three com-munities, therefore, provide a very clear example of theway in which speci¢c growth rates and biomass of PNare controlled in the plankton: predation coincides withhigh speci¢c growth rates and lower total biomass. Theabsence of predation is found in the opposite situation:slow growth and large biomass (see [17,47]).

Considering the whole food web in the three commun-ities, their structure and £uxes su¡er dramatic changes

along the salinity gradient (Figs. 3, 4, 6 and 7). The100^150x salinity community has an intricate web, in-cluding benthos, a large variety of phytoplankton taxonsand macroorganisms. As we proceed to the 250x salinitycommunity, the web has been dramatically simpli¢ed.There is only one primary producer D. salina and onlyone predator A. salina, forming a single, linear, and short`classical' food chain. In this community, bacterivores dis-appear and viruses play a small role. Finally, there areessentially no primary producers and no predators in thecrystallizers. Only two or three prokaryotes and their vi-ruses grow slowly and maintain a tremendous amount ofbiomass. Thus, the di¡erent ponds along the salterns pro-vide a whole range of ecosystems with food webs of widelydi¡erent complexities.

Fig. 7. Simpli¢ed scheme of the microbial food web at three points along the salinity gradient.

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Acknowledgements

This study was supported by DGICYT Grants PB91-075 and PB95-0222-C02-01. Dr. M.H. MacLean was sup-ported by fellowships from the Royal Society and theSpanish Ministerio de Educacion y Ciencia. We thankMr. Juan Duch from INFOSA SA and Mr. Miguel Cuer-vo-Arango for permission to work at La Trinitat and Brasdel Port salterns, respectively. Laura Ar|n determinedchlorophyll a values in the 1992 and 1993 visits to LaTrinitat and R. Guerrero greatly facilitated logistics dur-ing the same visits. F. Rodr|guez-Valera provided invalu-able help during the work at the Bras del Port salterns.

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The microbial food web along salinity gradients