a vigorous specialized microbial food web in the suboxic waters of a shallow subtropical coastal...

MICROBIOLOGY OF AQUATIC SYSTEMS

AVigorous Specialized Microbial Food Web in the SuboxicWaters of a Shallow Subtropical Coastal Lagoon

Maria Luiza S. Fontes & Paulo C. Abreu

Received: 28 July 2011 /Accepted: 3 March 2012 /Published online: 27 March 2012# Springer Science+Business Media, LLC 2012

Abstract To examine the extent of the microbial food webin suboxic waters of a shallow subtropical coastal lagoon,the density and biomass of bacteria and protozooplanktonwere quantified under different dissolved oxygen (DO) lev-els. In addition, bottom waters of a stratified site werecompared with bottom waters of a homogeneous site underperiods of high and low biological oxygen production/con-sumption in the lagoon. At the stratified site, microbialbiomass decreased with oxygen decline, from oxia to sub-oxia, with a recovery of the initial total biomass after a 20-day period of persistent suboxia. A peak in density andbiomass of purple sulfur bacteria (PSB) (90 μg C L−1)occurred in the suboxic waters 20 days prior to the peak inbiomass of ciliates >50 μm (Loxophyllum sp. of 150 μm)(160 μg C L−1), demonstrating a top down biomass control.Ciliates >50 μm were positively correlated with PSB andbacteriochlorophyll a (photosynthetic pigment of PSB).Total protozoan biomass reached 430 μg C L−1 in thesuboxic waters of the stratified site, with ciliates >50 μmaccounting for 90% of the total ciliate biomass and of 55 %of biomass of protozoa. At the homogeneous site, totalprotozoan biomass was only 66 μg C L−1, where flagellatesand ciliates <25 μm were the dominant microorganisms.

Therefore, as light is available for primary producers in thebottom waters of shallow stratified coastal lagoons orestuaries, one can expect that high primary productionof PSB may favor a specialized microbial food webcomposed by larger microorganisms, accessible to zoo-plankton that tolerate low DO levels.

Introduction

The exponentially increasing use of fertilizers, especiallynitrogen, in agriculture since 1960, has resulted on thegreening of the water column by accumulation of algal(algal blooms) and higher plant biomass in coastal aquaticecosystems in the last decades [13]. This blooming pro-motes dissolved oxygen (DO) consumption through aerobicrespiration by heterotrophic microbes in bottom waters,resulting in a depletion of DO under low ventilated andstable (stratified) water column. Diaz and Rosenberg [13]pointed DO as the variable of ecological importance that haschanged more drastically over such a short period of time,and lately, hypoxia and anoxia are among the most delete-rious anthropogenic influences on estuarine and marineenvironments, since aerobic metazoans (e.g., crustaceans,fish, mollusks) cannot tolerate long periods of hypoxia (or“dead zones” 0 lack of metazoan life due to DO <2–3 mg L−1)[63]. Therefore, specific microorganisms (bacteria, Archaea,flagellates, and ciliates) proliferate in these hypoxic–anoxicbottom waters, and as they are unavailable to higher trophiclevel predators, these zones function as an “oasis” formicrobes, accumulating biomass [15, 25, 26].

As half of the global coastal hypoxic waters is seasonallyformed (persist from summer to fall due to strong stratifica-tion caused by warming of surface waters) [13], these tem-porary suboxic–anoxic waters contain a great diversity of

M. L. S. Fontes (*) : P. C. AbreuInstitute of Oceanography, Federal University of Rio Grande,Av. Itália km 8, Campus Carreiros,Rio Grande, Rio Grande do Sul, Brazile-mail: [email protected]

Present Address:M. L. S. FontesLaboratório de Bioquímica e Biologia Molecular deMicroorganismos, Departamento de Ecologia e Zoologia,Universidade Federal de Santa Catarina,Campus Trindade,Florianópolis, Santa Catarina, Brazil

Microb Ecol (2012) 64:334–345DOI 10.1007/s00248-012-0040-z

ecological niches, with a periodic shift on the metabolism:from aerobic to anaerobic dominated [19, 41, 50]. Furthermore,in shallow aquatic systems, sunlight reaches the bottomhypoxic–suboxic waters that allows the co-existence ofoxygenic and anaerobic anoxygenic photoautotrophicbacteria [6, 49, 55, 64]. However, the role of prokaryoticprimary producers on the production of organic particu-late carbon and its fate through the microbial food web isunderexplored [25].

Conceição Lagoon (located in Southern Brazil) is anexample of a shallow subtropical coastal lagoon with tem-porary hypoxic–suboxic waters from the end of summer toearly winter in the Central sector. Consequently, bacterialcommunity structure in the bottom waters of the stratifiedsector changes from a cyanobacteria-dominated in earlysummer to a heterotrophic-dominated in fall–winter [21],and that primary production (PP) in these suboxic waterscan be 15 times higher than PP at surface waters [22]. Onthe other hand, at the Northern sector where no stratificationis formed throughout the year, bacterial community struc-ture is the same throughout the water column [22]. Thus,understanding the amount of carbon produced by microbes(bacteria, flagellates, and ciliates) under different conditions(oxygen and mixing) and its possible fates is fundamentalin order to estimate potential food webs in shallowsystems [24, 55]. With the global expansion of thesehypoxic–suboxic waters, it becomes essential to under-stand the production and fate of the organic carbon pro-duced by microbes and how this carbon can be transferredto the metazoans in shallow systems that undergo periodicmixing or not.

In this work, we aimed (1) to determine the existence of amicrobial food web that would benefit from the high prima-ry production in suboxic waters of Conceição lagoon and (2)to compare the microbial assemblage (abundance and bio-mass) of the bottom waters of the stratified location sub-jected to oxic/suboxic periods with a homogeneouspermanent oxygenated location.

Methods

Site Description

Conceição lagoon (27°34′ S and 48°27′ W) (Fig. 1), locatedin Southern Brazil, presents mean and maximum depths of1.7 and 8.7 m, respectively [46]. The lagoon has beendivided into four (Southern, Central-Southern, Central-Northern, and North) or three sectors (integrating Central-Northern with Northern sector). We collected water samplesfrom the homogeneous Northern sector and from the mer-omictic Central-Southern sector between January and Mayof 2007. Permanent stratification at Central sector is resulted

from its proximity to the connection with the sea, andtemporary suboxic–anoxic bottom waters develop seasonally,from the end of summer until early winter [21, 38].Consequently, hypolimnetic waters accumulate bacterialcells [21, 22].

Sample Collection and Analyses

Only the bottom water (hypolimnetic) microbial carbonflow is investigated in this research due to the representa-tiveness of local processes and to understand the dynamicsof oxygen along with major microbes. Surface water char-acteristics, biological production, and consumption of oxy-gen are presented elsewhere [22]. In order to demonstratesuch dynamics, water from 0.5 m above the sediment wascollected with a 3-L van Dorn bottle at two regions: at thestratified CS sector and at the homogeneous Northern sector,or sites 33 and 82, respectively (Fig. 1). Both sites are about

Figure 1 Location of sampling sites in the Conceição Lagoon, SouthernBrazil

Microbial Carbon in Suboxic Waters 335

the same depth (5.5–6.0 m). The reason why we havechosen to sample 0.5 m above the sediment was to avoidthe interference of the nepheloid layer present in the lagoon.Water was sampled at site 33 on January 29th, April 10th,April 17th, and May 7th of 2007 to evaluate the microbialdensity and biomass dynamics following DO decline, fromoxic (DO>3 mg L−1), to hypoxic (3>hypoxic<0.72 mg L−1),and suboxic (DO <0.72 mg L−1) conditions. At site 82,water was sampled only during two time periods, sinceoxygen did never reach levels <3 mg L−1 (January 24thand May 10th).

Salinity, DO, and temperature (T) were measured in situwith a YSI 33 SCT and a oximeter Metler Toledo MO 128calibrated by the Winkler method. Stratification index wascalculated as the difference between salinity of bottom andsurface waters. Aliquots of the collected samples (500 mL)were filtered in filters Whatman GF/F (47 mm) in the field.Filtered water (nutrients) and filters (pigments—chlorophylla (Chl a) and bacteriochlorophyll a (BChl a)) were storedon ice, brought to the laboratory, and frozen at −20°C untilfurther analysis.

Dissolved inorganic nitrogen (DIN 0 ammonium, nitrate+ nitrite) and dissolved inorganic phosphorus (phosphate)concentrations were determined according their specificmethods, i.e., UNESCO [61] for ammonium, and Grasshoffet al. [27] for the other nutrients. Photosynthetic pigments,Chl a (of oxygenic phototrophs) and BChl a (of anoxygenicphototrophs), were extracted from the GF/F filters withacetone 90 % for 24 h at 4°C in the dark. Pigment concen-trations were estimated from absorbance measurements at665 and 730 nm (turbidity correction) for Chl a and at 772and 880 nm (turbidity correction) for Bchl a, using aFEMTO 600 S UV–Vis (SP, Brazil) spectrophotometer[36, 60].

Total bacterial density was determined by immediatefiltration on board of the cells onto 0.2-μm pore size darkpolycarbonate filter (Millipore), labeled with the fluoro-chrome acridine orange (AO). The filters were mounted onglass slides and stored at −20°C until enumeration withepifluorescence microscopy [34] Zeiss Axioplan equippedwith a blue filter set (487709—BP 450-490, FT 510, LT520) and a CCD Watec (0.0003 lx sensibility). Coccoidcyanobacterial (CCY) density was enumerated on non-stained filters due to their phycoerythrin autofluorescence[42] using the green filter set (487715—BP 546/12 FT580 LP 590). Bacterial cells were classified into two groups:coccoid cyanobacteria and total heterotrophic bacteria. Thedensity of total heterotrophic bacteria was obtained by sub-tracting cyanobacterial density from the total AO counts. Inaddition, total heterotrophic bacteria were divided into twomorphotypes (coccus + rod-like cells (CB) and filamentouscells (HF)) according to their length/diameter ratio: coccus+rod-like cells <3> filamentous bacteria. Field images were

processed by the “UTHSCSA Image Tool” (University ofTexas Health Science Center, San Antonio, TX, USA)(available for download at http://ddsdx.uthscsa.edu/dig/download.html) using the program Capture X (Xtreme 98para Windows 98). Cell edges were detected following thefilter sequence of 1×Laplacian, 1×Gaussian, and 3×Mean[43]. A minimum of 200 cells were counted based on thecoefficient of variation (<30 %). Bacterial biovolume (incubic micrometers) was calculated from the algorithm sug-gested in Massana et al. [43] and bacterial biomass (infemtogram carbon per cell) was estimated using thefollowing algorithm based on the cellular biovolume:B0120×V0.72, where B 0 biomass, V 0 biovolume, and1200conversion factor in carbon (femtogram carbon percubic micrometer) [47]. Filamentous bacteria were enu-merated directly from untreated images due to the mis-interpretation of filamentous edges by the program. Aminimum of 30 filaments were measured and the samealgorithms were used to estimate their biomass and bio-volume. Purple sulfur bacteria (PSB—Chromatiaceae cf.)were not counted using the UTHSCSA Image Tool soft-ware due to their large length (5 μm). As PSB were noteasily differentiated from nanoflagellates under epifluor-escence, PSB density and biomass were determined usingan inverted microscope in Utermöhl sedimentation chambers[62]. PSB were then distinguished from other prokaryotes andnanoflagellates by their sulfur inclusions that are easily de-tectable under differential interference constrast (DIC). Inaddition to PSB, protozoa (flagellates and ciliates) were eval-uated from fixed samples (paraformaldehyde 4 % final con-centration (v/v)) in aliquots of 10 mL that were allowed tosettle for 24 h in sedimentation chambers and counted, and themajor axes were measured on an inverted ZEISSAxiovert-135 microscope equipped with DIC.

Flagellates were classified into four size classes: <7.5,7.5–25, 25–50, and >50 μm equivalent spherical diameter(ESD), while ciliates in three classes: 7.5–25, 25–50,and >50 μm ESD, in order to predict the carbon “link”through the microbial food web based on predator–preysize relationship. At least four diametric transects wereanalyzed under×100 magnification in order to count thedominant protozoa >25 μm. Density and cell size oforganisms <25 μm were estimated under×400 magnificationuntil the coefficient of variation achieved a value of <30 %.The mean biovolume of each group was calculated bymeasuring the linear dimensions of at least 30 cells persample using the geometric algorithm corresponding tothe cell shape [33]. Flagellate carbon biomass was calculatedusing the conversion factor of 0.216×V0.939 pg C per cell,according to Menden-Deuer and Lessard [45], while ciliatecarbon biomass was calculated using the conversion factor of0.14 pg C μm−3 [54] for formalin-fixed samples, but with amodification of 4 % formalin instead of 2 %.

336 M.L.S. Fontes, P.C. Abreu

http://ddsdx.uthscsa.edu/dig/download.html

http://ddsdx.uthscsa.edu/dig/download.html

Analysis of variance (one-way ANOVA) was applied inorder to compare the biomass of microorganisms in differenttime periods and stations. Correlation values were alsocalculated. If necessary, variables were transformed priorto analysis according to Zar [65]. The correspondence ca-nonical analysis was used to determine to what extent theenvironmental variables explained the trends of similarityon microbial assemblage composition (biomass of bacterio-plankton and protozooplankton). Microbial assemblagesfrom stations 33 and 82 were compared along the timeperiod. Thus, the Monte Carlo test with 999 permutationswas carried out in order to test the significance of each axis(CANOCO for Windows, v. 4, Center for Biometry, Wage-ningen, the Netherlands). The graphical vectors indicate thedirection of the maximum variance of a specific environ-mental variable and their length is proportional to the rate ofvariability.

Results

Characterization of Water

Bottom waters of sites 33 and 82 were chosen to representthe stratified and the homogeneous zones, respectively. Wedecided to use site numbers as descriptors of each zonethroughout the text. Photosynthetically active radiation(PAR) reaching the bottom waters never exceeded80 μmol quanta m−2 s−1, with a minimum of 3.92 μmolquanta m−2 s−1 (Table 1). From January to May, temperaturedecreased to 3.4°C in the bottom waters of site 33 and 7.1°Cin the bottomwaters of site 82, with a minimum of 19°C and amaximum of 27°C (Table 1). Salinity values were lower inJanuary (27.2 and 32.10) compared to May (30.9 and 34.10),

due to usual higher precipitation during summertime(January) [12]. Bottom DO was <3 mg L−1 only at site33, from April 10th onwards, reaching levels below0.72 mg L−1 on April 17th and May 7th (Fig. 2a). Site33 presented stratification indexes higher than 3, while site82 presented 0.2–0.5 (Fig. 2b). Ammonium (NH4

+) was thenutrient that presented the highest temporal variability,ranging from 3.24 (on January 29th—oxia) to 41.07 μmol L−1

(on April 17th—suboxia) at site 33 (Table 1) and contributingwith 87% of DIN in January and 99% of DIN on April 17th atsite 33. Nitrate (NO3

−) varied between 0.21 and 0.91μmol L−1

and phosphate between 0.24 and 0.96 μmol L−1 (Table 1).Site 82 presented the lowest concentration of Chl a

(pigment of oxygenic phototrophs) (2.03 μg L−1) in January,while the highest was observed at site 33 (11.46 μg L−1)under suboxia in May. This pattern was similar for BChl a(pigment of anoxygenic phototrophs), which varied from0.02 μg L−1 at site 82 in January to 10.41 μg L−1 at site33 in May (Table 1).

Bacterioplankton

CCY density ranged from 1.63×108 to 3.02×109 cells L−1

and biomass from 20 to 510 μg C L−1. CCY biomass wassignificantly higher (p<0.05) in oxic bottom waters(January 29th) (310±120 μg C L−1) compared to the otherperiods (Fig. 3a), with the lowest average on April 10th and17th (about 75 μg C L−1), which coincided with the sharpreduction on DO (Table 1, Fig. 3a). However, a small incre-ment of CCY density and biomass was detected inMay, undersuboxia (0.95×109 cells L−1 and 160±90 μg C L−1) (Fig. 3a).Biomass of CCY was significantly higher in the bottomwaters of site 33 compared to site 82, independent of oxygenconcentrations.

Table 1 Incident light (PARIo) and light in the bottom water (PARb,micromole quanta per square meter per second), temperature (T, degreeCelsius), salinity (Sal), water classification based on DO level,ammonium (NH4

+, micromole per liter), nitrate + nitrite (NO3− + NO2

−,

micromole per liter), phosphate (PO43−, micromole per liter), chlorophyll

a (Chl a, microgram per liter), and bacteriochlorophyll a (BChl a,microgram per liter)

Variables and parameters # 33 bottom # 82 bottom

January 29, 2007 April 10, 2007 April 17, 2007 May 7, 2007 January 24, 2007 May 10, 2007

PARb 12.74 70.80 78.79 3.92 68.44 11.75

T 27.00 24.50 24.60 23.60 26.10 19.00

Sal 32.10 34.10 33.90 33.80 27.20 30.90

Water class. Normoxia Hypoxia Suboxia Suboxia Normoxia Normoxia

NH4+ 3.24 7.02 41.07 17.93 4.30 37.54

NO3− + NO2

− 0.48 0.86 0.24 0.63 0.21 0.91

PO43− 0.73 0.63 0.24 0.53 0.96 0.24

Chl a 8.24 4.13 6.83 11.46 2.03 2.28

BChl a 5.32 1.43 6.27 10.41 0.02 1.95

All values were measured in the bottom waters of sites 33 (four time periods) and 82 (two time periods)


The density of heterotrophic coccus + rod-like bacteria(CB) varied between a minimum of 1.95×108 and a maximumof 6.30×109 cells L−1 and their biomass between 1 and 200 μgC L−1. The average biomass of CB at site 82 (130 μg C L−1)was similar to that at site 33 in January. In May, both CBdensity and biomass were significantly higher at site 33, with3.06±1.43×109 cells L−1 and 90±40 μg C L−1, respectively(Table 2, Fig. 3b), contributing with 72 % of the total bacterialdensity and 28.5 % of bacterial biomass in that period. Therelative low contribution of CB to total bacterial biomasscompared to their density was due to their small biovolume(0.18 μm3 of CB against 1.67 μm3 of CCY).

The density of heterotrophic filamentous bacteria (HF)ranged from 0.67×107 (site 82) to 5.72×108 cells L−1 (site 33),or 1 to 80 μg C L−1. The highest averages of HF biomass were

detected in January and May at site 33, with the lowest valuesin April, similar to the cyanobacterial pattern (Fig. 3c).

PSB (Chromatiaceae like) were present only whereDO values were below 3 mg L−1. PSB peaked with9.06×107 cells L−1, equivalent to 90 μg C L−1on April 17th,when DO was 0.3 mg L−1. As a result, PSB were inverselycorrelated with DO (n012, r200.88) (Fig. 3d). Regarding thecontribution of PSB to total bacterial density, it contributedwith 5.8 % of bacteria on April 17th, while in terms ofbiomass, PSB accounted for up to 37.7 % of total bacterialbiomass in the same period (April 17th), which is againattributed to their biovolume, which is large, averaging19.23 μm3 or 10 times higher than that of CCY.

In general, CCY were present at both sites and underoxic, hypoxic, and suboxic conditions, encompassing the

Figure 2 Dissolved oxygen(milligram per liter) in the bottomwater (a) and stratification index(b) [see [21] for details] of thestratified (site 33) andhomogeneous (site 82) zones.Dashed line represents thehypoxic upper limit of 3 mg L−1

and the dotted line the suboxicupper limit of 0.5 mg L−1 used inthis study

Figure 3 Biomass (mean ± standard error) of coccoid cyanobacteria(a), coccus + rod-like bacteria (b), filamentous heterotrophic bacteria(c), and purple sulfur bacteria (PSB) (Chromatiaceae like) (d) frombottom waters of stations 33 and 82. Lowercase letters represent theresult of homogeneous groups among four sampling times at site 33after ANOVA and Tukey post hoc test. n050–70 microscopic fields.

Asterisks represent significant difference by comparing sites 33 with 82after ANOVA and Tukey post hoc test (p<0.05). Average biovolumesof each bacterial group were 1.67 μm3 for cyanobacteria, 0.18 μm3 forcoccus + rod-heterotrophic bacteria, 1.93 μm3 for filamentous bacteria,and 19.23 μm3for PSB


majority of bacterial carbon compared to the other threemorphotypes (Fig. 3), contributing with 82 % of the totalbacterial biomass under oxia in January and with 43 and 50 %under suboxic levels in April and May, respectively, whentheir numbers were at least 3-fold smaller than those of CB.The opposite was observed at control site 82, where CB cellspredominated in January (with 56.5 % of total biomass) andCCY in May, with 55.5 % of bacterial biomass.

In January when DO levels were high at site 33, bacterialbiomass in the bottom waters was 1.8-fold higher than thatof site 82 (390 against 220 μg C L−1). However, in May,when suboxia persisted at site 33, the biomass of totalbacteria was 5-fold higher than that produced at site 82(350 against 78 μg C L−1, respectively).

Flagellates

The density of flagellates <7.5 μm oscillated between 8×105 (April 10th) and 10.83×106 cells L−1 (May 7), equiva-lent to a biomass of 4 to 67 μg C L−1. Their highest densityand biomass were detected in oxic waters in January andsuboxic waters in May, with the lowest biomass in April(Fig. 4a). At site 82, their biomass was higher in Januarythan in May, but biomass in January was similar to that of

site 33 on the same period. Under suboxia of site 33,biomass of flagellates <7.5 μm was 1.52-fold higher thanat site 82.

Flagellates of 7.5–25 μm size class had their densityranging from 0 (April 10th) to 2.71×106 cells L−1 (May7th), equivalent to a maximum biomass of 260 μg C L−1.These flagellates were dominant at site 33, regardless of DOlevels, and representing up to 85 % of total flagellate bio-mass on April 17th and 60 % on May 7th. The highestdensity (1.62×106±0.42×106 cells L−1) was responsiblefor the peak in biomass (140±30 μg C L−1) in the suboxicwaters of site 33 in May (Fig. 4b), which was 7.2-foldhigher than at site 82.

The density of flagellates of 25–50 μm ranged from 0 to7.8×104 cells L−1, with maximum in May, which wasequivalent to 47 μg C L−1. Their highest density and bio-mass averages (64.32×103±18.3×103 cells L−1 and 39±8 μg C L−1) occurred at site 33, in January and May(Fig. 4c), as observed for flagellates <7.5 μm. In the suboxicwaters in May, the biomass of this group of flagellates was8-fold higher than that at site 82.

The density of flagellates >50 μm oscillated from 0 to 9×103 cells L−1, corresponding to a maximum biomass of72 μg C L−1 (in January at site 33). The highest averages of

Table 2 Average (minimum and maximum) density of microorganisms (cells per liter) in the stratified (site 33) and homogeneous (site 82) areas ofthe lagoon during different time periods

Density of microorganisms 33 33 33 33 82 82January 29, 2007 April 10, 2007 April 17, 2007 May 7, 2007 January 24, 2007 May 10, 2007

PSB 0.0 1.6×107 7.6×107 4.8×107 0.0 0.0(0.8−2.4×107) (1.3−9.1×107) (2.7−7.6×107)

Cyanobacteria 1.8×109 5.1×108 5.7×108 9.8×108 5.8×108 4.2×108

(0.2−3.0×109) (0.2−1.2×109) (0.2−1.4×109) (0.2×2.2×109) (0.2−1.0×109) (0.2−2.2×109)

Heterotrophic coccoidbacteria

1.6×109 1.2×109 6.2×108 3.0×109 1.0×109 2.3×108

(0.2−3.9×109) (0.2−2.6×109) (0.2−4.2×109) (0.2−6.3×109) (0.2−4.6×109) (0.2−6.3×109)

Heterotrophic filamentousbacteria

1.6×108 6.1×107 5.5×107 1.3×108 2.2×107 3.3×107

(0.1−5.7×108) (0.1−2.5×108) (0.1−2.5×108) (0.1−4.2×108) (0.1−2.5×108) (0.1−4.2×108)

Flagellates <7.5 μm 5.1×106 2.3×106 2.4×106 5.2×106 5.4×106 4.4×106

(3.0−7.9×106) (0.8−4.1x106) (0.8−4.1×106) (1.6−10.8×106) (3.0−7.9×106) (2.4−7.9×106)

Flagellates 7.5–25 μm 7.3×105 1.4×105 4.3×105 1.6×106 2.9×105 2.9×105

(0.3−1.9×106) (0.0−0.8×106) (0.0−1.9×106) (0.8−2.7×106) (0.0−1.1×106) (0.0−1.1×106)

Flagellates 25–50 μm 4.36×104 9.53×103 8.85×103 6.43×104 5.28×103 5.62×103

(3.7−5.3×104) (0.4−1.4×104) (0.0−1.4×104) (5.1−7.8×104) (0.0−1.0×104) (2.0−8.2×103)

Flagellates >50 μm 3.40×103 2.72×103 0.00 8.17×103 0.51×103 1.53×103

(2.0−6.1×103) (0.0−6.1×103) (0.0−0.0) (8.2−9.0×103) (0.0−2.0×103) (0.0−2.0×103)

Ciliates 7.5–25 μm 9.03×103 0.14×103 0.07×103 6.32×104 1.81×104 9.03×103

(0.0−2.7×105) (0.0−2.0×103) (0.0−2.0×103) (0.0−2.7×105) (0.0−2.7×105) (0.0−2.7×105)

Ciliates 25–50 μm 7.49×103 2.72×103 2.72×103 3.06×103 3.06×103 0.51×103

(6.1−8.2×103) (0.0−4.1×103) (2.0−4.1×103) (2.0−4.1×103) (2.0−4.1×103) (0.0−2.0×103)

Ciliates >50 μm 0.0 0.0 0.68×103 1.50×103 0.0 0.0(0.0−2.0×103) (0.0−2.0×103)


biomass were reported in January andMay (ca. 50±7μg C L−1)(Fig. 4d), as previously observed for all flagellates, CCY, andHF bacteria. In the suboxic waters in May, the average biomassof this group was 6-fold higher at site 33 than at site 82.

Flagellates <7.5 μm dominated the flagellate communitybiomass at site 82 in January (58 %) and contributed with 36% of the total flagellate biomass in May. On the other hand,at the stratified site, flagellates of 7.5–25 μm predominated,representing 39 and 52 % of the total flagellate biomassunder oxia in January and suboxia in May, respectively. Thebiomass of all four flagellates together reached 170 μg C L−1

under oxia at site 33 against 47 μg C L−1 under oxia at site 82in January; inMay, their biomass reached 260 μg C L−1 at site33 (suboxia) against 60 μg C L−1 at site 82 (oxia).

Ciliates

The density of ciliates of 7.5–25μm ranged from 0 (April 10th)to 2.70×105 cells L−1 (at site 33, inMay), reaching a maximumbiomass of 16 μg C L−1. The biomass variability of this groupwas independent on DO, although the highest average wasdetected under suboxia in May (13±27 μg C L−1) (Fig. 5a).The average biomass of these ciliates was similar in both sites,but representing 95 % of the total ciliate biomass at site 82.

The density of ciliates of 25–50 μm ranged from 0 to 8×103 cells L−1, with a maximum biomass of 12 μg C L−1, notdiffering significantly between sites (Fig. 5b). The maximum

density of ciliates >50 μm was 2×103 cells L−1 in May,corresponding to a biomass of 320 μg C L−1. Ciliates>50 μm appeared only at site 33 and under suboxia (April17th and May 7th), as observed for PSB. The averages were,respectively, 680±1,170 cells L−1 and 110±190 μg C L−1 onApril 17th and 1.2×103±1.44×103 cells L−1 and 160±230 μgC L−1 onMay 7th (Fig. 5c), resulting in a contribution of up to90 % of total biomass of ciliates in the suboxic waters in May.Ciliates >50 μm were on average 150 μm long and corre-sponded to Loxophyllum sp.

Comparing the total biomass of ciliates at both sitesunder oxia, the biomass of ciliates reached 26 μg C L−1 atsite 33 (where 80 % of it was of ciliates <50 μm) and 14 μgC L−1 at site 82 (where 74 % of it was produced by ciliates<25 μm). Under contrasting oxygen levels (suboxia at site33×oxia at site 82), the total biomass of ciliates was 170 μgC L−1 at site 33, with the predominance of ciliates >50 μmlong, against a biomass of only 6 μg C L−1 at site 82, or a30-fold difference between locations.

The biomass of total bacteria was not correlated with thebiomass of the total flagellates or ciliates. However, weobserved statistically significant (p<0.05) relationshipswhen considering predator–prey size interactions, e.g.,flagellates of 25–50 μm were correlated with cyanobacteriaand filamentous bacteria (r00.85 and r00.94, respectively).In addition, flagellates >50 μm also correlated with fila-mentous bacteria (r00.92). Regarding ciliates, the biomass

Figure 4 Biomass (mean ± standard error) of flagellates grouped intosize classes of <7.5 (a), 7.5–25 (b), 25–50 (c), and >50 μm (d)collected from the bottom waters of stations 33 and 82. Lowercaseletters represent the result of homogeneous groups among four sam-pling times at site 33 after ANOVA and Tukey post hoc test. n030–40microscopic fields for cells <25 μm and n04 diametric transects for

cells >25 μm. Asterisks represent significant difference by comparingsites 33 with 82 after ANOVA and Tukey post hoc test (p<0.05).Average biovolumes of flagellates were 23.5 μm3 for flagellates<7.5 μm, 510.02 μm3 for flagellates 7.5–25 μm, 5,829.85 μm3 forflagellates 25–50 μm, and 53,547.9 μm3 for flagellates >50 μm


of size class 7.5–25 μm correlated with the biomass offlagellates <7.5 μm (r00.90), while those of 25–50 μm werepositively related to the biomass of cyanobacteria (r00.93),and the larger ciliates (>50 μm) correlated with PSB (r00.85).

PSB and ciliates >50 μm correlated significantly with the sameparameters: silicate (r00.91, 0.90) and DO (r0−0.96, −0.95).

Canonical correspondence analysis (CCA) showed that52 % of the variability in microbial carbon (bacteria,flagellates, and ciliates) was explained by the combina-tion of 10 environmental variables. Axis 1 separatedoxic–hypoxic from suboxic water samples (oxygen onthe negative side). Chl a, salinity, ammonium, and Bchla were related to suboxic samples of site 33 on April17th and May (positive axis 1), as well as with biomassof ciliates >50 μm and PSB (Fig. 6). PARz and phos-phate were the environmental variables more related tosamples of site 82, as well as with CCY, CB, andeukaryotic cells (flagellates <7.5 μm and ciliates 7.5–25 μm). Temperature and nitrate did not display animportant influence regarding any of the two axes. Figure 6depicted protozooplankton and bacteria into three groups:group 1—ciliate 3 with PSB; group 2—flagellate 4, flagellate3, flagellate 2 with HF bacteria and CCY; and group 3—flagellate 1, ciliate 1 with CB, while ciliate 2 was an isolatedgroup.

Figure 5 Biomass (mean ± standard error) of ciliates grouped into sizeclasses of 7.5–25 (a), 25–50 (b), and >50 μm (c) collected from thebottom waters of stations 33 and 82. Lowercase letters represent theresult of homogeneous groups among four sampling times at site 33after ANOVA and Tukey post hoc test. n030–40 microscopic fields forcells <25 μm and n04 diametric transects for cells >25 μm. Asterisksrepresent significant difference by comparing sites 33 with 82 afterANOVA and Tukey post hoc test (p<0.05). Average biovolumes ofciliates were 4,063.2 μm3 for ciliates 7.5–25 μm, 7,325.3 μm3 forciliates 25–50 μm, and 760,959.4 μm3 for ciliates >50 μm

Figure 6 CCA of environmental variables and biomass of bacteria,flagellates, and ciliates in the bottom waters of sites 33 and 82, whereJanuary 29 0 Jan, April 10 0 Apr10, April 17 0 Apr17, and May 7 0May of 2007. CCY biomass of cyanobacteria, CB biomass of totalheterotrophic bacteria, HF biomass of heterotrophic filamentous bac-teria, PSB biomass of purple sulfur bacteria, Ci ciliates (107.5–25 μm,2025–50 μm, 3 0 >50 μm), Fl flagellates (10<7.5 μm, 207.5–25 μm,3025–50 μm, 4 0 >50 μm). Samples were represented by whitediamonds, environmental variables by vectors, and biomass of micro-organisms by gray triangles. The right side of the CCA graph showedsuboxic samples (33 Apr17 and 33 May), while the left side, oxic andhypoxic samples


Discussion

Biomass and density of microorganisms in bottom waters ofthe stratified site (#33) changed significantly with time,whereas smaller variations occurred at site 82. The mostprominent drop on microbial biomass at site 33 followed theoxygen decay, from oxic to hypoxic conditions—January29th to April 10th. Cyanobacteria, heterotrophic filamentousbacteria, all groups of flagellates, and ciliates <50μmdeclinedin biomass and density. In contrast, PSB (Chromatiaceae cf.)were absent under oxic conditions, appearing on April 10th,followed by ciliates >50 μm on April 17th (under suboxia).

Thus, this first decrease in the majority of microbialbiomass at site 33 appears to be related to changes in watercharacteristics such as oxygen (as stated before), but alsotemperature, and salinity, indicating dilution of lagoonwaters with seawater. Increasing salinity from January toApril is usual for the lagoon, since seawater inflow is moreintense during the autumn–winter seasons, associated withfrequent cold fronts or southerly winds [12]. Furthermore,microbial dilution by seawater entrance can also be seen inthe Northern sector (with a time lag of weeks due to its distanceto the channel). The same pattern is observed in ChesapeakeBay where local bacterioplankton was shown to be diluted byseawater inflow [11]. The reduction of total microbialbiomass could consequently stimulate growth of newmicroorganisms or of those that were being outcompetedbefore [5, 32].

Bacterial biomass remained low from April 10th to April17th at site 33, when DO dropped from 3 mg L−1 to suboxiclevels; however, suboxia provided a niche for PSB, whichpeaked on April 17th, demonstrated by their abundance,biomass, and pigment (BChl a). Under 20 days of suboxia,from April 17th to May 7th, bacterial biomass at site 33increased to values comparable to the initial biomass underoxia in January.

The increment of bacterial biomass occurred at site 33 inMay after 1 month under low oxygen levels was alsoreported elsewhere [11, 53]. The amount of time necessaryfor bacterial community composition to change depends onhow long anoxia persists; thus, it will influence bacterial andbacterivorous biomass [24, 37, 58]. Consequently, excretionand egestion of organic matter by protozoa provide enoughsubstrate for heterotrophic bacteria to grow and stimulatethe microbial loop in the stratified bottom water, which canmaintain suboxia [4, 52].

In addition, ammonium was one order of magnitudehigher in April–May at both sites (#33 and 82). Regardlessof the stability of the water column, higher mineralization oforganic matter occurred in May, as reported previously [21].Ammonium might have been regenerated by heterotrophicbacteria or excreted by zooplankton: nano- and microzoo-plankton [30, 58] and mesozooplankton [51, 59]. However,

the decrease of 50 % of ammonium from April 17th to May7th at site 33 indicates that ammonium was also assimilated.As anoxygenic photosynthetic bacteria may utilize more ofthe regenerated nutrients than oxygenic photosynthetic bac-teria in the bottom waters of the lagoon [22], biomass oftheir specific predators should also increase, as supported bythis study and others [24, 55].

Gomes and Godinho [26] also reported that after stratifi-cation of the Monte Alegre lake, oxygen was completelydepleted in the bottom waters followed by an increase inNH4

+ and the appearance of sulfobacteria and ciliates. Over-all, comparing the microbial carbon produced under contin-uously mixing waters versus permanent stratified waters, wedemonstrated that carbon of fast growing smaller micro-organisms (CB, flagellates <7.5 μm, and ciliates <25 μm)predominated in the bottom waters of the homogeneous site,compared with the stratified site. On the other hand, at thestratified site, two different patterns were observed: (1)cyanobacteria were the dominant bacterial carbon sourceespecially under the oxic waters in January and, thus, sup-porting the larger three classes of flagellates; and (2) largemicroorganisms predominated (sulfur bacteria and ciliates>50 μm) under suboxia and anoxia in May. Consequently,nanoprotozooplankton (flagellates and ciliates of 2–20 μm)can be pointed out as the dominant predator of smallheterotrophic bacteria (CB) in oxic waters of the lagoon,as reported by previous studies [16, 18], while ciliatesare the main bacterivores in suboxic or in oxic–anoxicinterface waters [24, 26, 55].

In addition, at the stratified site, flagellates of 7.5–25, 25–50, and >50 μm correlated with heterotrophic filamentousbacteria and cyanobacteria and also with chlorophyll a, andthese flagellates can be pointed out as the main herbivores(grazing Chl a containing cells). This finding differs fromwhat was reported by Gobler et al. [25] where ciliates androtifers were the main herbivores, but similar to Sacca et al.[55], where both flagellated and ciliated protozoa benefitedfrom the photoautotrophic production in the oxic–anoxicinterface. This selective mortality partially relieves interspe-cific resource competition and allows weaker competitors toproliferate, as for sulfur bacteria [29, 35, 40].

The main difference observed in the microbial commu-nity of sites 33 and 82 was the presence of large ciliates,150 μm long (>50 μm), only at site 33, and of activepopulations of PSB, which accounted for up to 37.7 % ofthe total bacterial biomass. Ciliates such as Loxophyllum sp.(present in our samples) can survive if oxygen is present forsome periods of the day [17]. Bacterivorous ciliates canselect their preys, outcompeting flagellates for grazing oflarger bacteria of anoxic waters [8, 26, 29]. The relationshipbetween ciliates and sulfobacteria is frequent in other sub-oxic waters [24, 40], more specifically, e.g., between Spiro-stumum teres (a ciliate >50 μm) and Thiopedia rosea (a


purple sulfur bacterium) [2, 26], or between Caenomorphamedusula and Thiopedia sp. [28].

It is noteworthy that only 1.5 ciliates >50 μm mL−1 wereenough to produce 55 % of the total protozooplanktonbiomass in the suboxic water. This fact demonstrates theirsignificant role in concentrating particulate organic carbonin suboxic waters. As ciliates >50 μm (average 150 μmlong) were positively correlated with PSB and BChl a, theyare pointed as the main grazers of PSB. Saccà et al. [55]found a strong correlation between the pigment of sulfurbacteria and biomass of ciliates (biomass of ciliates peakedwith BChl a (94.27μg C L−1 and 48.59μg L−1, respectively)).The time lag between bacterial biomass and ciliates observedin our study (20 days) was similar to other hypolimnion ofmeromictic lakes, where high ciliate biomass occurred after 2–3 weeks of the peak of their prey biomass [29, 53]. If oneconsiders a doubling time of 1.5 days for PSB (minimumestimated for bacteria in Spanish lakes by Garcia-Cantizano etal. [23]), it should be expected to see an increase of 4.7-fold indensity in 7 days (from April 10th to April 17th—from appear-ance to peak), if there is no mortality. The increase in densityfrom 1.6×107 to 7.6×107 cells L−1 confirmed that hypothesis.However, after 20 days (from April 17th to May 7th), itshould be expected to have a 13-fold increase in density,which was not the case. These speculations reinforce theexistence of top down control of PSB by ciliates; hence,the particulate organic carbon produced by PSB wastransferred to the protozooplankton, resulting in highsecondary production. The total microbial carbon producedin the hypolimnetic suboxic waters is usually unavailablefor mesozooplankton due to the toxicity of these waters(low oxygen and high sulfide).

However, some of the microaerophilic ciliates (those thattolerate low oxygen levels) can move vertically from sedi-ment to the water column, demonstrating the ability totransport organic matter upwards [14, 20, 31]. In additionto protozooplankton, some copepods and Daphnia havebeen reported to migrate downwards in the water column,descending during daylight into low oxygen levels to feed orto escape predation and ascending at night hours [1, 3, 7, 39,44]. Thus, if ciliates migrate upwards (from anoxic–suboxicwaters to hypoxic waters) and zooplankton can tolerate lowoxygen levels as reported previously, there is a great chancethat the microbial biomass produced in the suboxic waters istransferred to aerobic mesozooplankton, or that the micro-bial food web “busts” the traditional planktonic food web, asobserved in oxic systems [56, 57].

Evidence of the accumulation of metazoans in the oxi-cline of CS sector of the lagoon was first pointed out in 1982by Odebrecht and Caruso [48] who observed a high densityof small fish feeding near the bottom of the oxicline. Inaddition, the amount of copepods and gastropod larvaeconcentrated with a trawling plankton net (80 μm) in the

oxicline (3.5–4.0 m) was ca. 5-fold higher in comparisonwith the surface layer (Fontes et al., in preparation), thusindicating the link between “bottom suboxic zone” and“top oxic zone”. Future studies using stable isotopes willbe carried out in the meromictic sector of the lagoon inorder to elucidate the connecting pathway between thesetwo zones.

Usually, studies reporting carbon fluxes in lakes andestuaries only take into account the oxic water layer, asreported by Cole et al. [9]. The authors reported a smallcontribution of bacteria to zooplankton carbon demand (ofonly 2 %) in the surface oxic waters, while Cole et al. [10]assumed that the zooplankton carbon demand was mainly ofterrestrial origin, since they did not consider the anoxicwaters in the carbon models (as they assumed that zooplank-ton were restricted to oxic waters). Our study indicates thatan autochthonous carbon produced in the hypolimneticwaters by the microbial food web was being ignored in thecarbon models by most of the researchers. Therefore, ifother studies start to quantify the amount of carbon pro-duced and transferred from the hypolimnetic to the oxicsurface waters, we believe that autochthonous contributionfor zooplankton would be much higher.

Concluding Remarks

The biomass of microbes of suboxic waters was at least 5-foldhigher than in the oxygenated bottom water, where it was5-fold higher for bacteria, 4.8-fold higher for flagellates,and 33-fold higher for ciliates. Thus, with our study, westate that the biomass of microorganisms produced in thebottom waters of meromictic shallow systems (6 m deep) ismuch higher than in the bottom waters of homogeneousshallow systems. We demonstrated the existence of aspecialized microbial food web in the hypolimnetic suboxicwaters, based on active anaerobic anoxygenic PSB, withciliates >50 μm, mainly Loxophyllum sp., as their predomi-nant grazer, while in oxic waters, nanoprotozoan werepredominantly grazing over heterotrophic bacteria. Therefore,this study provides evidence for the importance of suboxicbottom waters as places of high biomass production by themicrobial food web and, thus, of a possible source of carbonto mesozooplankton in shallow aquatic systems throughoutthe world.

Acknowledgments We are grateful to A. Shu, T. G. Rosa, and A. daSilva for sampling assistance and Dr. B. Spoganicz for sharing hislaboratory and the help of his undergraduate students to process watersamples. We wish to thank Clarisse Odebrecht for assistance withprotist identification and for reviewing the manuscript. MLSF had ashcholarship from CAPES - Brazilian Ministry of Education. PCA isResearch Fellow of the CNPq - Brazilian Ministry of Science, Deve-lopment and Innovation.


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a vigorous specialized microbial food web in the suboxic waters of a shallow subtropical coastal...

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