Abella, Carles

Montesinos, Emili

Ylla, J.

Revistes Catalanes amb Accés Obert

SCIENTIA gerundensis, 12:49-57 (1986) 
DISSIMILATORY SULFATE-REDUCING ACTIVITY IN 
LAKES OF BANYOLES KARSTIC AREA (GIRONA) 
Abella, c.A.', Ylla, J . ~  and E. Montesinos 1 i 2  
' Institut d'Ecologia Aquatica. Col.legi Universitari de Girona (UAB). Hospital, 6 .  
17071 - Girona. 
Departament dlAgricultura. Escola Universitaria Polittcnica de Girona (UPC). Av. Lluis 
Santaló. 17002 - Girona. 
Institut d'Ensenyament Mitja de Manlleu. Barcelona. 
RESUM 
Hem estudiat l'origen del sulfhidric en els estanys de la zona carstica de Banyoles. 
L'activitat reductora de sulfats s'ha mesurat mitjan~ant la transformació de sulfat ra- 
diactiu fins a sulfhídric radiactiu. La maxima activitat de reducció de sulfats es detecta 
en els primers 10-20 cm de sediment. Els valors de reducció integrats oscil.laven entre 
40 i 1245 nmol SO?-,m 2.dia-'. En les zones anaerbbies de la columna d'aigua l'acti- 
vitat fou generalment més baixa, entre 10 i 150 mmol SO,'-.m-'.dia-'. Una part 
substancial del sulfhídric produit per reducció de sulfat era retinguda en el sediment en 
forma de Fes  (37-9876) mentre que la resta difon a la columna d'aigua. S'ha observat 
una relació hiperbblica directa entre l'activitat sulfat-reductora i el pes sec del sediment. 
RESUMEN 
Se ha estudiado el origen del sulfhidrico en 10s lagos de la zona carstica de Banyoles. 
La actividad máxima de reducción de sulfatos se detecto en 10s primeros 10-20 cm del 
sedimento. Los valores de reducción integrados oscilaban entre 40 y 1245 mmol 
SO-42-Z.m- 2.dia-'. En las zonas anaerobias de la columna de agua la actividad general- 
mente fue mis baja, entre 10- y 150 m r n o l . ~ O ~ ~ - , m -  '.dia-'. Una parte sustancial del 
sulfhidrico producido por reducción de sulfatos se retenia en el sedimento en forma de 
sulfuros metálicos (37-98%) mientras que el resto difunde hacia la columna de agua. Se 
ha observado una relación hiperbólica directa entre la actividad sulfato-reductora y el 
peso sec0 del sedimento. 
ABSTRACT 
The origin of sulfide in lakes of Banyoles karstic area has been studied. The activity of 
sulfate reduction has been measured by means of transformation of radiolabelled sulfa- 
te 35S042- into 35S2-. The maxirnum activity of sulfate reduction was located in the first 
10-20 cm of sediment. Integrated values accounted for 40 to 1245 mmol SO: ' m' '.day-'. 
In the anaerobic zones of the water column the activity was generally lower accounting 
for 10-150 mmol SO:-,rn-'.day-'. A substantial arnount of the sulfide produced by 
50 ABELLA, C. A . ,  YLLA, J .  AND E. MONTESINOS 
sulfate reduction was retained into the sediment as Fes (37-98%) whereas the remai- 
ning diffused to the water column. A direct hyperbolic relationship has been observed 
between the activity of sulfate reduction and the dry weight of the mediment. 
Key words: Sulfate-reduction, karstic lakes, sediment, Desulfovibrio, activity. 
INTRODUCTION 
In lakes located in the Banyoles karstic area the most abundant anion is 
sulfate, because underground waters dissolve gypsum strata. Some of the 
lakes are crenogenic meromictic (Vilar and Banyoles 111). others are holo- 
mictic (Sisó and Nou). A few kilometers distance from lake Banyoles there 
are two small eutrophic lakes (Negre 1 and Coromines) in which carbonate 
is the dominant anion whereas the concentration of sulfate is 20 to 40 times 
lower. 
All of these lakes have in common the presence of (1) anaerobic layers 
of water, specially during summer stratification, which exhibit high concen- 
tration of sulfide (up to lOmM), and (2) anaerobic c<black sediments,, very 
rich,in decomposing organic matter. In some cases sulfide diffuses to the 
very surface of the lake. Till now nobody has studied the origin of sulfide 
in such lakes. It seems plausible at first sight that due to the abundance of 
sulfate, sulfate-reducing activity may substantially contribute to the produc- 
tion of sulfide. 
The present work tries to ascertain the contribution of sulfate-reducing 
bacteria to the production of sulfide and to know factors controlling this 
activity in lakes of the Banyoles karstic area. 
MATERIAL AND METHODS 
Limnological characteristics and physico-chemical analysis were assessed 
following the standard methods described in Strickland & Parsons (1968) 
and Goltermann et al. (1978). Primary productivity in the water column was 
measured in situ by the 14C method of Steeman-Nielsen (Vollenweider 1974). 
Sulfate-reduction activity was estimated by the method of Jorgensen 
(1978a) using 3 5 ~ 0 4 2 - .  Samples of anaerobic sediments have been collected 
with a home-made core sediment sampler. The cores were transported to 
the laboratory and at small depth intervals, injected with 50 pL  of a solu- 
tion of 3 5 ~ 0 4 2 -  (40 p ~ i . m ~ - ' ) .  Incubation was performed in the dark at 
20 "C during 24 hours. Afterwards the cores were freezed, cutted into small 
pieces of 1 cm thick, suspended in oxygen-free water and anaerobically 
purged with nitrogen gas. The resulting 3 5 ~ 2 -  was trapped into a solution of 
1 M zinc acetate. Radioactivity in the precipitate was measured by liquid 
scintillation counting. 
Sulfate- reducing bateria were cultivated in Postgate medium in environ- 
mental chambers (Postgate, 1979). 
DISSIMILATORY SULFATE-REDUCING 5 1 
Table 1. Physicochemical characteristics of Banyoles karstic lakes (holomictic group) 
in summer 1979. 
pH Cond. Eh SO~~-HCO;PO~~-  NO3' NH4+ H2S 
(mS/cm) (mV) (mM) (mM) (mM) (mM) (mM) (mM) 
Sisó Epilimnion 7.0 2.0 +I30 10.4 5.0 3.0 0.4 120 0.0 
Hipolimnion 6.8 2.1-310 7.3 7.5 0.0 0.0 250 7.4 
Nou Epilimnion 8.8 1.8 + 10 11.8 5.5 0.0 1.0 - 0.0 
Hipolimnion 6.8 2.1 -225 12.5 10.0 1.0 0.0 - 1.2 
Coromines Epilimnion 9.0 0.6 - 80 0.7 5.4 4.2 0.0 - 0.0 
Hipolimnion 7.8 0.8-380 0.3 7.3 21.3 0.0 - 1.5 
Negre 1 Epilimnion 8.5 0.6 -100 0.4 4.9 3.5 0.0 - 0.0 
Hipolimnion 8.0 0.8 -275 0.4 6.7 15.3 0.0 - 0.2 
Tabie 2. Pchysicohemical characteristics of Banyoles karstic lakes (meromictic 
group) during summer 1979. 
Banyoles Mixolim. 8.0 1.2+-275 5.2 3.5 0.6 2.0 5.0 0.0 
I11 Monimolim. 7.4 2.2-110 9.5 5.0 0.0 0.5 90.0 0.8 
Vilar Mixolim. 8.0 1.1 +I30 5,3 2.9 0.2 4.4 - 0.0 
Monimolim. 6.8 1.5-175 11.6 7.8 1.2 0.1 - 5.9 
RESULTS AND DISCUSSION 
1. Physicochemical and biologia1 characteristics, and sulfate-reduction 
activity in the water column 
Tables 1 and 2 show the main physicochemical characteristics of water from 
the six studied lakes. All the lakes have in common the presence of an 
hypolimnion or monimolimnion containing anoxic water with a red-ox po- 
tential between -110 and -380 mV, rich in reduced inorganic substances 
such as ammonium (up to 250 FM) and sulfide (up to 7.4 mM). In the lakes 
Banyoles 111, Vilar, Sisó and Nou, sulfate and bicarbonate are the dominant 
anions (5 to 12.5 and 5-10 mM respectively), whereas in the lakes Coromi- 
nes and Negre the concentration of sulfate was considerably lower ranging 
from 0.3-0.7 mM and being bicarbonate the most abundant (5.4 to 7.3 mM). 
Table 3 shows the integrated concentration of photosynthetic pigments, 
biomass of phototrophic sulfur bacteria and primary productivity for sum- 
mer 1979. The highest biomass of phototrophic bacteria has been recorded 
for Lake Sisó (98.3 g fresh weight.m * and 1900 mg Bchl.m2) whereas the 
lowest in Banyoles I11 (4. 2 g fresh weight.m and 150 mg Bch1.m 2) .  The 
highest productivity was observed in the lakes Nou and Vilar (1535 and 
52 ABELLA, C. A., YLLA, J .  AND E.  MONTESINOS 
Table 3. Biological parameters of Banyoles karstic lakes in summer 1979. 
Integrated Biomass Integrated 
photo- (fresh primary 
synthetic weight of productibn 
pigments phototrophic (mg.m- ' 
(Chl or bact. day-') 
Bchlor in (g.m- 2, 
mg.m- 2, 
Banyoles Mixolimnion 39.0 - 367 
Monimolimnion 150.0 4.2 53 
Vilar Mixolimnion 45.0 - 923 
Monimolimnion 203.0 17.4 57 
Sisó Epilimnion 10.0 - 406 
Hipolimnion 1900.0 98.3 458 
Nou Epilimnion 250.0 - 448 
Hipolimnion 580.0 13.2 1087 
Coromines Epilimnion 185.0 - 279 
Hipolimnion 450.0 27.5 59 
Negre 1 Epilimnion 138.0 - 683 
Hipolimnion 410.0 14.2 167 
Table 4. Integrated activity of sulfate-reduction (mmol ~ 0 ~ ~ - . m - ' . d a ~ " )  in the 
anaerobic water and sediments of severa1 lakes of Banyoles karstic area. 
Total sulfate 
Lake reduction 
activity 
Anaerobic Anaerobic 
water sediments. 
S i d  190 150 (79) a 40 (21) 
Nou 119 60 (50) 59 (50) 
Vilar 1365 120 (9) 1245 (91) 
Banyoles I11 850 65 (8) 785 (92) 
Coromines 376 15 (4) 361 (96) 
Negre 1 264 10 (4) 254 (96) 
a in brackets, percentage related to the total sulfate reduction values in the lake. 
DISSIMILATORY SULFATE-REDUCING 53 
980 mg C . m  2.day-1 respectively) whereas the lowest were in the Banyoles 
I11 and Coromines (420 and 338 mg C.m 2.day-1 respectively). 
The activity of sulfate reduction in the anaerobic waters is presented in 
table 4. The values ranged from 10 to 150 mmol  SO^-^,^- 2.day-1 sugges- 
ting an important contribution of this process in the water column to the 
total sulfate-reducing activity in certain lakes (Sid and Nou). 
2. Physicochemical characteristics and activity of sulfate reduction in 
anaerobic sediments 
Table 5 shows the main physicochemical characteristics of pore water (Eh 
and PH) and dry weight of anaerobic sediments as vell as tiie integrated 
sulfate reduction activity. In all cases redox potentials are negative, ranging 
from -110 to -400 mV and the pH from 6.5 to 7.2. Dry weight increa- 
sed from the top to the bottom parts of the cores. The highest activity of 
sulfate reduction was found for lakes Vilar and Banyoles I11 (1245 and 785 
mmol s02- .mT 2.day-1) whereas the lowest were observed in lake Sisó 
(40.3 mmol ~ 0 2 - . r n  2.day-1). 
Table 5. Sediment characteristics of Banyoles karstic lakes during summer 1979. 
Lake sediment Depth Eh pH Dry weight Integrated ~ 0 4 ' -  
(cm) (mV) (g.cm 3, reduction activity (mmol 
sd4-.m- '.day-') 
Banyoles I11 O -310 7.0 0.30 785.0 
10 -350 6.7 0.40 
20 - 4 0 0  6.5 0.60 
Vilar O -225 7.2 0.27 1245.00 
10 -275 7.1 0.41 
20 -275 7.1 0.55 
Sisó O -220 7.0 1.03 40.36 
10 -280 7.0 1.03 
20 -150 7.0 1.55 
Nou O -300 6.9 0.78 59.10 
10 -250 6.9 0.90 0.90 
20 -180 7.0 0.98 
Coromines O -150 6.6 0.31 361.36 
10 -110 6.6 0.50 
20 -110 6.7 0.60 
Negre 1 O -180 7.3 0.40 254.04 
10 -300 7.0 0.55 
20 -280 6.9 0.70 
54 ABELLA, C. A., YLLA', J .  AND E. MONTESINOS 
L ' . ' ' .  ' " . ' . ' l ' . ' - l  I 
0.2 0.4 0.6 0.8 0 2 4 6 8 10 1 2 3 4 
Dry weight ( 9 .  cmV3) H ~ s / s - ~  ("mol cm3) SULFAT€-REDUCTION RATE 
- (flmol S$-. cmaday) x103 
-200 -400 -600 
Eh (mV) 
Figure 1. Vertical distribution of red-ox potential of pore water (o), dry 
weight (e) (panel A); concentration of free sulfide (shaded area), acid 
volatile sulfide (empty area) (panel B)) and the activity of sulfate reduc- 
tion in anaerobic sediments of Banyoles basin 111 on june 29, 1979. 
Figure 1 shows the vertical distribution of red-ox potential and dry 
weight in relation to the concentration of sulfide and the activity of sulfate 
reduction in a core from Banyoles I11 taken on June 29, 1979. The profiles 
and patterns are essentially the same for the other lakes and fit well with 
that obtained by Jorgensen (1978b). Water content decreased with depth 
being about 75% in the very top, to 15% at 30 cm depth. The concentration 
of total sulfur is relatively constant in the first 30 cm. In the first 20 cm most 
of the sulfur is in the form of gaseous sulfide able to diffuse to the water 
column, whereas below 20 cm most of the sulfur is in the form of Fes. 
Depending on the lake the amount of sulfide retained into the sediment as 
Fes ranged from 37 to 98% of the total produced. The lowest values were 
found for lakes Vilar and Banyoles 111. In Coromines and Negre 1 more 
than 90% of sulfide produced was retained into the sediment. Most of the 
activity of sulfate-reduction was detected in all cases in the first 20 cm of 
sediment. 
An hyperbolic relationship has been observed between the integrated 
rate of sulfate-reduction and dry weight of the sediment (Fig. 2). These 
results are consistent with the hypothesis that sediment density, and hence 
porosity, is an important environmental factor controlling rates of dissimila- 
tory sulfate reduction. Sediment porosity determines not only the diffusion 
of sulfate (electron acceptor) and organic matter (carbon and energy sour- 
ce) from the water column, but also the free space that can be colonized by 
the microbial community. Thus the highest activity of sulfate reduction is 
found in sediments with low dry weight and high sulfate concentration. 
Only in lakes Coromies and Negres 1 sulfate concentration the water column 
(< 1 mM) can act as limiting factor for sulfate-reduction (see Fig. 3). On 
DISSIMILATORY SULFATE-REDUCING 
Figure 2. Hyperbolic relastionship between intgrated rate of sulfate re- 
duction and dry weight in anaerobic sediments collected from lakes of 
the Banyoles karstic area (1979). 
i o e I  , . . . , , , , , , . . . , .I 
5 10 15 20 25 3 0  35 4 0  45 50 55 60 6 5  70 75 
TlME (h) 
Figure 3. Growth of Desulfovibrio desulfuricans isolated from anaerobic 
sediments of Vilar lake, at different sulfate concentrations in Postgate 
rnedium. The kinetics of sulfide production follows the same pattern. 
56 ABELLA, C. A., YLLA, J .  AND E. MONTESINOS 
the basis of the results presented here, it is expected that the sediment 
density is the main limiting factor for sulfate-reduction at values higher than 
0.4 g.cm- 3. Below this values, sulfate concentration and primary production 
in the water column are threshold factors for sulfate-reduction. 
3. Isolation and growth, characteristics of sulfate-reduction bacteria 
Sulfate-reducing bacteria were isolated both from the anaerobic sediments 
and from water samples. In vivo absorption spectra of clarified supernatants 
obtained after sonication of cells, centrifugation and alcali treatment showed 
the presence of two absorption peaks corresponding to citochrome c3 (420 
nm) and desulfoviridin (630 nm). Fluorescence spectra confirmed also the 
presence of desulfoviridin, suggesting that the microorganisms pertain to 
genus Desulfovibrio (see Fig. 4). Nutritional requirements indicated that the 
bacteria ara able to growth in the presence of malate or piruvate as the only 
carbon and energy source and with sulfate as electron acceptor, thus produ- 
cing sulfide. Therefore it could be determined as D. desulfuricans (Krieg & 
Holt, 1984). Optimal temperature was between 20 to 30 "C. The maximum 
specific growth rate of 0.179 h-' (duplication time of 3.87 hours) was obtai- 
ned at 30 OC, 4-14 mM sulfate. The maximum sulfide production activity in 
Figure 4. Transmission electron micrography of cells of Desulfovibrio 
desulfuricans in stationary phase of growth. Empty cells and flagella are 
present. 
DISSIMILATORY SULFATE-REDLCING 57 
cultures was of about 2.35 mmo1.-l-l.h-'. In figure 3 it is shown the effect 
of sulfate concentration on growth of D. desulfuricans isolated from sedi- 
ments of lake Vilar. Concentrations below 4 mM sulfate are limiting for the 
development of D. desuifuricans. 
Both the activity of sulfate reduction and the presence of sulfate-redu- 
cing bacteria are consistent with the idea that sulfide in lakes of Banyoles 
karstic comes from a process of dissimilatory reduction of sulfate. 
Aknowlegments 
We are indebted to J. Turet and J. Garcia-Merchan for help and friendship. We also are 
grateful to Prof. R. Guerrero for help in many ways, to I. Esteve for the electron microscopy 
micrography of D. desuifuricans, and to J. Garcia-Gil for help in drawings. 
References 
GOLTERMANN, H.L. ; CLYMO., R.S. & OHNSTAD, M.A.M. (eds.) 1978. Methods for 
physical and chemical analysis of fresh waters. Blackwell, Oxford, 2n ed. 
JORGENSEN, B.B. (1978). a. A comparison of methods for the quantification of 
bacterial sulfate reduction in coastal marine sediments. I. Measurements with 
radiotracer techniques. Geomicrobiol. J. 1: 11-28. 
JORGENSEN, B.B. (1978). b. A comparison of methods for the quantification of 
bacterial sulfate reduction in coastal marine sediments. 111. Estimation from che- 
mica1 and bacteriological fie:ld data. Geomicrobiol. J .  1: 53-68 
KRIEG, N.R. & HOLT, J.G. (1984). Bergey's manual of systematic bacteriology. 
Vol. 1. Williams & Wilkins. Baltimore. 
POSTGATE, J.R. (1979). The sulphate-reducing bacteria. Cambridge University Press. 
STRICKLAND, J.D.H. & PARSONS, T.R. (1968). A practica1 handbook of seawater 
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Dissimulatory sulfate-reducing activity in lakes of Banyoles karstic area (Girona)

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