García Gil, L. J.

Sala Genoher, L.

Revistes Catalanes amb Accés Obert

ARTICLE SCIENTIA gerundensis, 14:Xl-92 (1988) 
INCIDENCE OF THE IRON CYCLE ON THE 
DEVELOPMENT OF SULPHUR PHOTOTROPHIC 
BACTERIA IN LAKE VILAR 
L. J. Garcia-Gil and L. Sala-Genoher. 
Institut d'Ecologia Aquatica. Estudi General de Girona (UAB). Hospital, 6. 17071-Girona. 
RESUM 
En el treball present s'estudia la desaparició d'una població de Chlorobium phaeobac- 
teroides i l'aparició simultania d'una de Chromatium minus a l'estanyol del Vilar a I'estiu 
de 1987. La presencia de Chlorobiaceae estava lligada a condicions de baixa concentra- 
ció de sufhidric (0.054 mM), mentre que la població de Chromatiaceae comen@ a desen- 
volupar-se quan la concentració de sulfhidric va augmentar (0.160 mM). Aixb va produir 
una limitació en la qualitat i en la quantitat de la llum que rebien les Chlorobiaceae, impe- 
dint-ne el creixement. Alhora vam detectar un increment en el contingut específic de fer- 
ro particular (SCPI), a mesura que l'efecte d'ombrejat de les Chromatiaceae es feia cada 
vegada mCs important. 
RESUMEN 
En este trabajo se estudia la desaparición de una población de Chlorobium phaeobacte- 
roides y la aparición simultánea de una de Chromatium minus en la laguna del Vilar en el 
verano de 1987. La presencia de Chlorobiaceae estaba ligada a condiciones de baja con- 
centración de sulfhidrico (0.054 mM), mientras que la población de Chromatiaceae em- 
pe26 a desarrollarse cuando la concentración de sulfhidrico aumentó (0.160 mM). Este 
hecho produjo una limitación en la calidad y en la cantidad de luz que recibian las Chloro- 
biaceae, 10 que impidó su crecimiento. Al mismo tiempo, se detectó un incremento en el 
contenido especifico de hierro particulado (SCPI), a medida que el efecto de sombreado 
producido por las Chromatiaceae se hacia cada vez mas importante. 
ABSTRACT 
The present paper deals with the disppearence of a population of Chlorobium phaeobac- 
teroides and the simultaneous appearence of a population of Chrornatium minuts in the 
lake Vilar during the summer of 1987. The presence of Chlorobiaceae was related to con- 
ditions of low sulphide concentration (0.054 mM); Chromatiaceae started to develop 
when the sulphide concentration increased (0.160 mM). This produced a limitation in the 
quality and quantity of the light received by the Chlorobiaceae, and therefore, they could 
not keep on growing. At the same time, an increase in the specific content of particulated 
iron (SCPI) in Chlorobiaceae was detected, while the shadowing effect due to Chroma- 
tiaceae was becoming more important. 
Key words: Fes, light competition, phototrophic bacteria, poblational succession. 
L. J. GARCIA-GIL I L. SALA-GENOHER 
INTRODUCTION 
Recent papers have dealt with the relation between the iron cycle and photo- 
trophic bacteria in Banyoles lake, a quite new aspect of the eco-physiology of 
these microorganisms (Garcia-Gil & Abella 1988, Garcia-Gil et al., 1988). Fe- 
rrous iron is released mainly from both the compact sediment, by leaching 
processes, and the suspended sediment of the bottom springs, by the incoming 
water. This water contains dissolved ferrous iron, which diffusses through the 
hypolimnion or monimolimnion (depending on the kind of lake, holomictic or 
meromictic, respectively). When iron reaches the oxygenated layers is then 
oxidized to ferric iron which forms insoluble yellowish hydroxides. If there is a 
high flux of incoming water, the supply of dissolved oxigen (1 m g . ~ - l  is high 
enough to oxidize all the ferrous iron; this happens in the bottom springs 1 
and 7 of the Banyoles lake, in which ferrous iron is almost inexistent (unpu- 
blished data). 
In the same way, in lake Vilar it is not possible to find free ferrous iron in 
the monimolimnion, due to the precipitation with the sulphide produced 
by the sulphate-reducing activity of the sediment. Sulphide concentration, 3.8 
mM (Montesinos et al., 1983), is much higher than iron concentration, 7.8 
PM. Therefore, sulphide combines with ferrous iron to produce amorphous 
insoluble iron sulphide particles. 
It has been reported by severa1 authors (Van Gemerden 1984, Brugada 
1986) that green sulphur bacteria (Chlorobiaceae) of the genus Chlorobiun to- 
lerate the highest sulphide concentration (4-8 mM), in front of the purple 
sulphur bacteria (Chromatiaceae) of the general Chromatium, Thiocystis and 
Thiocapsa (0.8-4 mM) and the purple non-sulphur bacteria (Rhodospirilla- 
ceae, 0.4-4 mM) (Pfennig, 1975). Moreover, it is well known that species of 
the genus Chlorobium have high affinities for sulphide (around 70 
h-lmmol-I), specially Chlorobium phaeobacteroides, which reaches values 
of 120 h-'.mmol-' (Van Gemerden, 1984). 
The dominant species in the monimolimnion of the lake Vilar is the brown 
freshwater Chlorobiaceae Chlorobium phaeobacteroides, reflecting the light 
limitation produced by the algae living in the mixolimnion. These algae act 
as a natural filter and have a strong absorption in the wavelenghts bet- 
ween 400 and 500 nm; this kind of light is absorbed by brown Chlorobiaceae 
(maximum peaks at 470 and 520 nm), but not by green Chlorobiaceae (maxi- 
mum at 440-450 nm) which cannot develop under these conditions (Montesi- 
nos et al., 1983). Therefore, the bacterial population and its dynamics in a cer- 
tain moment are controlled mainly by the dynamics of algae, because of its ef- 
fect on the light transmission properties through the water column (Montesi- 
nos & Esteve, 1984). Other sulphur phototrophic bacteria which can be found 
in this lake after enrichment cultures are Chlorobium limicola and Chroma- 
tium minus (Montesinos et al., 1983). 
INCIDENCE OF THE IRON CYCLE 83 
The aim of this work is to study the establishment of a dense population of 
Chlorobiumphaeobacteroides in lake Vilar during the late spring and the early 
summer, with conditions of extremely low sulphide concentrations in the mo- 
nimolimnion (0.054 mM) as a consequence of both a total mixing period ocu- 
rred last winter, and the presence of soluble iron, which combined with the 
low sulphide produced in the sediment (Garcia-Gil et al., 1988). Another im- 
portant subject studied in this paper are the reasons for the progressive disap- 
pearence of this population and its replacement by a population of Chroma- 
tium minus along the summer. It took place parallelly with the fact that the 
concentration of sulphide in the water was increasing, exceeding the soluble 
iron concentration. 
MATERIALS AND METHODS 
Lake Vilar is located in the Banyoles karstic area in the NE of Spain (42" 
08'N, 2" 45'E), near the town of Girona. It is composed by two basins each 
with one bottom spring feeding the lake (see figure 1). 
Samples were taken along the summer in the maximal depth point of the 
basin one. Temperature, conductivity, oxygen and light extinction were mea- 
sured in situ. For temperature measurements a Crison T-637 thermistor was 
used. Conductivity was recorded with an YSI model 33 and corrected for tem- 
perature. Oxygen concentration was measured with an YSI oxymeter. Light 
extinction was measured with a selenium photoelectric cell. These measure- 
ments were performed in order to determine the location of the redoxcline 
and the bacterial layers before starting the sampling. 
Water samples from different depths were taken with a pumping system 
and were stored in hermetically closed bottles. All analysis were carried out in 
the laboratory within 24 hours after sampling. Redox potential was determi- 
ned with a platinum Metrohm 6.041.100 (JB) electrode. pH was measured 
with an Orion glass electrode (ORION 81-85). Total iron was determined ac- 
cording to the thiocianate method (Vogel, 1978). Iron sulphide was measured 
in the following way: water samples were filtered, and the filter was kept for 
one hour in acidified water (950 mL of destilled water + 50 mL of sulphuric 
acid). Because of this treatment, iron solubilized in the form of iron (11), 
which was analysed by the o-phenantroline method (9.5 mL of treated sample 
+ 0.5 mL o-phenantroline, measuring absorption at a 510 nm wavelength) 
(Golterman et al., 1978). 
Samples for sulphide analysis were stored in screw capped tubes and pre- 
served from oxidation by addition of a known volume of SAOB I1 (sulphide 
antioxidant buffer). Analysis were performed by potentiometric methods 
with an Orion 94-16 sulphide selective electrode and an Orion 90-92 double 
junction reference electrode. 
Pigments were extracted from cells retained in membrane filters with a 
layer of MgCo3 with 90 % acetone according to the method of Montesinos 
84 L. J .  GARCIA-GIL I L. SALA-GENOHER 
Figure 1. Location of lake Vilar. 
. . .  
800 1000 1200 l& ' 1600 ' 1800 . 2000 Cond. ( ~ ~ S . c m l )  
Figure 2. Physicochemical data of lake Vilar measured in August lOth, 1987. 
INCIDENCE OF THE IRON CYCLE 85 
(1982) also within 24 hours. Pigment concentration was calculated in ug.L -1 
using Bchlor e = 10.2 X (A650-A830) X f and Bchlor a = 25.2 X (A775-A830) x 
x f, where f = v1V.d and v is the estract volume in mililiters, V is the sample 
volume (filtered) in liters, and d is the pathlength of the used cuvette (Smith & 
Benitez 1955, Takahashi & Ichimura 1970, Gloe et al. 1975). 
RESULTS 
The physico-chemical data (Fig. 2) show a thermocline between 4 and 6 m 
depth, and a sharp redoxcline between 5 and 6 m depth. Oxygen is completely 
depleted below 5 m, where sulphide is formed. Light penetration is less 
than 0.8 % at 5 m depth, as a consequence of the strong algal absorption. 
When Fes and bacteriochlorophyll ccew (Bchlor cceu) values corresponding 
to the early summer are plotted together, a close relationship can be observed 
(figure 3a). Profiles present similar shapes, with both peaks almost coinciding. 
This relation disappears with depth due to the light limitation, and also with 
time, because of the disappearence of the Chlorobium phaeobacteroides po- 
pulation along the summer. Chromatium cells, which become dominant, do 
not show the same feature (figure 3b). Therefore, at the end of the summer it 
is possible to distinguish clearly the bchlor a peak from the Fes peak, which is 
found below. Tridimensional plots of Fes, bchlor e and bchlor a were perfor- 
med in order to visualize their spatial-temporal evolution (figures 4,5 and 6). 
In these graphics the coincidence between the appearence of Chromatiaceae 
and the decreasing of Chlorobiaceae populations is observable. 
Sulphide concentration reaches its maximum value (0.16 mM) (see ta- 
ble l )  at the end of July at 5.75 m depth, and moves downwards to 6 m depth, 
due to the activity of the dense Chromatium population, which established 
between 4.75 and 5.25 m depth. A portion of the iron-combined sulphide has 
to be included in these data of sulphide concentration, because of the respon- 
se of the selective electrode. This feature will be discussed below. 
DISCUSSION 
Relation between iron and phototrophic bacteria 
Our data agree with those in which iron and bchlor e (or bchlor c) peaks coin- 
cide (see figure 7). In these cases iron acts with valence 2+, independently of 
its chemical form, which can be soluble free cation (ferrous iron) on Fes. 
Population dynamics 
Tridimensional plots of pigments versus both time and depth show clearly the 
establihment of the population of Chromatium minus and the disappearence 
of the population of Chlorobiumphaeobacteroides. It is due mainly to the clo- 
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INCIDENCE OF THE IRON CYCLE 
P a r t  l c u  1 a  t e d  l r o n  
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Figure 4. Spatial-temporal distribution of particulate iron (mainly Fes) from July 6th, 
1987 (week number 1) till August 24 th, 1987 (week number 8). 
B e c t e r  l o c h l  o r o p h y  1 I " e "  
. .-- -. . 
Figure 5. Spatial-temporal distribirtion of bacteriochlorophyll e. The decrease of this 
parameter in the top of the monimolimnion coincides with the start of the develop- 
ment of Chromatiaceae. 
88 L. J. GARCIA-GIL I L. SALA-GENOHER 
Bec ter l o c h  l o r o p h y  l I 
"Q." m 
Figure 6. Spatial-temporal distribution of bacteriochlorophyll a. The high concentra- 
tion of Chromatiaceae produces a strong shading effect, affecting to the development 
of Chlorobiaceae. 
Table 1. Free sulphide concentration in lake Vilar at different moments 
Time Fes peak [S2-] (free) Dominant species 
1983 +? 3.800 mM Chlorobiaceae 
Winter 1987 - . T o t a l  mixing period 
June 1987 + 0.054 mM Chlorobiaceae 
Auaust 1987 - 0.160 mM Chromatiaceae 
sing of the 540 nm wavelenght window, which was still opened and was used 
by Chlorobium cells. There is no possibility for the stablishment of a popula- 
tion of green Chlorobiaceae as it uses to happen many times (Montesinos 
et al. 1983), because of the dense alga1 population living in the mixolimnion. 
Fes profiles 
The shape of the first Fes profile, corresponding to the early summer, (figu- 
re 8, solid line) allows us to suppose that it is the result of the addition of two 
INCIDENCE OF THE IRON CYCLE 
12 Bchlor 
16 r O PIGMENTS I ( u ~ .  2 L-' I 3
Figure 7. Vertical distribution of iron I1 and pigments of phototrophic bacteria 
in C-111, C-IV and C-VI of Banyoles lake. Bchlor e corresponds to Chlorobiumphaeo- 
bacteroides and Bchlor c represents the population of Chlorobium limicola. 
90 L. J. GARCIA-GIL I L. SALA-GENOHER 
Figure 8. Typical iron sulphide profile (solid line) at date August 4th, 1987. At this 
moment the population of Chlorobiumphaeobacteroides was completely developped. 
This profile corresponds to the measured data. FeS sedimentation profile (disconti- 
nuous line I), and BFeS profile (discontinuous line 2) are both supposed. 
independent profiles: the first is the typical profile of the Fes sedimenta- 
tion (discontinuous line I), and the second is the profile of the <<bacterial)) fer- 
rous sulphide BFeS, (discontinuous line 2), this is, the Fes which accumulates 
in the Chlorobium plate. In each case we can calculate this BFeS as the 
measured Fes value minus an estimated value which would correspond to the 
one of the sedimentation profile. 
This BFeS reduces as the population of Chromatium minus increases, just 
the same as bchlor e. We used a coefficient (SCPI, specific content of particu- 
lated iron) to give a numeric value to this feature. SCPI was calculated divi- 
ding the integrated values (along the plate) of BFeS (PM) by those of bchlor ((e,, 
(pg. L-'), which gave the following units: pmol BFeS pg bchlor e -I. The re- 
duction rate did not appear to be the same for BFeS that for bchlor e; in fact, 
bchlor e reduced faster than BFeS. For this reason SCPI increased the 
summer while the light was becoming the most important limiting factor 
(Fig. 9). The shading effect of the Chromatiaceae cells seems to collapse Chlo- 
robiaceae cells with ferrous sulphide (BFeS) in their surroundings. 
INCIDENCE OF THE IRON CYCLE 91 
- 
o Bchlor'h" (ug.L X100) 7-- 
c SCPI (urnol Fe/ug Bchlor ) 
6- -  Bchlor "eU(ug. L X 100) 
5 -- 
4 -- 
1 - -  
__------ -0 
Figure 9. Population and SCPI dynamics along the summer of '87. These data are the 
integrated values in the bacterial plate. Notice the direct relation between SCPI and 
bacteriochlorophyll a (shading effect). Plotted values for the SCPI are 
one hundred times higher. 
Role of the BFeS in the metabolism of Chlorobium 
Recent data obtained in our laboratory show the ability of Chlorobiumphaeo- 
bacteroides to use iron sulphide as electron donor for photosynthesis (Garcia- 
Gil & Abella, 1988). Light is required by Chlorobiun cells for the release of 
sulphide from Fes, before its usual well-known uptake. When there is no light 
available, BFeS cannot be removed by the cell, and remains in its externa1 
part. This hypothesis could explain our field data presented in figures 3 and 9. 
Sulphide measurements 
Although the absolute concentration values are between 10 and 15 times hig- 
her for free sulphide, the sulphide and Fes profiles appear to take similar sha- 
pes. We must consider that the sulphide concentration measured does not co- 
rrespond to free s2- concentration only, because it includes part of the Fes. It 
has also been tested in our laboratory that the selective electrode responses 
proportionally to Fes (unpublished datz). This electrode contains silver, 
which has a higher affinity for sulphide than iron, and this is the reason why a 
portion of the sulphide is removed from the iron and measured as free sulphi- 
de. So, we conclude that the real free sulphide concentration values must be 
lower than the measured. Nevertheless, the shape of the sulphide profile is 
not modified. 
92 L. J .  GARCIA-GIL I L. SALA-GENOHER 
Acknowledgements 
We wish to thank Josep Esteva and David Brugadafor their unselfish help during sampling in the 
field and performing the analysis in the laboratory. We are also indebted with C.A. Abella for his 
helpful discussion on the manuscript. 
References 
BRUGADA,, D. (1986). Metabolisme del sofre i funció destoxificadora dels bacteris de 
l'estanyol del Vilar (Banyoles). Tesina de llicenciatura, U.A.B. 
GARCIA-GIL, L.J. & ABELLA, C.A. (1988). Iron sulfide as electron donor for green 
phototrophic bacteria in natural condicions, Abstracts of the VI Symposium of Pho- 
totrophic Prokaryotes, Noordwijkerhout, The Netherlands. 
GARCIA-GIL, L.J., BRUNET, R.C., SALA-GENOHER, L1, & ABELLA, C.A. (1988) Po- 
pulation dynamics of green and purple sulfur bacteria in realtion to chemical proces- 
ses between ~e~~ ans s2+ in lake Vilar (Banyoles, Spain), Abstracts of the VI Sym- 
posium of Phototrophic Prokaryotes, Noordwijkerhout, The Netherlands. 
GLOE, A.,  PFENNING, N., BROCKMANN, H. & TROWITZSCH, W. (1975). A new bacte- 
riochlorophyll from brown coloured Chlorobiaceae, Arch. Microbiol., 102: 103- 
109. 
GOLTERMAN, H.L., CLYMO, R.S. & OHNSTAD, M.A.M. (1978). Methods for Physical 
& Chemical Analysis of Fresh Waters, IBP Handbook No 8, Blackwell Scientific Pu- 
blications. 
MONTESINOS, E.  (1982). Ecofisiologia de la fotosíntesis bacteriana. Tesis Doctoral, 
UAB . 
MONTESINOS, E., GUERRERO, R., ABELLA, C. & ESTEVE, I. (1983). Ecology and phy- 
siology of the competition for light between Clorobium limicola and Chlorobium 
phaeobacteroides in natural habitats, Applied and Environmental Microbiology, 
Vol. 46, No. 5: 1007-1016. 
MONTESINOS, E.  & ESTEVE, I. (1984). Effect of alga1 shading on the net growth and 
production of phototrophic sulfur bacteria in lakes of the Banyoles karstic area, 
Verh. Internat. Verein. Limnol., Vol. 22: 1102-1105. 
PFENNIG, N. (1975). The phototrophic bacteria and their role in the sulfur cycle, Plant 
and soil, 43: 1-16. 
SMITH, J.H. & BENITEZ, A. (1955). Chlorophylls. Analysis in plant materials. In: Mo- 
dern methods of plant analysis, V.H. Springer Verlag Heidelberg. 
TAKAHASHI, M. & ICHIMURA, S. (1970). Photosynthetic properties and growth of 
phototrophic bacteria in lakes, Limnol. Oceanogr. 15: 924-944. 
VAN GEMERDEN, H. (1984). The sulphide affinity of phototrophic bacteria in relation 
to the location of elemental sulfur, Arch. Microbiol., 139: 289-294. 
VOGEL, A.I. (1978). Text-book of quantitative inorganic analysis, 4th edition, 
Longman. 


Incidence of the iron cycle on the development of sulphur phototrophic bacteria in lake Vilar

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