Abella, Carles

Pibernat, I. V.

Revistes Catalanes amb Accés Obert

SCZENTL4 gerundensis, 19: 35-46 (1993) 
EVOLUTION OF THE PHYSICO-CHEMICAL AND 
BIOLOGICAL PARAMETERS IN A MODIFIED 
WINOGRADSKY COLUMN 
I.V. Pibernat and C.A. Abell& 
Institut d'Ecologia Aquitica. Departament de Biologia. 
Universitat de Girona. Pl. Hospital, 6. 17071 Girona 
RESUM 
S'estudia la dinimica microbiana en una columna de Winogradsky modificada en relació als 
parimetres biolbgics i físico-químics que s'esdevenen en la columna d'aigua. 
Chlorobium limicola, C phaeobacteroides i Chromatium minus són bacteris fototrbfics del 
sofre amb una dinimica poblacional ben estudiada en condicions de camp, en els estanyols 
del Vilar i del Sisó (Banyoles, Girona). En aquest treball es compara la dinimica d'aquestes 
poblacions entre el període de mixima estratificació en l'estanyol i en la columna de labora- 
tori. Aquesta columna de laboratori permet controlar la temperatura de l'aigua i el cicle 
d'il.luminaci6. 
En condicions naturals s'estudia la competencia entre bacteris verds del sofre i bacteris por- 
pres en base a raons fisiolbgiques i d'adaptació pigmentiria a la llum disponible. En la 
columna de Winogradsky modificada s'observa una primera situació de competbncia entre 
dues especies de bacteris verds del sofre; al final de l'estudi, la competbncia es produeix de 
forma similar a les observades en condicions de camp. Aquest fet seria provocat pel caricter 
més oportunista dels bacteris fototrbfics verds del sofre enfront les Cromatiicies. 
RESUMEN 
Se estudia la dinámica microbiana entre poblaciones de bacterias fototróficas del azufre en 
una columna de Winogradsky modificada. 
Chlorobium limicola, Cphaeobacteroides y Chromatium minus son bacterias fototróficas del 
azufie con una dinámica poblacional bien estudiada en condiciones de campo. En este trabajo 
se compara la dinámica de estas poblaciones en el período de máxima estratificación en el 
campo y en la columna de Winogradsky modificada. Esta columna de laboratorio permite 
controlar el ciclo de iluminación y la temperatura del agua. 
En estudios de campo, la competencia entre bacterias del azufre se establece entre poblacio- 
nes de bacterias verdes y bacterias púrpuras o Cromatiáceas. En la columna de laboratorio, la 
competencia se establece en un primer momento, entre especies de bacterias verdes del azu- 
fre y finaliza con la coexistencia entre poblaciones de bacterias verdes del azufre y Cromatiá- 
ceas. Las poblaciones de bacterias verdes del azufre se manifestan más oportunistas que las 
Cromatiáceas. 
ABSTRACT 
The microbial dynamics of phototrophic sulfur bacteria was studied in a modified Wino- 
gradsky column. Chlorobium limicola, Cphaeobacteroides and Chromatium minus are pho- 
totrophic sulfur bacteria which population dynamics were well know on lake conditions in 
Sisó and Vilar lakes (Banyoles, Girona). 
Evolution of these populations were studied in order to compare stratification both in lake 
and laboratory column. Laboratory water column was stratified in similar way to lake 
36 EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 
column. Modified Winogradsky column has and electrical mechanism to stablish a thermal 
gradient and i l l~minatio~cycle control. 
In lake conditions, ecological competition were stablished between green and purple sulfur 
bacteria. In this work, we found previous competence between both Chlorobium (C. limicola 
and C. phaeobacteroides) and before the following competence were with Chromatium 
minus and Chlorobium limicola. These were due to mixing sediments to Vilar lake with 
Chromatium minus and Chlorobium limicola and Sisó lake with Chromatium minus and 
Chlorobium phaeobacteroides as phototrophic sulfur bacteria. The green sulfur bacteria was 
more fast to useful physico-chemical factors for grown than Chromatiaceae. 
Keywords: Laboratory column experiments, Chlorobium limicola, Phototrophic Sulphur Bacteria, 
Chlorobium Phaeobacteroides, Chromatium minus competencia, Physico-chemical factors. 
INTRODUCTION 
Physico-chemical conditions of aquatic environments determine the presence of 
phototrophic sulfur bacteria populations (Abella & Garcia-Gil 1988). Laboratory 
columns for microbial studies of bacteria were developed by Sergei Winogradsky 
in 1880 (Winogradsky 1949). 
The Winogradsky column consists of a glass cylinder tube (usually 50 cm x 15 
cm). It can be prepared by filling one-third full with sediment rich in organic mat- 
ter, preferably sulphide containing. The mud is covered with lake, pond or ditch 
water and placed in a north window so as to receive adequate sunlight. As a result 
of the production of sulphide, purple and green sulfur bacteria develop on the outer 
layer of the mud exposed to light. For isolation and enrichment of purple and green 
sulfur bacteria, the Winogradsky column has traditionally been used (Broock and 
Madigan 1988). 
Recently a new large (1 m, 2 m , 20 m) laboratory columns have been develo- 
ped for studying phytoplankton ecophysiology, Margalef (1963), Strickland et al. 
(1969), Booker et al. (1976), Booker & Walsby (1979, 1981), Heaney et al. (1989), 
Andersen and Nival (1989), how described the importance of a population enriched 
in a laboratory column to the limnological studies of aquatic systems in their natural 
environments. 
In this work, we describe evolution of physico-chemicals factors as well as bio- 
logical factors which occurred in a modified Winogradsky column. This column 
130 cm in length enable us to control temperature and the illumination cycle. 
MATERIAL AND METHODS 
The column utilized was described previously by Pibernat et al (1991) with an illu- 
mination cycle about 12 hours lightldark. Thermical gradient occured at 50 cm and 
the intensity of gradient was selected about 14 "Clm. 
Sediment used was formed by mixing half parts of compact and suspended sedi- 
ment from Sisó and Vilar lakes respectively. The sediment was also mixed with 1% 
nutritive broth and 9% Caso4 as a source of sulphate (Schegel 1987). Water was 
taken out of "Riera Castellana" stream (Banyoles, Girona). 
Water samples were collected at week periodicity with a peristaltic pump. Sam- 
pling rate was 0.4 - 0.6,l/h in order to keep water stratification. 
EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 37 
Measures: 
Temperature and conductivity were measured with a Crison thermometer T-637 and 
conductivimeter 523 respectively. Density was calculated by Bürher and Ambühl 
(1975) formula. Light intensities were measured by a photocell Megatron with lec- 
tures were on luxs. Sulphide was measured by a methyl blue method (Pachmayer 
1960). The pH was measured by a pHmeter Crison 506, redox power was measured 
by a glass electrode with Pt Metrom 6.0401.100 and oxygen was measured by a 
Yellow Spring model 57 oxymeter. 
Biological parameters as photosynthetic pigments samplings were studied follo- 
owing the methods described by Montesinos (1982). Cells were observed through a 
Zeiss microscope. 
RESULTS 
Evolution of physico-chemical factors of water were studied in a modified Wino- 
gradsky column together with photosynthetic pigments of phototrophic bacteria. 
Figures 1, 2 and 3 shows time-depth representation of temperature (OC), conduc- 
tivity (mS.cm-1) and density (g.cm-3) respectively in a modified Winogradsky 
column. These parameters appeared as density discontinuities such thermocline 
with an intensity gradient of 14 OC. m-1 and chemocline with 2 mS.cm-1.m-1. 
Modified Winogradsky column was stratified in similar way as meromictic lake 
water column. 
35 70 91 
TlME (days) 
Figure 1. Time-depth representation of temperature (OC) in a modified Winogradsky 
column. 
38 EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 
Conductivity (mSlcm) 
- 
- 
4.0 
- 
- 
I 
- 
I  I  I  I  I  I  I  I  I I I  
O 35 70 9 1 
TlME (days) 
Figure 2. Time-depth representation of conductivity (mS/cm) in a modified Winogradsky 
column. 
35 70 91 
TlME (days) 
Density (glcm3) 
Figure 3. Time-depth representation of density (glcm3) in a modified Winogradsky 
column. 
- 
0.9950 
EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 39 
. . . . . . . . . . . . . . . . . . . . . . . . 
1 1 o 1 O0 1 O00 1 O000 
LlGTH penetration 
Figure 4. Representation of ligth penetration in water column on time. 
Bacterial photosynthesis depends closely on light intensity and quality as well 
as H-donor, nutrients and temperature. In aquatic environments, light intensity 
decreases exponentially with depth, and the energy distribution of light also chan- 
ges with depth. Figure 4 shows light penetration in a water laboratory column. The 
light profile in the column is also modified by photosynthetic pigments of photo- 
trophic sulphur bacteria. Light extinction coefficient (2) shows the variation in the 
hypolimnetical zone. The 2 = 90 cm-1 at first time, between 20 and 30 cm-1 when 
populations of photosynthetic sulfur bacteria grew and 2 = 125 cm-1 at the end of 
this study. 
Time-depth evolution of PH, redox potential and sulphide concentration were 
shown at figures 5,6 and 7 respectively. These factors describe main chemical con- 
ditions which explain the establishment of phototrophic sulfur bacteria and the 
following evolution. 
PH: 
Water original came from Banyoles lake area and has a shightly alcaly PH, 7.6. The 
pH we found in Winogradsky column 7.2 and 7.4 at the half water column is due to 
sulphide concentration. Sulphide is a weak acid and it's formed by sulphate reduc- 
tion metabolism. 
Near to sediment pH was 8.0 because was formed an accumulation area by concen- 
tration of sedimented particles and diffusion of substractes from sediment (Riera 1987). 
40 EVOLUTION OF THE PHYSICO-CHEMICP3, AND BIOLOGICAL 
35 70 9 1 
TlME (days) 
Figure 5. Time-depth representation of pH in a modified Winogradsky column. 
35 70 9 1 
TlME (days) 
Figure 6. Time-depth representation of redox potential (mV) in a modified Winogradsky 
column. 
EVOLUTION OF THE PHYSICO-CHEMICAL AND IBIOLOGICAL 41 
O S2- (mM) 
1 o 
20 
30 
40 
50 
60 
70 
80 
90 
100 
O 35 70 9 1 
TlME (days) 
Figure 7. Time-depth representation of sulfide concentration (mM) in a modified Wino- 
gradsky column. 
Redox: 
The redox potential decreased in depth with time. Decreasing is due to bacterial metabo- 
lisms and presence of sulphide in the water column. The upper part of column rested 
aerobic but the bottom part remained anaerobic. The zone between aerobic and anaere 
bic life is formed by density gradient and indicated by line of + 200 mV redox potential. 
Sulphide: 
Sulphide appeared in the column after 7 days from the beginning or the study and 
suddenly high concentration were measured. When phototrophic sulfur bacteria 
developed, sulphide concentration decreased by consumption. Presence of sulphide 
in the water column occured during 91 days. 
Figure 8 showed time-depth representation of photosynthetic pigments of Chlo- 
robium phaeobacteriodes a brown phototrophic sulfur bacteria. Figure 9 showed 
photosynthetic pigments of Chlorobium limicola, a green sulfur bacteria; and figure 
10 showed time-depth representation of Bclor-a and okenone concentration to 
Chromatium minus, purple sulfur bacteria. 
DISCUSSION 
- PHYSICO-CHEMICAL FACTORS: 
In aquatic systems vertical heterogenity is created by density discontinuities such 
as thermocline and chemocline. These barriers result in a very effective differentia- 
tion of the water masses above and below the discontinuity. In stratified lakes, the 
42 EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 
O 35 70 9 1 
o 
TlME (days) 
10 - lsorenieratene 
20 - 
30 - 
- 40 - 
50 - 
I 
60 - 
W 
0 70 - 
80 - 
90 - 
1 O0 I I 
35 
TlME (days) 
Figure 8. Time-depth representation of Bacteriochlorophyll-e (d) and Isorenieratene 
concentration (U.A.11) in a modified Winogradsky column. Photosynthetic pigments of 
Chlorobium phaeobacteroides. 
hypolimnion frequently becomes anaerobic. In the water laboratory column used, 
density discontinuities were created according to natural environments. Density gra- 
dient was stronger than natural ecosystem because the depth of the column was 
shorter (1.30 m in the laboratory column to 13 m in lake water column). 
Chemical factors had been evolutioned in relation to prevail metabolism. Accor- 
ding to the modified chemistry of the environment the population of microorganisms 
EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 43 
4 Bchlor - c 
35 
TlME (days) 
4 Chlorobactene 
35 70 91 
TlME (days) 
Figure 9. Time-depth representation of Bacteriochlorophyll-c (@i) and Chlorobactene 
concentration (U.A./l) in a modified Winogradsky colurnn. Photosynthetic pigments of 
Chlorobium limicola. 
was changed. When sulphide was present in the water column, the redox potential 
was negative, and the population of phototrophic sulfur bacteria .began to develop. 
- BIOLOGICAL FACTORS: 
Populations of phototrophic sulfur bacteria developed were Chlorobium limicola, 
C. phaeobacteroides and Chromatium minus in the modified Winogradsky cslumn. 
Green phototrophic sulfur bacteria appeared 20 days before Chromatiaceae. In 
lake conditions, maxima values of Chromatiaceae and green sulfur bacteria were 
44 EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 
Bchlor-a 
- 
- 
- 
- 
- 
- 
- 
- o 
- 
I I I  I I I I I  I I I  
O 35 70 
TlME (days) 
O 35 70 91 
TlME (days) 
Figure 10. Time-depth representation of Bacteriochlorophyll-a (&I) and Okenone con- 
centration (U.A.11) in a modified Winogradsky column. Photosynthetic pigments of Chro- 
matium minus. 
shift 15 - 30 days (Montesinos 1982). Growth of green phototrophic bacteria was 
fast 14 - 20 days on Vilar lake (Montesinos 1982) and 16 days in CIV of Banyoles 
lake (Garcia-Gil et al. 1987). Generation time of Chromatiaceae at lake conditions 
was 23 days on Vilar lake (Montesinos 1982). 
Three bacterial populations developed in the modified Winogradsky column 
appeared in lake environment of which the sediment was used (Abella 1980). 
The main difference between lake dynamics and laboratory column population 
dynamic was the simultaneous presence of Chlorobium limicola and C. phaeobacte- 
EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 45 
roides. In Sisó lake, Chromatium minus grewd during summer whereas Chlorobium 
phaeobacteroides had the minimum population. In Vilar lake, the same population 
dynamics had been described (Montesinos 1982, Abella 1980, Guerrero et al 1980), 
but Chlorobium limicola appeared when the algal population of epilimnion were 
not abundant. 
C. limicola and Chromatium minus coexist under laboratory conditions (van 
Gemerden and Beeftink 1981) because don't light competition for their photosynthesis. 
In this work the population of green phototrophic bacteria, C. phaeobacteroides 
and C. limicola coexist in competition during approximately 20 days. These bacte- 
ria present a physiological competition for sulphide and for available light. C. limi- 
cola occuped the upper part of hypolimnion and C. phaeobacteroides, the bottom 
part, near to sediment where sulphide concentration was maxima. After a few days, 
Chromatium minus appeared at the upper part of hypolimnion and the brown sulfur 
bacteria disappeared for light competition and coexist green sulfur bacteria at the 
bottom part of column with Chromatiaceae at the upper part. 
Purple sulfur bacteria (Chromatiaceae) and green sulfur bacteria thrive in the 
anaerobic parts of many ponds and lakes. Under suitable conditions, these bacteria 
develop profusely. Such blooms of phototrophic bacteria are often reported to be 
mixed population of green and purple sulfur bacteria (Takahashi and Ichimura 
1970, Gorlenko et al. 1975, Guerrero and Abella 1978). For the composition of 
such mixed natural blooms a variety of parameters can be expected to be of impor- 
tance. In this study we focused on the dynamics of population under physico-che- 
mica1 conditions. 
ACKNOWLEDGMENTS 
This work was supported by Fundació Caixa de Barcelona 1991. 
References 
AB EL^, C.A. 1980. Dinamica poblacional comparada de bacterias fotosintéticas 
planctónicas. PhoThesis. UAB. 
ABELLA, C.A. and GARCIA-GIL LJ. 1988. Diel migration as a mechanism for 
enrichment of natural population of branching species of Pelodictyon In: Green 
Photosynthetic bacteria. Olson, Ormerod, Amesz, Stackebrandt and Triiper (eds). 
Plenum Press. phg. 269-286. 
ANDERSEN, V. and P. NIVAL. 1989. Modelling of phytoplankton population 
dynamics in an enclosure water column. J. mar. biol. Ass. U.K. 69: 625-646. 
BOOKER, M.J., M.T. DINSDALE and A.E. WALSBY. 1976. A continuously 
monitored column for the study of stratification by planktonic microorganisms. 
Lirnnol. Oceanog. 21 (6): 915-919. 
BOOKER, M.J. and A.E. WALSBY. 1979.   he relative form resistence of straight 
and helical blue-green alga1 filaments. Br. Phycol. J. 14: 141-150. 
BOOKER, M.J. and A.E. WALSBY. 1981. Bloom formation and stratification by a 
planktonic blue-green alga in an experimental water column. Br. Phycol. J. 16: 
411-421. 
46 EVOLUTION OF THE PHYSICO-CHEMICAL AND BIOLOGICAL 
BURHER J. and J. AMBUHL. 1975. Einleintung von abwasser in Seen. Schweiz. 
2. Hydrol. 37: 347-369. 
GORLENKO U.M. CHEBOTAREV E.N. and KACHALKIN V.I. (1975). Partici- 
pation of microorganisms in the circulation of sulfur in Pomyaretskoe Lake. 
Microbiology 43: 772 - 776. 
GARCIA-GIL, L.J., R BRUNET and C.A. ABELLA. 1987. Incidencias de la ines- 
tabilidad de la meromixis de C-IV (Lago de Banyoles, Girona) en la dinámica 
poblacional de bacterias fototróficas del azufre. Actas del N Congreso Español 
de Limnologia. pig.: 85-94. 
GUERRERO R. and ABELLA C.A. 1978. Dinámica espacio-temporal de las pobla- 
ciones bacterianas fotosintéticas en una laguna anaeróbica de aguas sulfurosas. 
OecolAquatica. 3: 193 - 205. 
GUERRERO, R., E. MONTESINOS, I. ESTEVE and C.A. ABELLA. 1980. Phy- 
siological adaptation and growth of purple and green sulfur bacteria in a mero- 
mictic lake (Sisó). Developments in Hydrobiology. (M. Dokulil, H. Metz i D. 
Jewson eds) 3: 161-192. 
HEANEY, S.I., M.C. DAVEY and A.S. BROOKS. 1989. Formation of sub-surface 
maxima of a diatom within a stratified lake and in a laboratory column. J. of 
Plankton Research, 11 (6): 1169-1184. 
MARGALEF, R. ,1963. Modelos simplificados del ambiente marino para el 
estudio de la sucesion y distribución del fitoplancton y del valor indicado de sus 
pigmentos. Invest. Pesq. 23: 11-52. 
MONTESINOS, E. 1982. Ecofisiologia de la fotosíntesis bacteriana. Tesi doctoral, 
UAB. 
PACHMAYR, F. 1960. Vorkmmen and bestimmung von Schefelverbindunger in 
Mineralwasser. Ph. D. Thesis. Univ. München. 
PIBERNAT, I.V., J. GARCIA-GIL and C.A. ABELLA. 1991. Descripció d'un 
model experimental de columna de Winogradsky. Parimetres físics i químics. 
Scientia gerundensis 17: 59-68. 
RIERA, X.G. 1987. Cicle limnoldgic i ecologia de les poblacions de bacteris 
fototrdfics en la llacuna costanera de "La Massona" (Alt Empordi, Girona). 
Tesina de Llicenciatura. Univ. Autbn. Barcelona. 
SCHEGEL, H.G. 1987. General Microbiology. Cambridge University Press. 6th 
edition. Cambridge. 
STRICKLAND, J.D., O. HOLM-HANSEN, R.W. EPPLEY and R.T. LINN, 1969. 
The use of a deep tank in phytoplankton ecology. 1. Studies of growth and com- 
position of phytoplankton groups at low nutrients levels. Limnol. Oceanogr. 14: 
23-34. 
TAKAHASHI M. and ICHIMURA K.S. (1970). Photosynthetic properties and 
growth of photosynthetic sulfur bacteria in lakes. Limnol. Oceanogr. 15: 929 - 
944. 
VAN GEMERDEN, H. and H.H. BEEFTINK, 1981. Coexistence of Chlorobium 
and Chromatium in a sulfide-limited continuous culture. Arch. Microbiol. 129: 
32-34. 
WINOGRADSKY, S. 1949. Microbiologie du sol. Probl2mes et méthodes. Masson 
et Cie Éditeurs. Paris. 


Evolution of the physico-chemical and biological parameters in a modified winogradsky column

Abstract

Similar works

Full text

Available Versions

Revistes Catalanes amb Accés Obert