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A Benthic Gradient Chamber for culturing phototrophic sulfur bacteria on reconstituted sediments
ELSEVIER
FEMS Microbiology
Ecology 20 (1996) 237-250
A Benthic Gradient Chamber for culturing phototrophic sulfur bacteria on reconstituted sediments Olivier Pringault, Rutger de Wit *, Pierre Caumette Laborotoire d’Oc&mogmphie Biologique, Unirersit~ de Bordeaux 1. C.N.R.S.-U.R.A. 197, 2. rue du Pro$ Jolyet. F-33120 Arcackon. France Received
19 January
1996: revised 16 April 1996; accepted
17 April 1996
Abstract The growth of phototrophic sulfur bacteria in benthic systems is restricted to well-defined layers within the sedimentary oxygen, sulfide, pH and light gradients. In order to culture these microorganisms under more ecologically relevant conditions. we have developed a Benthic Gradient Chamber (BGC) in which phototrophic sulfur bacteria can be grown within experimentally imposed solute and light gradients. The new autoclavable device is composed of a reconstituted sand core sandwiched in between a lower anoxic sulfide-containing compartment and an upper oxic compartment. The core can be illuminated from above by a collimated light beam. An axenic biofilm of Thiocupsa roseopersicina strain EP 2204 developed from a tiny inoculum within the sand core, using a 5-week incubation period and a 16:8 h light/dark illumination regime. The metabolic activities in this biofilm were inferred from the analyses of oxygen, sulfide and pH profiles, and their shifts during light-dark cycles. Keywords: Thiocapsa roseopersicirza; oxidation; Minielectrode
Oxygen:
Sulfide
gradient:
1. Introduction Microbial life can occur in spatial gradients of solutes, light intensity, temperature and viscosity. Several microbes occupy remarkably small zones within these gradients, which is often the result from the fact that their growth depends on more than one substrate diffusing from opposite directions. For example, the purple and green sulfur phototrophs form well-defined zones of growth in the water column of
* Corresponding 83 51 04.
author. Tel: +33
0168-6496/96/$15.00 Copyright PII SO168-6496(96)00035-9
56 22 39 09: Fax: +33
0 1996 Federation
56
of European
Light
gradient;
Axenic
biofilm;
Diffusion-limited
growth;
Sulfide
stratified lakes in response to gradients of light, sulfide, oxygen and pH [I]. Similar gradients are frequently observed in benthic environments where these parameters vary within millimeters. In these benthic gradients phototrophic microorganisms build microbial mats which can reach several millimeters of thickness [2-41. The role and importance of the diffusion process in microbial growth was recognized by Beijerinck [5] as early as 1889. However, most systems that have been used for the isolation, cultivation and study of microorganisms in the laboratory are homogeneous and thus do not mimic the dynamic gradients observed in the natural environments. Consequently, Microbiological
Societies.
Published
by Elsevier Science B.V.
238
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et al. / FEMS
Microbiolog!:
we have only a limited understanding of the physiology and behaviour of microbes confronted with multiple environmental gradients [6,7]. Only more recently have techniques been developed which mimic the spatial and temporal heterogeneity of natural habitats [8- 161. These incIude solute gradients created in agar tubes [ 12.131 or more complicated diffusion chambers containing gel-stabilized media upon which one- or two-dimensional solute gradients can be imposed [9- 11,14- 161. Such systems have been successfully applied for the study of benthic microbial ecology using both axenic or defined mixed microbial cultures as well as in situ microbial communities [ 17,l S]. Another approach is incorporating specific polymers which slowly release solutes into ordinary agar plates, thus creating long-term gradients [19]. Recently, a gradient plate has been developed that allows the rapid short-term establishment of two-dimensional diffusion gradients in a thin agar slab [201. Most gradient cultures are based on a spatial separation of solute sources and sinks; hence, while the water matrix is stabilized by added polymers, solute gradients are generated by molecular diffusion. In an effort to extend the development of gradient systems for experimental microbial ecology, we have developed a Benthic Gradient Chamber (BGC) capable of establishing long-term continuous one-dimensional gradients of small solutes and light. However, rather than using polymers, we have used a reconstituted sediment column. The attractiveness of the BGC described in this paper is that this device has been used to cultivate purple sulfur and green sulfur bacteria, largely represented in microbial mats where they stratify within steep light-, oxygen-, sulfide- and pH-gradients [2 1,221. The use of fine quartz sand allows an excellent mimicking of benthic conditions observed in these natural environments [23]. In addition, in the BGC we have also reproduced the major physicochemical gradients. Thus, the organisms have been cultured under conditions similar to those observed in their natural habitats. Their metabolic activities and thus their growth capacities have been inferred from oxygen, sulfide and pH profiles and their shifts during die1 cycles. The aim of the BGC is to obtain a better understanding of the metabolic response of bacteria when their growth and survival depend on diffusion processes.
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Xl ClYY6i
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2. Materials and methods 2. I. Bacterial strain Thincapsa roseopersicina strain EP 2204 was cultured in the BGC. This strain from the culture coltection of our laboratory is a nonmotile bacterium able to grow by photolithotrophy with sulfide, thiosulfate or elemental sulfur as electron donor [24]. Furthermore, this species is able to grow also chemolithotrophically with oxygen as the terminal electron acceptor [24,25]. During the assemblage of the BGC, the top centimeter of the sand column was inoculated with a small volume (IO ml> from a batch culture in exponential growth phase. 2.2. Benthic gradient mental setup
chamber
design
and experi-
2.2.1. Benthic Gradient Chamber design The BGC is made of glass (Premont, Bordeaux France); its design is depicted in Fig. 1. The culturing device is composed of an upper chamber and a lower chamber fitted by a slightly conical ground glass joint. As shown in Fig. 1, the upper and lower chambers communicate through a constricted part which represents a 5 cm long cylinder of 48 mm inner diameter. This part houses a 45 mm long black core tube containing a reconstituted sediment of pure fine quartz sand. Two O-rings between the black wall of the core tube and the inner glass wall provide water-tight seals; hence. molecular diffusion of solutes between the chambers takes place only through the sediment column. The 45 mm outer diameter core tube is made of black nylon (Nylon Ertalon 66 SA. Erta Engineering, USA), to prevent light penetration from the side. The bottom of the core tube consists of a fine mesh (63 pm pore size) which carries the sand. This core is steam-sterilized separately. The quartz sand with a granulometry of 125-250 pm was obtained from Merck, Germany (Ref. 77 12.1000). The upper compartment has a volume of I.500 ml. From a bent-tube inlet, sterile air is bubbled through an air-lift which has two functions: (i) to saturate the upper medium with oxygen (230 PM final concentration at the ambient temperature and salinity); and (ii) to create a gentle mixing of medium in the upper
0. Pringaultet al./ FEM.5MicrobiologyEcology 20 ( 19961237-250
Open
chimney with sterile fffter
II
-7 Sterile air-Iiff
z
A UPPER
0
-
:
0 J
CfP2Jlated
MEDIUM
oxygen
= 230 PM
F&YC
8 _
0 0
with bacteria
2.4
Fig. 1. Schematic (cross-section).
LOWER
MEDIUM
Sulfide
= 40 mM
representation
I Erlenmeyer
ffask
of the Benthic Gradient Chamber
chamber. We found that this way of gentle mixing did not create hydraulic pressure gradients. The upper reservoir and the first centimeter of the sand are thermostated by water at 20°C which circulates through an outer mantle. The lower reservoir has a volume of 2400 ml. The lower medium is stirred gently to maintain homogeneous conditions in the source. 2.2.2. Media composition and assemblage The media in the upper and lower chambers partially had a common composition, comprising filtered (0.2 pm) sea water, NH,Cl (5 mM), KH?PO, (0.5 mM), SL12B without ethylenediaminetetraacetic acid. (1 ml 1-l) [26] and the vitamin solution V7 (1 ml 1-l ) [26]. The upper medium was oxic (230 PM 02) and the lower medium anoxic with 40 mM of total dissolved free sulfide. Hence, in
239
the sedimentary column, opposed gradients of oxygen and sulfide were created by molecular diffusion. The neutralized upper medium contained 4 mM NaHCO,, being purged with air, its pH equilibrated to pH = 8.3. NaHCO, was added to the lower chamber at a concentration of 80 mM to prevent a CO, limitation for the bacteria. This way a bicarbonate gradient was created that was parallel to the sulfide gradient. The ratio of bicarbonate to sulfide fluxes was approximately 2: 1, which is in agreement with the stoichiometry of a complete oxidation of sulfide to sulfate by photolithotrophy (i.e. 2 HCO,+ lH,S + 2 [CH?O] + 1 SO:-). The lower medium was prepared carefully and aseptically under an atmosphere of sterile N?. Sulfide was added from a 1 M Na,S stock solution in small portions only, to prevent precipitation of CaCO, due to increase of pH. After each addition, the pH was carefully back titrated to pH = 7.2-7.6. Once the final concentration of 40 mM was achieved the pH was set at 7.5. The lower medium was prepared directly in the lower compartment of the BGC. The two compartments were then assembled and the black core tube with sand was inserted under aseptic conditions. After a period of 24 h, necessary to allow the establishment of a sulfide gradient, the sand column was inoculated homogeneously throughout the top one cm and then the upper compartment was filled with sterile oxic medium. 2.2.3. Light conditions The BGC was illuminated from above by a collimated light beam from an incandescent lamp (100 W, Super Philux R80 Philips). The light regimen was 16:8 h light/dark. The spectral composition of the light (360- 1000 nm) was measured with a fiberoptic radiance probe connected to a spectroradiometer (Spectron SE590, Spectron Engineering, USA). Fig. 2 shows how the spectral composition of the radiance was modified by the 10 cm column of the medium in the upper chamber above the sediment. The downwelling it-radiance across the sediment surface was 300 PEin rn-* s- ’ at 400-700 nm, quantified with a cosinus corrected PAR irradiance sensor (OL-2000Q Quantum sensor, Delta Lys Optik, Denmark). The spectral composition measured with the radiance probe showed that the ratio of near infrared (NIR) photons (700- 1000 nm) to PAR photons
0. Pringaultet al. / FEMS MicrobiologyEC&~ 20 f lYY6)237-250
240
H+ and the calibration curves exhibited a slope of 53 to 58 mV/pH unit and a 90% response time of < 20 to 30 s.
560
760 wavelength (nm)
900
Fig. 2. Spectral composition of the downwelling radiance (perpendicular to the sediment surface), below air (dotted line), and within the BGC at the sediment surface below a water column of 10 cm upper medium (continuous line).
(400-700 nm) was 8:3. Hence, we estimated that downwelling NIR irradiance across the water sediment interface was 800 PEin rn-’ s- ’ , and total downwelling irradiance (400-1000 nm) was 1100 PEin m-’ s-‘. 2.3. Minielectrodes Concentration profiles of oxygen, pH and sulfide in the sand were measured with needle minielectrodes. 2.3. I. Oxygen minielectrode The oxygen concentration was measured polarographically using a minielectrode with a cathode of approximately 10 pm [4] connected to a picoammeter (Keithley 485, Keithley Instruments, USA) with a reference electrode (Ag/AgCl). A two-points calibration of the electrode was performed in airsaturated medium above the sand and in the anaerobic zone of the sand. 2.3.2. pH Minielectrode pH was measured with a pH glass minielectrode with a pH glass bulb of 400 pm diameter (Microelectrode Inc., MI 407 b, USA) against a calomel reference electrode (Tacussel XR 100) using a highimpedance millivoltmeter (Crison pH 2002, Spain). The electrode couple was calibrated at different pH values in 0.2 M phosphate-buffered solutions containing 35 g 1-l NaCl at room temperature (20°C). The pH minielectrode had a log-linear response for
2.3.3. Sulfide minielectrode Sulfide concentrations were measured with a custom-made sulfide minielectrode with a tip diameter of 70 pm [4]. The measuring circuit was the same as for the pH measurements. Calibrations and calculations of total sulfide (sum of H,S, HS and S’-) were performed as described by Klhl and Jorgensen [27]. The sum of H,S, HS and S’- will be designated as sulfide or H,S, in the rest of the paper. Calibrations were done in a sulfide dilution series prepared in anoxic saline solution (35 g lP ’ NaCl) buffered with 0.2 M phosphate close to the pH observed in the BGC sediment column. Sulfide concentrations in these solutions were determined by the methylene blue technique [28], and related to the electrode readings. The calibration curves thus obtained exhibited a log-linear response for 2 X 10-j to IO-’ M H,S with a slope of 28 to 35 mV per decade. The electrode response times were dependent on the H,S concentration and varied from 30 s for the highest total sulfide concentrations to 15 min for the lowest. It was necessary to correct for the variation of pH along the profile. The total sulfide concentration. S, , in an aqueous solution can be expressed as follows [29]:
s,=[P-]
(1)
1+%+$ i
I 1
-1
where K; and Kb are the first and second dissociation constants of the sulfide equilibrium system, respectively, [S’-] is the sulfide concentration, and aH + is the proton activity. We used the following dissociation constants for sulfide. expressed as pK values: pK{ = 7.05 [30] and pKk = 17.1 [31]. 2.3.4. Minielectrode meaLsurements Measurements were made with oxygen, sulfide and pH minielectrodes mounted on the same micromanipulator. At the end of the incubation period (five weeks), the glass cover of the upper chamber was moved a few centimeters to provide entry for the electrodes while leaving the light source and collimator in place. The three electrodes were intro-
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duced sidewise from above and penetrated the sand under an angle of 30” with the vertical. This way the light path remained undisturbed at the measurement spots. The electrodes were moved in steps of 100 pm through the sand. Several profiles were measured at different times during a full die1 cycle as indicated in Section 3, Results. Unfortunately, the measurements were not possible under completely aseptical conditions. However, microscopic observations showed that contamination was negligible even after the 36 h measurement periods. 2.4. Depth distributions and calculations The depth distribution of metabolic processes and molecular diffusion were inferred from the steadystate O2 and H,S profiles. 2.4.1. Rates of 0,
respiration
and H,S
oxidation in
the reaction zone
In the BGC the transport of solutes occurs by molecular diffusion [32,33]. The shape of solute concentration profiles in the biofims reflects the combined action of production (P), consumption (K) and diffusion as described by Fick’s second law of one dimensional diffusion applied to the sediment [32-361:
6C( XJ) at
S’C( x,t) =cbXDs
6x*
+P(x)
-K(x)
(2)
Ecology
20 (19961237-250
241
lop5 cm* s-i and 4 X Ds(0,) = 0.694 X lo-’ cm* s- I. At steady state, Eq. (2) can be rearranged to: P(X)
-K(x)
= -+XDs
6’C( x) 6x2
(4)
The specific reaction rate for the net result of production and consumption of a solute (P(x)-K(x) ) can thus be expressed as the product of the diffusion coefficient and the second derivative (curvature) of the steady-state concentration profile [27]. Integration of Eq. (4), shows that steady-state concentration profiles are parabolic in layers where diffusion coefficients and reaction rates are uniformly distributed and independent of the actual solute concentration (zero-order kinetics) [33,35]. Depth distributions of oxygen respiration and sulfide oxidation were obtained by manually fitting parabolic functions to the curved section of the measured profiles as described by Nielsen et al. [39] and Klhl and Jorgensen [27]. Total rates of oxygen respiration and sulfide oxidation per unit area were then calculated by multiplying the zero-order rates with the thickness of the reaction zone. 2.4.2. Fluxes
of 0,
and H,S
towards the reaction
zone O2 and
H,S mass flow along the gradient is described by Fick’s first law as applied to the sediment [32]: J(X)
= -_rbxDsgx
6C( x)
(5)
where C is the solute concentration at position x and time t , D, is the diffusion coefficient of the solute in the sediment and 4 the porosity of the sand. The porosity was 0.5, determined from the volume ratio of dry sand to wet sand. Ds is related to the diffusion coefficient in free solution (Do) according to:
For the oxygen flux, the gradient across the water-sediment interface was estimated as described by Rasmussen and Jorgensen [35]. The sulfide flux was measured from the linear part of the HZ S gradient below the reaction zone.
In which 13’ represents a correction term accounting for the tortuosity of the sediment. 19’ averaged 1.5 for a large number of sediments [37]. Therefore, we assumed that 4 X Ds = 0.333 X Do. The diffusion coefficient for oxygen and sulfide determined at ZO”C, by Broecker and Peng [38] have been used in the calculations. Hence, 4 X Ds(H,S) = 0.528 X
2.4.3. Transient state measurements Short-term transient state anoxygenic photosynthesis was measured upon dark-light shifts. A sulfide and pH electrode were positioned at the same depth and the two signals were monitored during a short-term experiment. Between successive measurements at different depths, sufficiently long dark periods were intercalated to reestablish steady-state dark conditions. Taking a similar approach to that de-
242
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initial decrease of the sulfide concentration equals the rate of anoxygenic photooxidation of sulfide. The spatial resolution of this type of measurement is
scribed by Revsbech and Jorgensen [40], we assumed that diffusion and respiration remained initially unchanged when the light was switched on. Hence, the Oxygen respiration (pmol cmJ h-l) 0
0.1
0.2
0.3
0.4
Oxygen respiration (pm01 cm9 h-l) 0.5
0.1
0
100
150
0.3
0.4
0.5
Oxygen (pm01 1-l)
Oxygen (pm01 1-l) 50
0.2
200
250
50
0
100
150
2 hours
200
250
of light
6
D 0
0.5
1.0
1.5
2.0
2.5
0
8 hours 0.5
0.5
1.0
Sulfide oxidation 7.0
1.5
2.0
(pmol cme3 h-l) 8.0
PH
1.5
2.0
2 5
Sulfide (mmol 1-l)
Sulfide (mmol 1-l) 0
1.0
of light
9.0
2.5
0
0.5
1.0
Sulfide oxidation 7.0
1.5
2.0
2.5
(pmol cme3 h-l) 8.0
9.0
PH
Fig. 3. Changes of oxygen (0). sulfide (0) and pH (+) profiles during the light period within the biofdm of Tbiocapsa roseopersicina strain EP 2204. The strain was cultured in the BGC and the measurements were performed after five weeks of incubation. The microzonation and zero-order O,-respiration and H,S-oxidation reaction rates were calculated from curve-fitting of the steady state profiles and are indicated by boxes. The Iines represent the fitted curves.
0. Pringault
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described by the Einstein-Smoluchowski (cited in [40]):
where s is the standard deviation distance of the sulfide molecule distribution expressed in millimeters. The minimum time required to measure a significant decrease of the sulfide concentration was 4
equation
s=(2x+XDst)+
(6)
Oxygen respiration (Fmol cm” 0
0.1
0.2
0
50
100
I
I
0.3
Oxygen respiration (pmol cme3 h-l)
h-l)
0.4
0.5
0.1
0
A 6,
C 6
4 hours 1
0
200
250
0
0.5
1
I
of dark
1
I
I
I
1.0
1.5
2.0
50
100
I
I
B
End of light period
I
0.5
1.0
Sulfide oxidation 7.0
1.5
PH Fig. 4. Changes of oxygen (0). sulfide (0) and pH (+) strain EP 2204. See legend of Fig. 3 for details.
0.4
0.5
D
2.5
0
150
200
250
I
I
8 hours of dark 015
IlO
115
;.o
2
2.0
2.5
Sulfide (mmol 1-l) 2.0
(kmol cm3 h-l) 8.0
0.3
2 hours of dark
Sulfide (mmol 1-l) 0
0.2
Oxygen (pm01 1-l)
Oxygen (pm01 1-l) 150
243
Ecology 20 (1996) 237-250
9.0
2.5
0
0.5
1.0
Sulfide oxidation 7.0
1.5
(pmol cm” 8.0
h-l) 9.0
PH profiles during the dark period within the biofilm of Thiocapsa roseopersicina
244
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FEMS
Microbiolo,q
min, with a standard diffusion distance s equal to 0.410 mm (Eq. (5). Therefore, the spatial resolution of transient-state anoxygenic photosynthetic profiles was approximately 0.5 mm.
3. Results 3.1. Biojilm formation and pH profiles
and vertical
oxygen,
sulfide
After five weeks of incubation in the BGC, a biofilm of T. roseopersicina strain EP 2204 had developed. Microscopic observations revealed only the unique morphology of the Thiocupsa-like cells indicating that axenic conditions had been maintained throughout the incubation period. Unfortunately, it was not possible to obtain a high-resolution depth profile of the biomass distribution. Nevertheless, the metabolic activities of the biofilm could be inferred from the vertical oxygen, sulfide and pH profiles and their variations during light and dark periods, which are shown in Figs. 3 and 4, respectively. A representation of the maximum oxygen penetration and the sulfide front (i.e., the highest sediment horizon at which sulfide was detected) throughout the die1 cycle is shown in Fig. 5. At the end of the dark period (Fig. 3A, 5). oxygen penetrated down to 1.73 mm and sulfide ascended up to 0.95 mm. Thus, these compounds coexisted within a 0.8 mm-thick layer. When the light was switched on, the penetration depth of O2 increased only
Ecolog!
20 (19961
237-250
slightly and stabilized at 2.1 mm after 5 h of light. Simultaneously, the sulfide profile changed dramatically as the sulfide front shifted downwards over several millimeters, reaching 3.2 and 3.5 mm after 2 and 5 h of light. subsequently (Fig. 5). From 2 h of light onwards, a typical zone (2.1-3.7 mm) was found where neither oxygen nor sulfide was present. However, when the light was turned off and photosynthetic activity stopped, the sulfide front rose again and stabilized, after 4 h of darkness, at 1.7 mm below the sediment surface (Figs. 4 and 5). at the same depth as measured at the end of the previous dark period. Simultaneously, the maximum penetration of oxygen decreased slightly, to reach 1.75 mm after 4 h of darkness. During the first hours of both light and dark periods, the profiles were shifting due to the fact that the diffusive delivery of sulfide from the source below was not in balance with its oxidation. Thus, during the first hours of light, photosynthetic and chemosynthetic sulfide oxidation exceeded the diffusive flux (sulfide front shifted downwards), and during the first hours of the dark period, sulfide oxidation stopped completely until it reached the oxic zone (sulfide front shifted upwards). However, after 5 and 4 h for light and dark periods, respectively, steady states were established during which the oxidation of sulfide equalled the diffusive delivery and the profiles of both oxygen and sulfide remained constant.
3.2. Reaction rates and diffbsir’e fluxes ,frorn steady state profiles
Time (h,
Fig. 5. Die1 variations of the oxygen penetration depth (0) into the sand and the sulfide front (0) within the biofilm of Thiocupsn ro.reopersicina strain EP 2204 (see text).
calculated
For steady state conditions, the metabolic rates were calculated in the different depth layers, assuming zero-order kinetics in the reaction zone, using the curve-fitting approach described in Section 2, Materials and methods. The depth distributions of the metabolic activities are shown in Figs. 3 and 4. As mentioned above, the two profiles performed after 2 h of light (Fig. 3B) and 2 h of dark (Fig. 4B) were not in steady-state. From 5 h of light onwards, photosynthetic oxidation of sulfide was detected from 3.7 up to 4.8 mm depth under anoxic conditions, at a rate of 1.5 pm01 cm-j hh ’ In this zone a slight increase of pH was observed. During the light pe-
0. Pringault
et al./ FEMS Microbiology
riod, 0, respiration took place in the top 2 mm at a low rate of 0.21 prnol cme3 hh’. In these top 2 mm Thiocupsa cells contained intracellular sulfur globules as revealed by microscopic observations. Conceivably, the observed respiration may be due to the oxidation of the sulfur globules to sulfate. During the dark period, oxygen and sulfide profiles overlapped from about 3 h of darkness onwards. The profiles at 4 and 8 h of darkness were in steady state. The sulfide oxidation now took place under oxic conditions from 0.9 to 1.7 mm depth at a rate of 2.05 pmol cmm3 h-‘. Thus, during darkness sulfide removal was due to aerobic chemosynthesis or abiotic processes. In comparison with the light period, the oxygen penetration was shallower because 0, respiration in the oxic zone had increased up to 0.3 pmol cmm3 h-‘. Thus, in the oxygen-sulfide coexistence zone, the ratio of sulfide oxidation to oxygen consumption was 7:l. In order to compare the steady state metabolic activities in the reaction zone with the diffusive fluxes towards these zones, we have integrated the consumption rates over the entire reaction zone. The results are listed in Table 1, which shows that the consumption processes for both sulfide and oxygen
Table 1 Oxygen and sulfide diffusion fluxes and zero-order rosromrsicinu strain EP 2204 Time
End of dark period
Substrate
1
reaction
Reaction zone (mm)
Ecology 20 (1996) 237-250
245
in the reaction zone were indeed balanced diffusive fluxes. 3.3. Measurements tion rates
of transient
by their
state sulfide oxida-
In between 1.7 mm and 3.7 mm depth, sulfide oxidation took place only during a short transient period upon the switch-on of the light period. To characterize this process, we have performed shortterm dark-light shift experiments which are shown in Fig. 6. For each measurement, the switch-on of the light was immediately followed by a decrease of sulfide concentration and an increase of pH. The strong increase of the pH indicated that CO, fixation was not balanced by proton production; thus during transient state, H,S was only oxidized to an intermediate product and likely stored as intracellular sulfur. Intracellular stored sulfur may serve as an electron donor for photosynthesis after depletion of sulfide, but this process was not traced by the minielectrode measurements. At the end of the light period (4 min), sulfide was always present except at 1.5 mm depth where it was depleted after 4 min of light. The anoxygenic photo-
rates of 0,
respiration
and H2S oxidation
within a biofilm
of Thiocapsa
Zero-order rate ( pm01 cm-j hh’l
Integrated rate in reaction zone ( pm01 cm-’ hh’)
Diffusive flux toward reaction zone ( pmol cm- ’ h
Oxygen Sulfide
O-l.73 0.95-1.65
0.28 2.25
0.048 0.158
0.047 0.160
5 hours of light
Oxygen Sulfide
O-2.08 3.65-4.68
0.23 1.50
0.048 0.154
0.045 0.156
8 hours of light
Oxygen Sulfide
O-2.10 3.70-4.72
0.2 1 1.51
0.045 0.154
0.043 0.162
End of light period
Oxygen Sulfide
O-2.10 3.70-4.85
0.22 1.38
0.046 0.156
0.044 0.158
4 hours of dark
Oxygen Sulfide
o- 1.73 0.93- 1.68
0.30 2.07
0.05 1 0.155
0.052 0.159
End of dark period 2
Oxygen Sulfide
o- 1.75 0.94- 1.7 1
0.27 2.05
0.048 0.161
0.05 1 0.161
’)
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synthetic activity, estimated from the linear part of the sulfide concentration decrease, increased with depth, and was 3.5, 7, 16 and 27 pmol H,S cm-3 h- ’ at 1.5, 2, 2.5 and 3 mm depth, respectively.
4. Discussion 4.1. General features
of the BGC
Using the new Benthic Gradient Chamber, we have succeeded in culturing an axenic biofilm of the purple sulfur bacterium T. roseopersicina strain EP 2204 upon a sedimentary support and with vertical oxygen, sulfide, pH and light gradients that are very similar to those observed in natural benthic environments [3,4,42]. In conjunction with measurements of
Ecology 20 (19961237-250
the physicochemical gradients within the cultured biofilm by using mini- or microsensors, the BGC permits the calculation of different metabolic activities of the bacterial population. The BGC is autoclaved in parts and relatively easy to assemble and inoculate aseptically. The choice of incandescent illumination of high intensity rich in infrared wavelengths was motivated by the observations that benthic phototrophic bacteria in exposed and shallow water sediments rely mainly on NIR wavelengths [41,42]. T. roseopersicina strain EP 2204 has characteristic NIR absorption maxima at 798 and 856 nm corresponding to the bacteriochlorophyll a-antenna light-harvesting units; the 10 cm water column in the upper chamber caused an attenuation of these wavelengths by only 10 and
0.8
1.5 mm of depth _ b
0.6
8.6 8.4 8.2 8.0 7.8 7.6
0.0 0
2
4
6
8
10
0
2
Time (min) Fig. 6. Time courses of sulfide concentrations (0) and pH (0) during short-term biofilm of Thiocapsa roseopersicina strain EP 2204 (see text).
4
6
8
10
Time (min) dark-light
shift experiments
at different depths within the
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26%, respectively (see Fig. 2). Thus, sufficient radiant energy of the appropriate wavelengths arrived at the sediment surface to sustain phototrophic growth within the sediment. In sterile wet sand of the same granulometry as used in the BGC, the reported NIR attenuation coefficient equals 1.2 mm-’ [43] meaning that NIR light is attenuated to 2.7%, 0.82% and 0.25% of the surface value at 3, 4 and 5 mm depth, respectively. The upper chamber was a constant source of oxygen (230 PM). The lower chamber was the source of sulfide and bicarbonate. Although its medium was not refreshed during the experiment, it approximated constant source conditions, because of the combination of a long diffusion path length (4.5 cm) and a large volume (2.4 1). Hence, the sulfide loss from this lower chamber by diffusion was estimated at 3% of the initial value after five weeks of incubation. For comparison, with a chamber of 0.5 1 the loss will reach 10% after the same incubation period. Another advantage of the long diffusion path is that it minimizes boundary effects which have been described for semi-permeable membranes used in several gradient culture devices [7,16]. During the die1 cycle, we measured eight oxygen, sulfide and pH profiles, successively (see Figs. 3 and 4). Six of these represented steady state conditions,
Ecology 20 (19961237-250
241
which allowed to calculate the depth distribution of the net metabolic rates including sulfide and oxygen consumption. The mathematics of this approach were described by Bouldin [33]; it is based on the application of Ficks diffusion laws and assumes zero-order kinetics in the reaction zone. Hence, a curve-fitting approach [39] (see Section 2, Materials and methods) has been employed more recently on a wide range of benthic and biofilm systems in the natural environment [27,35,36,39]. The BGC enables the accuracy of this technique to be checked, because the diffusive supply towards the reaction zone is experimentally controlled. In this experiment, the sulfide gradient was 0.850 mM mm-‘, which resulted in a sulfide flux of 0.160 prnol H,S cm-’ hh’. This value corresponded well (+4%) with both the integrated rate in the reaction zone and with the diffusive flux calculated from the linear part of the profile (see Table 1). Moreover, this sulfide flux is comparable to the rates of sulfide formation due to sulfate reduction measured in coastal sediments [44,45]. 4.2. Depth distribution Thiocapsa biofilm
of metabolic
activities
in the
Most studies concerning the species T. roseopersicina have been performed in homogeneous liquid
I 6
12
16
20
Time(h)
Fig. 7. Conceptual scheme of the metaboIism of Thiocapsa roseopersicina strain EP 2704 cultivated in the BGC during a die1 cycle. Light energy is symbolised by h v. The O2 maximum penetration depth (dotted line) and the H,S front (dashed line) are also indicated.
248
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batch or continuous cultures, and a substantial amount of ecophysiological knowledge has been accumulated on this species [3,25,46-481. This bacterium is, however, particularly common in coastal benthic environments [3]. Therefore, it was attractive to use the BGC to cultivate T. roseopersicina, in order to study its metabolic responses when exposed to solute and light gradients. The cells of T. roseopersicina are nonmotile; hence, growth and mode of sulfide oxidation were determined by the inoculation distribution (homogeneous throughout the top 1 cm) and particularly by the distributions of sulfide, oxygen and light. The final biofilm observed after five weeks of incubation was the net result of these processes. From the data presented in Figs. 3 and 4, 5 and 6, we infer that the biofilm extended from the sediment surface up to almost 5 mm depth. Furthermore, we propose the conceptual scheme depicted in Fig. 7 to describe the stratification and alternations of metabolic processes during the studied light dark cycle by comparison with previously published ecophysiological data on the same species [3,25,46-481. In the top part of the biofilm up to 1.7 mm depth, the oxidation of sulfide and sulfur was due to chemosynthesis because of the pemlanent presence of oxygen [25]. Of course, chemical oxidation is also possible, but this process is very slow [49] compared with bacterial oxidation, Bacteriochlorophyll a syntheses is repressed by oxygen [25,46,47.50], and therefore, photosynthesis did not occur in the top I .7 mm. After five weeks of incubation. sulfide was not detected during the entire die1 cycle in the first millimeters, which showed a faint milky white appearance. Obviously, the bacteria present in this layer had developed during previous periods. while sulfide still reached the upper layers. In the absence of sulfide, these bacteria continued to respire O? with intracellular sulfur as the likely electron donor. Between 0.9-1.7 mm, the presence of sulfide was dependent on the dark conditions, it became detectable after 3 h of darkness. Therefore, the bacteria living in this layer grew by chemotrophy, but the electron donor changed throughout the die1 cycle. Using homogeneous batch and continuous cultures of T. rosenpersiciw strain MI. it was reported that intracellular sulfur is oxidized to sulfate while sulfide is depleted, and that sulfide is mainly oxidized
Ecolo,~
20
I IS%,61 237-250
to intermediate products and stored as sulfur in the bacterial cell when sulfide flux is high [25,47]. In a small zone between 1.7 to 2.1 mm depth, the bacterium was exposed to oxic/anoxic alternations that corresponded with light and dark periods, respectively. T. voseopersicina strain Ml has been experimentally exposed to such alternating conditions in continuous cultures [46], which showed that bacteriochlorophyll synthesis during anoxic dark periods may be sufficient to sustain photosynthetic utilisation of sulfide and sulfur during the oxic light periods. Below 2.1 mm depth, oxygen was not detected during the entire die1 cycle and, consequently, bacteriochlorophyll and carotenoids syntheses were not repressed. Bacteria living in this layer had apparently synthesized high pigment contents as the layer was clearly pink colored visible to the naked eye. Their metabolism depended strictly on the presence or absence of light and sulfide [46,47]. Between 2 and 3.5 mm depth, sulfide was quickly oxidized by photosynthetic activity during a short transient period upon the onset of the light (cf. Fig. 6). The increase in pH indicated the transient formation of intracellular sulfur representing an electron donor that was likely used subsequent the disappearance of sulfide 1471. As a result. between 2 and 3.5 mm below the surface, a typical zone without sulfide and oxygen was observed from the second hour to the end of the light period (cf. Fig. 5). Below this zone, bacteria were continuously exposed to sulfide during the light, consequently, photosynthetic sulfide oxidation is likely to be incomplete and sulfur is stored in cell material as described previously [47]. During the dark period, chemosynthetic sulfide oxidation was not possible below the maximum penetration depth of oxygen (i.e., 1.7 mm). It has been reported that under those conditions, this species may catabolize glycogen to acetate which is coupled to the reduction of intracellular sulfur to sulfide (1 glycosyl unit + 4 S + 2 acetate + 2 CO, + 4 H.S), likely representing a rather efficient dark energy generation mechanism [51]. However, sulfide production was not detectable from the steady state dark profiles.
The new BGC is a valuable tool with which to study the physiology and behaviour of benthic mi-
0. Pringault
et al. / FEMS Microbiology
croorganisms when cultivated in physicochemical gradients similar to those observed in their natural habitats. Potentially, a wide range of long-term solute gradients can be imposed and the granulometry of the filling can be changed. Thus, different types of benthic environments can be simulated with the BGC. Both axenic and defined mixed cultures can be grown. For example, we have recently obtained a biofilm composed of the purple sulfur bacterium T. roseopersicina strain EP 2204 and green sulfur bacterium Prosthecochloris sp. strain CE 2401, in which the former stratified on top of the latter. Other likely candidates for cultivation in this system include colourless sulfur bacteria and cyanobacteria with the final aim to reconstitute the microbial mat community in the laboratory.
Acknowledgements We are grateful to M. Guy Avenaud for his excellent technical assistance during the development of the BGC and to Dr. Jean-Marie Froidefond for help with light measurements. Special thanks are also due to Prof. Niels-Peter Revsbech (Aarhus University, Denmark), who stimulated our interest in gradient cultures. Financial support for the gradient cultures was provided from the county council of Aquitaine (Bordeaux). Olivier Pringault was supported by a PhD fellowship from the Ministry of Higher Education and Research (Paris).
References []I Pfennig, N. (1989) Ecology of phototrophic
purple and green sulfur bacteria. In: Autotrophic Bacteria (Schlegel. H.G. and Bowien. B., Eds.1. pp. 97-l 16. Science Tech Publishers, Madison, Wisconsin. 121Stal, L.J., Van Ciemerden. H. and Krumbein, WE. (19851 Structure and development of a benthic marine microbial mat. FEMS Microbial. Ecol. 31, 11 l-125. [31 Van Gemerden. H.. De Wit, R., Tughan. C.S. and Herbert. R.A. (1989) Development of mass blooms of Thiocapsa roseopersicina on sheltered beaches on the Orkney Islands. FEMS Microbial. Ecol. 62, 11 I- 118. 141 Van Gemerden, H., Tughan. C.S.. De Wit, R. and Herbert. R.A. (1989) Laminated microbial ecosystems on sheltered beaches in Scapa flow, Orkney Islands, FEMS Microbial. Ecol. 62. 87-102.
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[37] Ullman. W.S. and Aller, R.C. (19821 Diffusion coefficients in nearshore marine sediments. Limnol. Oceanogr. 27, 552556. [38] Broecker. W.S. and Peng, T.H. (19741 Gas exchange rates between air and see. Tellus 26. 21-35. [39] Nielsen. L.P., Christensen, P.B.. Revsbech. N.P. and Sorensen. J. (1990) Denitrification and photosynthesis in a stream sediment studied with microsensor and whole-core techniques. Limnol. Oceanogr. 35, 1135-l 144. [40] Revsbech. N.P. and Jorgensen. B.B. (1983) Photosynthesis of benthic microflora measured with high spatial resolution by the oxygen microprofile method: capabilities and limitations of the method. Limnol. Oceanogr. 28. 749-756. 1411 Lassen. C.. Ploug, H. and Jorgensen, B.B. (19921 A fibreoptic scalar itradiance microsensor: application for spectral light measurements in sediments. FEMS Microbial. Ecol. 86. 247-254. [42] Jorgensen. B.B. and Des Marais, D.J. (19861 Competition for sulfide among colorless and purple sulfur bacteria in cyanobacterial mats. FEMS. Microbial. Ecol. 38, 179-186. ]43] Kuhl. M.. Lassen, C. and Jorgensen B.B. (19941 Light penetration and light intensity in sandy marine sediments measured with irradiance and scalar irradiance fiber-optic microprobes. Mar. Ecol. Prog. Ser. 105. 139-148. 1441 Ingvorsen, K. and Jorgensen, B.B. (1982) Seasonal variation in Hz S emission to the atmosphere from intertidal sediments in Denmark. Atmos. Environ. 16, 855-865. [45] Howarth. R.W. and Jorgensen. B.B. (19841 Formation of pyrite and elemental sulfur in coastal marine sediments (Limfjorden and Kysing Fjord. Denmark) during short-term ‘sS sulfate reduction measurements. Geochim. Cosmochim. Acta 48, 1807-1818. [46] De Wit. R. and Van Gemerden, H. (19901 Growth and metabolism of the purple sulfur bacterium Thiocopsa roseopersicina under combined light/dark and oxic/anoxic regimens Arch. Microbial. 154. 459-464. [47] De Wit, R. and Van Gemerden. H. (1990) Growth and metabolism of the purple sulfur bacterium Thiocupsa roseopersicina under oxic/anoxic regimens in the light. FEMS Microbial. Ecol. 73, 69-76. [48] Visscher, P.T.. Nijburg. J.W. and Van Gemerden. H. (19901 Polysulfide utilization by Thiocapsa roseopersicino. Arch. Microbial. 155, 75-81. [49] Millero, F.J. (19911 The oxidation of H,S in Framvaren Fjord. Limnol. Oceanogr. 36, 1007-1014. 1501 Cohen-Bazire. G., Sistrom. W.R. and Stanier, R.Y. (19571 Kinetics studies of pigment synthesis by non-sulfur purple bacteria. J. Cell. Comp. Physiol. 49. 25-68. [51] De Wit. R. (19891 Interactions between phototrophic bacteria in marine sediments. PhD thesis. University of Groningen.
FEMS Microbiology
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A Benthic Gradient Chamber for culturing phototrophic sulfur bacteria on reconstituted sediments Olivier Pringault, Rutger de Wit *, Pierre Caumette Laborotoire d’Oc&mogmphie Biologique, Unirersit~ de Bordeaux 1. C.N.R.S.-U.R.A. 197, 2. rue du Pro$ Jolyet. F-33120 Arcackon. France Received
19 January
1996: revised 16 April 1996; accepted
17 April 1996
Abstract The growth of phototrophic sulfur bacteria in benthic systems is restricted to well-defined layers within the sedimentary oxygen, sulfide, pH and light gradients. In order to culture these microorganisms under more ecologically relevant conditions. we have developed a Benthic Gradient Chamber (BGC) in which phototrophic sulfur bacteria can be grown within experimentally imposed solute and light gradients. The new autoclavable device is composed of a reconstituted sand core sandwiched in between a lower anoxic sulfide-containing compartment and an upper oxic compartment. The core can be illuminated from above by a collimated light beam. An axenic biofilm of Thiocupsa roseopersicina strain EP 2204 developed from a tiny inoculum within the sand core, using a 5-week incubation period and a 16:8 h light/dark illumination regime. The metabolic activities in this biofilm were inferred from the analyses of oxygen, sulfide and pH profiles, and their shifts during light-dark cycles. Keywords: Thiocapsa roseopersicirza; oxidation; Minielectrode
Oxygen:
Sulfide
gradient:
1. Introduction Microbial life can occur in spatial gradients of solutes, light intensity, temperature and viscosity. Several microbes occupy remarkably small zones within these gradients, which is often the result from the fact that their growth depends on more than one substrate diffusing from opposite directions. For example, the purple and green sulfur phototrophs form well-defined zones of growth in the water column of
* Corresponding 83 51 04.
author. Tel: +33
0168-6496/96/$15.00 Copyright PII SO168-6496(96)00035-9
56 22 39 09: Fax: +33
0 1996 Federation
56
of European
Light
gradient;
Axenic
biofilm;
Diffusion-limited
growth;
Sulfide
stratified lakes in response to gradients of light, sulfide, oxygen and pH [I]. Similar gradients are frequently observed in benthic environments where these parameters vary within millimeters. In these benthic gradients phototrophic microorganisms build microbial mats which can reach several millimeters of thickness [2-41. The role and importance of the diffusion process in microbial growth was recognized by Beijerinck [5] as early as 1889. However, most systems that have been used for the isolation, cultivation and study of microorganisms in the laboratory are homogeneous and thus do not mimic the dynamic gradients observed in the natural environments. Consequently, Microbiological
Societies.
Published
by Elsevier Science B.V.
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Microbiolog!:
we have only a limited understanding of the physiology and behaviour of microbes confronted with multiple environmental gradients [6,7]. Only more recently have techniques been developed which mimic the spatial and temporal heterogeneity of natural habitats [8- 161. These incIude solute gradients created in agar tubes [ 12.131 or more complicated diffusion chambers containing gel-stabilized media upon which one- or two-dimensional solute gradients can be imposed [9- 11,14- 161. Such systems have been successfully applied for the study of benthic microbial ecology using both axenic or defined mixed microbial cultures as well as in situ microbial communities [ 17,l S]. Another approach is incorporating specific polymers which slowly release solutes into ordinary agar plates, thus creating long-term gradients [19]. Recently, a gradient plate has been developed that allows the rapid short-term establishment of two-dimensional diffusion gradients in a thin agar slab [201. Most gradient cultures are based on a spatial separation of solute sources and sinks; hence, while the water matrix is stabilized by added polymers, solute gradients are generated by molecular diffusion. In an effort to extend the development of gradient systems for experimental microbial ecology, we have developed a Benthic Gradient Chamber (BGC) capable of establishing long-term continuous one-dimensional gradients of small solutes and light. However, rather than using polymers, we have used a reconstituted sediment column. The attractiveness of the BGC described in this paper is that this device has been used to cultivate purple sulfur and green sulfur bacteria, largely represented in microbial mats where they stratify within steep light-, oxygen-, sulfide- and pH-gradients [2 1,221. The use of fine quartz sand allows an excellent mimicking of benthic conditions observed in these natural environments [23]. In addition, in the BGC we have also reproduced the major physicochemical gradients. Thus, the organisms have been cultured under conditions similar to those observed in their natural habitats. Their metabolic activities and thus their growth capacities have been inferred from oxygen, sulfide and pH profiles and their shifts during die1 cycles. The aim of the BGC is to obtain a better understanding of the metabolic response of bacteria when their growth and survival depend on diffusion processes.
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2. Materials and methods 2. I. Bacterial strain Thincapsa roseopersicina strain EP 2204 was cultured in the BGC. This strain from the culture coltection of our laboratory is a nonmotile bacterium able to grow by photolithotrophy with sulfide, thiosulfate or elemental sulfur as electron donor [24]. Furthermore, this species is able to grow also chemolithotrophically with oxygen as the terminal electron acceptor [24,25]. During the assemblage of the BGC, the top centimeter of the sand column was inoculated with a small volume (IO ml> from a batch culture in exponential growth phase. 2.2. Benthic gradient mental setup
chamber
design
and experi-
2.2.1. Benthic Gradient Chamber design The BGC is made of glass (Premont, Bordeaux France); its design is depicted in Fig. 1. The culturing device is composed of an upper chamber and a lower chamber fitted by a slightly conical ground glass joint. As shown in Fig. 1, the upper and lower chambers communicate through a constricted part which represents a 5 cm long cylinder of 48 mm inner diameter. This part houses a 45 mm long black core tube containing a reconstituted sediment of pure fine quartz sand. Two O-rings between the black wall of the core tube and the inner glass wall provide water-tight seals; hence. molecular diffusion of solutes between the chambers takes place only through the sediment column. The 45 mm outer diameter core tube is made of black nylon (Nylon Ertalon 66 SA. Erta Engineering, USA), to prevent light penetration from the side. The bottom of the core tube consists of a fine mesh (63 pm pore size) which carries the sand. This core is steam-sterilized separately. The quartz sand with a granulometry of 125-250 pm was obtained from Merck, Germany (Ref. 77 12.1000). The upper compartment has a volume of I.500 ml. From a bent-tube inlet, sterile air is bubbled through an air-lift which has two functions: (i) to saturate the upper medium with oxygen (230 PM final concentration at the ambient temperature and salinity); and (ii) to create a gentle mixing of medium in the upper
0. Pringaultet al./ FEM.5MicrobiologyEcology 20 ( 19961237-250
Open
chimney with sterile fffter
II
-7 Sterile air-Iiff
z
A UPPER
0
-
:
0 J
CfP2Jlated
MEDIUM
oxygen
= 230 PM
F&YC
8 _
0 0
with bacteria
2.4
Fig. 1. Schematic (cross-section).
LOWER
MEDIUM
Sulfide
= 40 mM
representation
I Erlenmeyer
ffask
of the Benthic Gradient Chamber
chamber. We found that this way of gentle mixing did not create hydraulic pressure gradients. The upper reservoir and the first centimeter of the sand are thermostated by water at 20°C which circulates through an outer mantle. The lower reservoir has a volume of 2400 ml. The lower medium is stirred gently to maintain homogeneous conditions in the source. 2.2.2. Media composition and assemblage The media in the upper and lower chambers partially had a common composition, comprising filtered (0.2 pm) sea water, NH,Cl (5 mM), KH?PO, (0.5 mM), SL12B without ethylenediaminetetraacetic acid. (1 ml 1-l) [26] and the vitamin solution V7 (1 ml 1-l ) [26]. The upper medium was oxic (230 PM 02) and the lower medium anoxic with 40 mM of total dissolved free sulfide. Hence, in
239
the sedimentary column, opposed gradients of oxygen and sulfide were created by molecular diffusion. The neutralized upper medium contained 4 mM NaHCO,, being purged with air, its pH equilibrated to pH = 8.3. NaHCO, was added to the lower chamber at a concentration of 80 mM to prevent a CO, limitation for the bacteria. This way a bicarbonate gradient was created that was parallel to the sulfide gradient. The ratio of bicarbonate to sulfide fluxes was approximately 2: 1, which is in agreement with the stoichiometry of a complete oxidation of sulfide to sulfate by photolithotrophy (i.e. 2 HCO,+ lH,S + 2 [CH?O] + 1 SO:-). The lower medium was prepared carefully and aseptically under an atmosphere of sterile N?. Sulfide was added from a 1 M Na,S stock solution in small portions only, to prevent precipitation of CaCO, due to increase of pH. After each addition, the pH was carefully back titrated to pH = 7.2-7.6. Once the final concentration of 40 mM was achieved the pH was set at 7.5. The lower medium was prepared directly in the lower compartment of the BGC. The two compartments were then assembled and the black core tube with sand was inserted under aseptic conditions. After a period of 24 h, necessary to allow the establishment of a sulfide gradient, the sand column was inoculated homogeneously throughout the top one cm and then the upper compartment was filled with sterile oxic medium. 2.2.3. Light conditions The BGC was illuminated from above by a collimated light beam from an incandescent lamp (100 W, Super Philux R80 Philips). The light regimen was 16:8 h light/dark. The spectral composition of the light (360- 1000 nm) was measured with a fiberoptic radiance probe connected to a spectroradiometer (Spectron SE590, Spectron Engineering, USA). Fig. 2 shows how the spectral composition of the radiance was modified by the 10 cm column of the medium in the upper chamber above the sediment. The downwelling it-radiance across the sediment surface was 300 PEin rn-* s- ’ at 400-700 nm, quantified with a cosinus corrected PAR irradiance sensor (OL-2000Q Quantum sensor, Delta Lys Optik, Denmark). The spectral composition measured with the radiance probe showed that the ratio of near infrared (NIR) photons (700- 1000 nm) to PAR photons
0. Pringaultet al. / FEMS MicrobiologyEC&~ 20 f lYY6)237-250
240
H+ and the calibration curves exhibited a slope of 53 to 58 mV/pH unit and a 90% response time of < 20 to 30 s.
560
760 wavelength (nm)
900
Fig. 2. Spectral composition of the downwelling radiance (perpendicular to the sediment surface), below air (dotted line), and within the BGC at the sediment surface below a water column of 10 cm upper medium (continuous line).
(400-700 nm) was 8:3. Hence, we estimated that downwelling NIR irradiance across the water sediment interface was 800 PEin rn-’ s- ’ , and total downwelling irradiance (400-1000 nm) was 1100 PEin m-’ s-‘. 2.3. Minielectrodes Concentration profiles of oxygen, pH and sulfide in the sand were measured with needle minielectrodes. 2.3. I. Oxygen minielectrode The oxygen concentration was measured polarographically using a minielectrode with a cathode of approximately 10 pm [4] connected to a picoammeter (Keithley 485, Keithley Instruments, USA) with a reference electrode (Ag/AgCl). A two-points calibration of the electrode was performed in airsaturated medium above the sand and in the anaerobic zone of the sand. 2.3.2. pH Minielectrode pH was measured with a pH glass minielectrode with a pH glass bulb of 400 pm diameter (Microelectrode Inc., MI 407 b, USA) against a calomel reference electrode (Tacussel XR 100) using a highimpedance millivoltmeter (Crison pH 2002, Spain). The electrode couple was calibrated at different pH values in 0.2 M phosphate-buffered solutions containing 35 g 1-l NaCl at room temperature (20°C). The pH minielectrode had a log-linear response for
2.3.3. Sulfide minielectrode Sulfide concentrations were measured with a custom-made sulfide minielectrode with a tip diameter of 70 pm [4]. The measuring circuit was the same as for the pH measurements. Calibrations and calculations of total sulfide (sum of H,S, HS and S’-) were performed as described by Klhl and Jorgensen [27]. The sum of H,S, HS and S’- will be designated as sulfide or H,S, in the rest of the paper. Calibrations were done in a sulfide dilution series prepared in anoxic saline solution (35 g lP ’ NaCl) buffered with 0.2 M phosphate close to the pH observed in the BGC sediment column. Sulfide concentrations in these solutions were determined by the methylene blue technique [28], and related to the electrode readings. The calibration curves thus obtained exhibited a log-linear response for 2 X 10-j to IO-’ M H,S with a slope of 28 to 35 mV per decade. The electrode response times were dependent on the H,S concentration and varied from 30 s for the highest total sulfide concentrations to 15 min for the lowest. It was necessary to correct for the variation of pH along the profile. The total sulfide concentration. S, , in an aqueous solution can be expressed as follows [29]:
s,=[P-]
(1)
1+%+$ i
I 1
-1
where K; and Kb are the first and second dissociation constants of the sulfide equilibrium system, respectively, [S’-] is the sulfide concentration, and aH + is the proton activity. We used the following dissociation constants for sulfide. expressed as pK values: pK{ = 7.05 [30] and pKk = 17.1 [31]. 2.3.4. Minielectrode meaLsurements Measurements were made with oxygen, sulfide and pH minielectrodes mounted on the same micromanipulator. At the end of the incubation period (five weeks), the glass cover of the upper chamber was moved a few centimeters to provide entry for the electrodes while leaving the light source and collimator in place. The three electrodes were intro-
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duced sidewise from above and penetrated the sand under an angle of 30” with the vertical. This way the light path remained undisturbed at the measurement spots. The electrodes were moved in steps of 100 pm through the sand. Several profiles were measured at different times during a full die1 cycle as indicated in Section 3, Results. Unfortunately, the measurements were not possible under completely aseptical conditions. However, microscopic observations showed that contamination was negligible even after the 36 h measurement periods. 2.4. Depth distributions and calculations The depth distribution of metabolic processes and molecular diffusion were inferred from the steadystate O2 and H,S profiles. 2.4.1. Rates of 0,
respiration
and H,S
oxidation in
the reaction zone
In the BGC the transport of solutes occurs by molecular diffusion [32,33]. The shape of solute concentration profiles in the biofims reflects the combined action of production (P), consumption (K) and diffusion as described by Fick’s second law of one dimensional diffusion applied to the sediment [32-361:
6C( XJ) at
S’C( x,t) =cbXDs
6x*
+P(x)
-K(x)
(2)
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20 (19961237-250
241
lop5 cm* s-i and 4 X Ds(0,) = 0.694 X lo-’ cm* s- I. At steady state, Eq. (2) can be rearranged to: P(X)
-K(x)
= -+XDs
6’C( x) 6x2
(4)
The specific reaction rate for the net result of production and consumption of a solute (P(x)-K(x) ) can thus be expressed as the product of the diffusion coefficient and the second derivative (curvature) of the steady-state concentration profile [27]. Integration of Eq. (4), shows that steady-state concentration profiles are parabolic in layers where diffusion coefficients and reaction rates are uniformly distributed and independent of the actual solute concentration (zero-order kinetics) [33,35]. Depth distributions of oxygen respiration and sulfide oxidation were obtained by manually fitting parabolic functions to the curved section of the measured profiles as described by Nielsen et al. [39] and Klhl and Jorgensen [27]. Total rates of oxygen respiration and sulfide oxidation per unit area were then calculated by multiplying the zero-order rates with the thickness of the reaction zone. 2.4.2. Fluxes
of 0,
and H,S
towards the reaction
zone O2 and
H,S mass flow along the gradient is described by Fick’s first law as applied to the sediment [32]: J(X)
= -_rbxDsgx
6C( x)
(5)
where C is the solute concentration at position x and time t , D, is the diffusion coefficient of the solute in the sediment and 4 the porosity of the sand. The porosity was 0.5, determined from the volume ratio of dry sand to wet sand. Ds is related to the diffusion coefficient in free solution (Do) according to:
For the oxygen flux, the gradient across the water-sediment interface was estimated as described by Rasmussen and Jorgensen [35]. The sulfide flux was measured from the linear part of the HZ S gradient below the reaction zone.
In which 13’ represents a correction term accounting for the tortuosity of the sediment. 19’ averaged 1.5 for a large number of sediments [37]. Therefore, we assumed that 4 X Ds = 0.333 X Do. The diffusion coefficient for oxygen and sulfide determined at ZO”C, by Broecker and Peng [38] have been used in the calculations. Hence, 4 X Ds(H,S) = 0.528 X
2.4.3. Transient state measurements Short-term transient state anoxygenic photosynthesis was measured upon dark-light shifts. A sulfide and pH electrode were positioned at the same depth and the two signals were monitored during a short-term experiment. Between successive measurements at different depths, sufficiently long dark periods were intercalated to reestablish steady-state dark conditions. Taking a similar approach to that de-
242
0. Pringaulr et al. / FEMS Microbiology Ecology 20 f 1996) 237-250
initial decrease of the sulfide concentration equals the rate of anoxygenic photooxidation of sulfide. The spatial resolution of this type of measurement is
scribed by Revsbech and Jorgensen [40], we assumed that diffusion and respiration remained initially unchanged when the light was switched on. Hence, the Oxygen respiration (pmol cmJ h-l) 0
0.1
0.2
0.3
0.4
Oxygen respiration (pm01 cm9 h-l) 0.5
0.1
0
100
150
0.3
0.4
0.5
Oxygen (pm01 1-l)
Oxygen (pm01 1-l) 50
0.2
200
250
50
0
100
150
2 hours
200
250
of light
6
D 0
0.5
1.0
1.5
2.0
2.5
0
8 hours 0.5
0.5
1.0
Sulfide oxidation 7.0
1.5
2.0
(pmol cme3 h-l) 8.0
PH
1.5
2.0
2 5
Sulfide (mmol 1-l)
Sulfide (mmol 1-l) 0
1.0
of light
9.0
2.5
0
0.5
1.0
Sulfide oxidation 7.0
1.5
2.0
2.5
(pmol cme3 h-l) 8.0
9.0
PH
Fig. 3. Changes of oxygen (0). sulfide (0) and pH (+) profiles during the light period within the biofdm of Tbiocapsa roseopersicina strain EP 2204. The strain was cultured in the BGC and the measurements were performed after five weeks of incubation. The microzonation and zero-order O,-respiration and H,S-oxidation reaction rates were calculated from curve-fitting of the steady state profiles and are indicated by boxes. The Iines represent the fitted curves.
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described by the Einstein-Smoluchowski (cited in [40]):
where s is the standard deviation distance of the sulfide molecule distribution expressed in millimeters. The minimum time required to measure a significant decrease of the sulfide concentration was 4
equation
s=(2x+XDst)+
(6)
Oxygen respiration (Fmol cm” 0
0.1
0.2
0
50
100
I
I
0.3
Oxygen respiration (pmol cme3 h-l)
h-l)
0.4
0.5
0.1
0
A 6,
C 6
4 hours 1
0
200
250
0
0.5
1
I
of dark
1
I
I
I
1.0
1.5
2.0
50
100
I
I
B
End of light period
I
0.5
1.0
Sulfide oxidation 7.0
1.5
PH Fig. 4. Changes of oxygen (0). sulfide (0) and pH (+) strain EP 2204. See legend of Fig. 3 for details.
0.4
0.5
D
2.5
0
150
200
250
I
I
8 hours of dark 015
IlO
115
;.o
2
2.0
2.5
Sulfide (mmol 1-l) 2.0
(kmol cm3 h-l) 8.0
0.3
2 hours of dark
Sulfide (mmol 1-l) 0
0.2
Oxygen (pm01 1-l)
Oxygen (pm01 1-l) 150
243
Ecology 20 (1996) 237-250
9.0
2.5
0
0.5
1.0
Sulfide oxidation 7.0
1.5
(pmol cm” 8.0
h-l) 9.0
PH profiles during the dark period within the biofilm of Thiocapsa roseopersicina
244
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et al./
FEMS
Microbiolo,q
min, with a standard diffusion distance s equal to 0.410 mm (Eq. (5). Therefore, the spatial resolution of transient-state anoxygenic photosynthetic profiles was approximately 0.5 mm.
3. Results 3.1. Biojilm formation and pH profiles
and vertical
oxygen,
sulfide
After five weeks of incubation in the BGC, a biofilm of T. roseopersicina strain EP 2204 had developed. Microscopic observations revealed only the unique morphology of the Thiocupsa-like cells indicating that axenic conditions had been maintained throughout the incubation period. Unfortunately, it was not possible to obtain a high-resolution depth profile of the biomass distribution. Nevertheless, the metabolic activities of the biofilm could be inferred from the vertical oxygen, sulfide and pH profiles and their variations during light and dark periods, which are shown in Figs. 3 and 4, respectively. A representation of the maximum oxygen penetration and the sulfide front (i.e., the highest sediment horizon at which sulfide was detected) throughout the die1 cycle is shown in Fig. 5. At the end of the dark period (Fig. 3A, 5). oxygen penetrated down to 1.73 mm and sulfide ascended up to 0.95 mm. Thus, these compounds coexisted within a 0.8 mm-thick layer. When the light was switched on, the penetration depth of O2 increased only
Ecolog!
20 (19961
237-250
slightly and stabilized at 2.1 mm after 5 h of light. Simultaneously, the sulfide profile changed dramatically as the sulfide front shifted downwards over several millimeters, reaching 3.2 and 3.5 mm after 2 and 5 h of light. subsequently (Fig. 5). From 2 h of light onwards, a typical zone (2.1-3.7 mm) was found where neither oxygen nor sulfide was present. However, when the light was turned off and photosynthetic activity stopped, the sulfide front rose again and stabilized, after 4 h of darkness, at 1.7 mm below the sediment surface (Figs. 4 and 5). at the same depth as measured at the end of the previous dark period. Simultaneously, the maximum penetration of oxygen decreased slightly, to reach 1.75 mm after 4 h of darkness. During the first hours of both light and dark periods, the profiles were shifting due to the fact that the diffusive delivery of sulfide from the source below was not in balance with its oxidation. Thus, during the first hours of light, photosynthetic and chemosynthetic sulfide oxidation exceeded the diffusive flux (sulfide front shifted downwards), and during the first hours of the dark period, sulfide oxidation stopped completely until it reached the oxic zone (sulfide front shifted upwards). However, after 5 and 4 h for light and dark periods, respectively, steady states were established during which the oxidation of sulfide equalled the diffusive delivery and the profiles of both oxygen and sulfide remained constant.
3.2. Reaction rates and diffbsir’e fluxes ,frorn steady state profiles
Time (h,
Fig. 5. Die1 variations of the oxygen penetration depth (0) into the sand and the sulfide front (0) within the biofilm of Thiocupsn ro.reopersicina strain EP 2204 (see text).
calculated
For steady state conditions, the metabolic rates were calculated in the different depth layers, assuming zero-order kinetics in the reaction zone, using the curve-fitting approach described in Section 2, Materials and methods. The depth distributions of the metabolic activities are shown in Figs. 3 and 4. As mentioned above, the two profiles performed after 2 h of light (Fig. 3B) and 2 h of dark (Fig. 4B) were not in steady-state. From 5 h of light onwards, photosynthetic oxidation of sulfide was detected from 3.7 up to 4.8 mm depth under anoxic conditions, at a rate of 1.5 pm01 cm-j hh ’ In this zone a slight increase of pH was observed. During the light pe-
0. Pringault
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riod, 0, respiration took place in the top 2 mm at a low rate of 0.21 prnol cme3 hh’. In these top 2 mm Thiocupsa cells contained intracellular sulfur globules as revealed by microscopic observations. Conceivably, the observed respiration may be due to the oxidation of the sulfur globules to sulfate. During the dark period, oxygen and sulfide profiles overlapped from about 3 h of darkness onwards. The profiles at 4 and 8 h of darkness were in steady state. The sulfide oxidation now took place under oxic conditions from 0.9 to 1.7 mm depth at a rate of 2.05 pmol cmm3 h-‘. Thus, during darkness sulfide removal was due to aerobic chemosynthesis or abiotic processes. In comparison with the light period, the oxygen penetration was shallower because 0, respiration in the oxic zone had increased up to 0.3 pmol cmm3 h-‘. Thus, in the oxygen-sulfide coexistence zone, the ratio of sulfide oxidation to oxygen consumption was 7:l. In order to compare the steady state metabolic activities in the reaction zone with the diffusive fluxes towards these zones, we have integrated the consumption rates over the entire reaction zone. The results are listed in Table 1, which shows that the consumption processes for both sulfide and oxygen
Table 1 Oxygen and sulfide diffusion fluxes and zero-order rosromrsicinu strain EP 2204 Time
End of dark period
Substrate
1
reaction
Reaction zone (mm)
Ecology 20 (1996) 237-250
245
in the reaction zone were indeed balanced diffusive fluxes. 3.3. Measurements tion rates
of transient
by their
state sulfide oxida-
In between 1.7 mm and 3.7 mm depth, sulfide oxidation took place only during a short transient period upon the switch-on of the light period. To characterize this process, we have performed shortterm dark-light shift experiments which are shown in Fig. 6. For each measurement, the switch-on of the light was immediately followed by a decrease of sulfide concentration and an increase of pH. The strong increase of the pH indicated that CO, fixation was not balanced by proton production; thus during transient state, H,S was only oxidized to an intermediate product and likely stored as intracellular sulfur. Intracellular stored sulfur may serve as an electron donor for photosynthesis after depletion of sulfide, but this process was not traced by the minielectrode measurements. At the end of the light period (4 min), sulfide was always present except at 1.5 mm depth where it was depleted after 4 min of light. The anoxygenic photo-
rates of 0,
respiration
and H2S oxidation
within a biofilm
of Thiocapsa
Zero-order rate ( pm01 cm-j hh’l
Integrated rate in reaction zone ( pm01 cm-’ hh’)
Diffusive flux toward reaction zone ( pmol cm- ’ h
Oxygen Sulfide
O-l.73 0.95-1.65
0.28 2.25
0.048 0.158
0.047 0.160
5 hours of light
Oxygen Sulfide
O-2.08 3.65-4.68
0.23 1.50
0.048 0.154
0.045 0.156
8 hours of light
Oxygen Sulfide
O-2.10 3.70-4.72
0.2 1 1.51
0.045 0.154
0.043 0.162
End of light period
Oxygen Sulfide
O-2.10 3.70-4.85
0.22 1.38
0.046 0.156
0.044 0.158
4 hours of dark
Oxygen Sulfide
o- 1.73 0.93- 1.68
0.30 2.07
0.05 1 0.155
0.052 0.159
End of dark period 2
Oxygen Sulfide
o- 1.75 0.94- 1.7 1
0.27 2.05
0.048 0.161
0.05 1 0.161
’)
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0. Pringadt
et al. / FEMS Microbiology
synthetic activity, estimated from the linear part of the sulfide concentration decrease, increased with depth, and was 3.5, 7, 16 and 27 pmol H,S cm-3 h- ’ at 1.5, 2, 2.5 and 3 mm depth, respectively.
4. Discussion 4.1. General features
of the BGC
Using the new Benthic Gradient Chamber, we have succeeded in culturing an axenic biofilm of the purple sulfur bacterium T. roseopersicina strain EP 2204 upon a sedimentary support and with vertical oxygen, sulfide, pH and light gradients that are very similar to those observed in natural benthic environments [3,4,42]. In conjunction with measurements of
Ecology 20 (19961237-250
the physicochemical gradients within the cultured biofilm by using mini- or microsensors, the BGC permits the calculation of different metabolic activities of the bacterial population. The BGC is autoclaved in parts and relatively easy to assemble and inoculate aseptically. The choice of incandescent illumination of high intensity rich in infrared wavelengths was motivated by the observations that benthic phototrophic bacteria in exposed and shallow water sediments rely mainly on NIR wavelengths [41,42]. T. roseopersicina strain EP 2204 has characteristic NIR absorption maxima at 798 and 856 nm corresponding to the bacteriochlorophyll a-antenna light-harvesting units; the 10 cm water column in the upper chamber caused an attenuation of these wavelengths by only 10 and
0.8
1.5 mm of depth _ b
0.6
8.6 8.4 8.2 8.0 7.8 7.6
0.0 0
2
4
6
8
10
0
2
Time (min) Fig. 6. Time courses of sulfide concentrations (0) and pH (0) during short-term biofilm of Thiocapsa roseopersicina strain EP 2204 (see text).
4
6
8
10
Time (min) dark-light
shift experiments
at different depths within the
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26%, respectively (see Fig. 2). Thus, sufficient radiant energy of the appropriate wavelengths arrived at the sediment surface to sustain phototrophic growth within the sediment. In sterile wet sand of the same granulometry as used in the BGC, the reported NIR attenuation coefficient equals 1.2 mm-’ [43] meaning that NIR light is attenuated to 2.7%, 0.82% and 0.25% of the surface value at 3, 4 and 5 mm depth, respectively. The upper chamber was a constant source of oxygen (230 PM). The lower chamber was the source of sulfide and bicarbonate. Although its medium was not refreshed during the experiment, it approximated constant source conditions, because of the combination of a long diffusion path length (4.5 cm) and a large volume (2.4 1). Hence, the sulfide loss from this lower chamber by diffusion was estimated at 3% of the initial value after five weeks of incubation. For comparison, with a chamber of 0.5 1 the loss will reach 10% after the same incubation period. Another advantage of the long diffusion path is that it minimizes boundary effects which have been described for semi-permeable membranes used in several gradient culture devices [7,16]. During the die1 cycle, we measured eight oxygen, sulfide and pH profiles, successively (see Figs. 3 and 4). Six of these represented steady state conditions,
Ecology 20 (19961237-250
241
which allowed to calculate the depth distribution of the net metabolic rates including sulfide and oxygen consumption. The mathematics of this approach were described by Bouldin [33]; it is based on the application of Ficks diffusion laws and assumes zero-order kinetics in the reaction zone. Hence, a curve-fitting approach [39] (see Section 2, Materials and methods) has been employed more recently on a wide range of benthic and biofilm systems in the natural environment [27,35,36,39]. The BGC enables the accuracy of this technique to be checked, because the diffusive supply towards the reaction zone is experimentally controlled. In this experiment, the sulfide gradient was 0.850 mM mm-‘, which resulted in a sulfide flux of 0.160 prnol H,S cm-’ hh’. This value corresponded well (+4%) with both the integrated rate in the reaction zone and with the diffusive flux calculated from the linear part of the profile (see Table 1). Moreover, this sulfide flux is comparable to the rates of sulfide formation due to sulfate reduction measured in coastal sediments [44,45]. 4.2. Depth distribution Thiocapsa biofilm
of metabolic
activities
in the
Most studies concerning the species T. roseopersicina have been performed in homogeneous liquid
I 6
12
16
20
Time(h)
Fig. 7. Conceptual scheme of the metaboIism of Thiocapsa roseopersicina strain EP 2704 cultivated in the BGC during a die1 cycle. Light energy is symbolised by h v. The O2 maximum penetration depth (dotted line) and the H,S front (dashed line) are also indicated.
248
0. Prirzguult et al. / FEM.5 Microbiology
batch or continuous cultures, and a substantial amount of ecophysiological knowledge has been accumulated on this species [3,25,46-481. This bacterium is, however, particularly common in coastal benthic environments [3]. Therefore, it was attractive to use the BGC to cultivate T. roseopersicina, in order to study its metabolic responses when exposed to solute and light gradients. The cells of T. roseopersicina are nonmotile; hence, growth and mode of sulfide oxidation were determined by the inoculation distribution (homogeneous throughout the top 1 cm) and particularly by the distributions of sulfide, oxygen and light. The final biofilm observed after five weeks of incubation was the net result of these processes. From the data presented in Figs. 3 and 4, 5 and 6, we infer that the biofilm extended from the sediment surface up to almost 5 mm depth. Furthermore, we propose the conceptual scheme depicted in Fig. 7 to describe the stratification and alternations of metabolic processes during the studied light dark cycle by comparison with previously published ecophysiological data on the same species [3,25,46-481. In the top part of the biofilm up to 1.7 mm depth, the oxidation of sulfide and sulfur was due to chemosynthesis because of the pemlanent presence of oxygen [25]. Of course, chemical oxidation is also possible, but this process is very slow [49] compared with bacterial oxidation, Bacteriochlorophyll a syntheses is repressed by oxygen [25,46,47.50], and therefore, photosynthesis did not occur in the top I .7 mm. After five weeks of incubation. sulfide was not detected during the entire die1 cycle in the first millimeters, which showed a faint milky white appearance. Obviously, the bacteria present in this layer had developed during previous periods. while sulfide still reached the upper layers. In the absence of sulfide, these bacteria continued to respire O? with intracellular sulfur as the likely electron donor. Between 0.9-1.7 mm, the presence of sulfide was dependent on the dark conditions, it became detectable after 3 h of darkness. Therefore, the bacteria living in this layer grew by chemotrophy, but the electron donor changed throughout the die1 cycle. Using homogeneous batch and continuous cultures of T. rosenpersiciw strain MI. it was reported that intracellular sulfur is oxidized to sulfate while sulfide is depleted, and that sulfide is mainly oxidized
Ecolo,~
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I IS%,61 237-250
to intermediate products and stored as sulfur in the bacterial cell when sulfide flux is high [25,47]. In a small zone between 1.7 to 2.1 mm depth, the bacterium was exposed to oxic/anoxic alternations that corresponded with light and dark periods, respectively. T. voseopersicina strain Ml has been experimentally exposed to such alternating conditions in continuous cultures [46], which showed that bacteriochlorophyll synthesis during anoxic dark periods may be sufficient to sustain photosynthetic utilisation of sulfide and sulfur during the oxic light periods. Below 2.1 mm depth, oxygen was not detected during the entire die1 cycle and, consequently, bacteriochlorophyll and carotenoids syntheses were not repressed. Bacteria living in this layer had apparently synthesized high pigment contents as the layer was clearly pink colored visible to the naked eye. Their metabolism depended strictly on the presence or absence of light and sulfide [46,47]. Between 2 and 3.5 mm depth, sulfide was quickly oxidized by photosynthetic activity during a short transient period upon the onset of the light (cf. Fig. 6). The increase in pH indicated the transient formation of intracellular sulfur representing an electron donor that was likely used subsequent the disappearance of sulfide 1471. As a result. between 2 and 3.5 mm below the surface, a typical zone without sulfide and oxygen was observed from the second hour to the end of the light period (cf. Fig. 5). Below this zone, bacteria were continuously exposed to sulfide during the light, consequently, photosynthetic sulfide oxidation is likely to be incomplete and sulfur is stored in cell material as described previously [47]. During the dark period, chemosynthetic sulfide oxidation was not possible below the maximum penetration depth of oxygen (i.e., 1.7 mm). It has been reported that under those conditions, this species may catabolize glycogen to acetate which is coupled to the reduction of intracellular sulfur to sulfide (1 glycosyl unit + 4 S + 2 acetate + 2 CO, + 4 H.S), likely representing a rather efficient dark energy generation mechanism [51]. However, sulfide production was not detectable from the steady state dark profiles.
The new BGC is a valuable tool with which to study the physiology and behaviour of benthic mi-
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croorganisms when cultivated in physicochemical gradients similar to those observed in their natural habitats. Potentially, a wide range of long-term solute gradients can be imposed and the granulometry of the filling can be changed. Thus, different types of benthic environments can be simulated with the BGC. Both axenic and defined mixed cultures can be grown. For example, we have recently obtained a biofilm composed of the purple sulfur bacterium T. roseopersicina strain EP 2204 and green sulfur bacterium Prosthecochloris sp. strain CE 2401, in which the former stratified on top of the latter. Other likely candidates for cultivation in this system include colourless sulfur bacteria and cyanobacteria with the final aim to reconstitute the microbial mat community in the laboratory.
Acknowledgements We are grateful to M. Guy Avenaud for his excellent technical assistance during the development of the BGC and to Dr. Jean-Marie Froidefond for help with light measurements. Special thanks are also due to Prof. Niels-Peter Revsbech (Aarhus University, Denmark), who stimulated our interest in gradient cultures. Financial support for the gradient cultures was provided from the county council of Aquitaine (Bordeaux). Olivier Pringault was supported by a PhD fellowship from the Ministry of Higher Education and Research (Paris).
References []I Pfennig, N. (1989) Ecology of phototrophic
purple and green sulfur bacteria. In: Autotrophic Bacteria (Schlegel. H.G. and Bowien. B., Eds.1. pp. 97-l 16. Science Tech Publishers, Madison, Wisconsin. 121Stal, L.J., Van Ciemerden. H. and Krumbein, WE. (19851 Structure and development of a benthic marine microbial mat. FEMS Microbial. Ecol. 31, 11 l-125. [31 Van Gemerden. H.. De Wit, R., Tughan. C.S. and Herbert. R.A. (1989) Development of mass blooms of Thiocapsa roseopersicina on sheltered beaches on the Orkney Islands. FEMS Microbial. Ecol. 62, 11 I- 118. 141 Van Gemerden, H., Tughan. C.S.. De Wit, R. and Herbert. R.A. (1989) Laminated microbial ecosystems on sheltered beaches in Scapa flow, Orkney Islands, FEMS Microbial. Ecol. 62. 87-102.
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