APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1983, p. 1007-10160099-2240/83/111007-10$02.00/0Copyright © 1983, American Society for Microbiology

Vol. 46, No. 5

Ecology and Physiology of the Competition for Light BetweenChlorobium limicola and Chlorobium phaeobacteroides in

Natural HabitatsEMILIO MONTESINOS,* RICARDO GUERRERO, CARLOS ABELLA, AND ISABEL ESTEVE

Department ofMicrobiology and Institute for Fundamental Biology, Autonomous University of Barcelona,Bellaterra (Barcelona), Spain

Received 7 February 1983/Accepted 1 July 1983

Accurate studies of the pigment composition and isolation in pure cultures ofChlorobiaceae from samples of eight Spanish lakes show that there are two maincoexisting groups of green and brown Chlorobium spp. represented respectivelyby Chlorobium limicola and Chlorobium phaeobacteroides. Laboratory experi-ments with pure and mixed cultures of the isolated strains show that light qualityplays a selective role on the species composition among Chlorobiaceae. Thisselection depends on the pigment composition which determines the in vivoabsorption spectrum of the cells as well as on their ability to adjust theintracellular concentration of light-harvesting pigments to the spectral distributionand energy of light. Correlation analysis performed with field data resulted insignificant, but low, correlation coefficients. Nevertheless, they were consistentwith laboratory data showing that brown Chlorobiaceae were dominant in deeplayers in meromictic lakes, whereas green Chlorobiaceae dominated in layersnearer the surface or underneath plates of Chromatiaceae. The combination oflaboratory and field observations stress the role of biological light filtering indetermining the species composition among Chlorobiaceae in lakes.

Purple and green phototrophic sulfur bacteriarequire light as energy source and suitable elec-tron donors, such as hydrogen sulfide. In naturalhabitats, both light energy and light qualitychange as a result of physical properties ofwater, chemicals in solution, suspended parti-cles, and biological filtering by phototrophicmicroorganisms (15).

Early works already proposed that light quali-ty might be an important factor in determiningthe species composition among phototrophicbacteria in lakes (22, 28). Depending on thespectral composition of light the organisms canbe selected according to their ability to capturelight of different wavelengths for photosyntheticpurposes. This selection depends on the actionspectrum of the cells, which is very close to itsin vivo absorption spectrum, and is determinedmainly by the pigment composition.

It has been demonstrated in the laboratorythat green or yellow light is a selective agent forenrichment of purple sulfur bacteria, whereasblue light favors the growth of green sulfurbacteria (14, 20). These experiments are inagreement with the fact that light absorbed bythe carotenoids is photosynthetically active andwill support growth, although the efficiency inenergy transfer to the reaction center lies be-tween 30 and 98% relative to the antenna bacte-

riochlorophylls (Bchls), depending on the spe-cies (8, 19).

In natural habitats it has been also shown thatlight quality determines the species compositionamong phototrophic bacteria. Parkin and Brock(20) compared the dominant species of phototro-phic bacteria among lakes which differ in theabsorption spectral properties of light due to thepresence of dissolved organic chemicals (humicand tannic acids). They concluded that red andgreen light acted as selective agents, explainingthe dominance of green or purple sulfur bacteriain some lakes.

Nevertheless, in many natural habitats wherephototrophic sulfur bacteria develop, light quali-ty is mostly affected by biological factors, suchas filtering by algae (especially in meromicticlakes) and by Chromatiaceae and Chlorobia-ceae, which distribute themselves differentiallywithin the water column.To date, little attention has been devoted to

the effect of biological light filtering. The pur-pose of the present study is to determine theeffect of light quality as a result of biologicallight filtering on the species composition ofChlorobiaceae. Our study shows that competi-tion for light results in the selection of greenChlorobiaceae when growing in the upper layersor underneath plates of Chromatiaceae, where-

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as brown Chlorobiaceae dominate in deeperlayers or in meromictic lakes below dense popu-lations of algae or cyanobacteria.

MATERIALS AND METHODSStudy areas. Field samples were taken from eight

Spanish lakes in which dense populations of photosyn-thetic bacteria develop. There is a wide variety ofenvironmental conditions among the lakes, thus per-mitting a comparative study. Lakes Vilar, Cis6, Ban-yoles (basih III), Nou, Coromines, and Negre arelocated in the Banyoles karstic area (42°08' N, 2045'E), near the town of Girona. Lakes Estanya (42010' N,0°20' W) and Moncortes (42020' N, 2040' W) arelocated in the provinces of Huesca and Barcelona,respectively.

Field measurements and sample collection. Beforesampling, vertical profiles of temperature, conductiv-ity, turbidity, and light energy were taken to locate thebacterial plates and select appropriate depths for sam-pling. Conductivity and temperature were measuredwith a combined meter YSI 33 (Yellow Springs Instru-ments). Turbidity was measured with a submersiblevertical light transmissometer made in the course ofthis work (13). Light energy between 400 and 700 nmwas determined with a radiometer Crump model 550(Crump Scientific Instruments). The spectral distribu-tion of light was recorded measuring vertical profilesof light penetration by interposing over the sensordifferent Kodak Wratten color filters (numbers 25, 50,73, 74, and 75). The results are expressed as percentlight relative to surface levels, taking as representativethe wavelength that corresponds to the maximumtransmission of each filter (30). Samples were collect-ed with a 1.6-liter Ruttner bottle or by means of aperistaltic pump (WAB LPA-2; W. A. Bochofen,Basel) depending on the degree of water stratification.Chemical assays. H2S was measured by the methyl-

ene blue method (9).Total numbers. Total bacterial numbers were quanti-

fied by direct microscopy by the acridine orangeepifluorescence method (31).

In vivo absorption spectrum. In vivo absorptionspectra of samples and pure cultures were done by twodifferent methods. Cells were harvested at 5,000 x gfor 15 min, and the pellet was placed between twomicroscope cover slips. When there was interferencedue to abundant detritus or mineral particles, cellswere washed twice in Tris-hydrochloride buffer (25mM, pH 7.0), broken by sonic treatment, and centri-fuged at 20,000 x g for 30 min; then the clear superna-tant containing the chromatophores was used for theabsorption analysis. The absorption spectra of thepellets and supematants were recorded between 350and 850 nm in a Pye Unicam SP-1700 spectrophotome-ter.

Laboratory cultures and growth conditions. Photo-trophic bacteria were isolated as individual colonieswithout previous enrichment by means of the agarshake method. The composition and preparation of thebasal medium was as described previously (3); sodiumacetate (2.2 mM) was added to increase yields. Cul-tures were incubated at 25°C in a Refriterm chamber(Struers Co., Sweden) fitted with continuous illumina-tion provided by a bank of fluorescent lamps. Differentlight intensities were obtained by inserting neutral

filters between the light sources and the cultures.Identification of the strains was made by the method ofPfennig (23). For selection experiments in the labora-tory, we devised a liquid chemical light filter whichmimics the combined absorption spectrum of Chroma-tium minus and water. The composition was as fol-lows: copper sulfate (5%), acid fuchsine (0.1%), andmethylene blue (0.01%). Chlorobium vibrioforme 8327was used for comparison purposes in pigment biosyn-thesis experiments.Pigment analysis. Generally, for field samples, vol-

umes of 200 to 500 ml were filtered through membranefilters (Sartorius; 47-mm filter diameter, 0.45-,um porediameter) previously covered with a thin layer ofMgCO3. Laboratory cultures were harvested at 5,000x g for 15 min. The MgCO3 layer containing cells, orthe pellet from centrifugation, was suspended in 5 mlof 90% acetone, and cells were disrupted by sonictreatment. Extraction of pigments continued for 24 hat 4°C. Afterward, the extracts were centrifuged at10,000 x g for 10 min, and the clear supernatant wasused for pigment analysis. Extracts were evaporatedby flushing N2 gas and saponified with 20% (wt/vol)KOH-methanol (5% final concentration). Pigmentswere transferred to diethyl ether after the addition of10% NaCl, and the pigitent containing the epiphasewas washed twice with distilled water. The etherextract was centrifuged at 4,000 x g for 10 min toremove the remaining water. Carotenoids were recov-ered by extraction. with petroleum ether (60 to 80°Cboiling range). The resulting diethyl ether extractcontaining Bchls. was bubbled with N2 gas over anhy-drous Na2SO4. The concentrated extracts were useddirectly for thin-layer chromatography analysis ortransferred to 90% acetone (in the case of Bchl) or topetroleum ether (in the case of carotenoids), and theabsorption spectra were recorded between 350 and 850nm. Thin-layer chromatographic separation was car-ried out on plates coated with Silica Gel (Merck) (20 by20 cm) activated for 30 min at 120°C. Separation wasperformed using a solvent mixture of benzene-petro-leum ether-acetone (10:3:2, vol/vol). The colored spotswere scraped off, the pigments were dissolved with theappropriate solvent, and then the usual spectrophoto-metric assays were made from 350 to 850 nm. Carot-enoids in petroleum ether solution were quantified byusing an extinction coefficient E"j. of 3,000 for themiddle central absorption peak (16, 17, 25). Bchls inacetone solution were calculated from the absorptionat 664 nm (Bchl c) or 654 nm (Bchl d and e) by usingspecific absorption coefficients of 92.6 and 98.0 li-ters * g-1 * cm-', respectively (26). The relative pro-portion of Bchl c plus Bchl d and Bchl e in fieldsamples and mixed cultures of Chlorobiaceae wascalculated directly from the acetone-purified extractcontaining Bchls by using the ratio between absor-bances at 428 nm (Bchl c plus Bchl d) and 468 nm (Bchle) in the Soret band. Absorbances at 428 and 468 nmwere corrected for the contribution of the absorbanceof Bchl a at these wavelengths. The resulting ratio wascalled R428 and was used as a measure of the relativeproportion between green and brown Chlorobium spp.

RESULTSStudy areas. Figure 1 illustrates the typical

physicochemical characteristics of temperature,

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COMPETITION FOR LIGHT AMONG CHLOROBIACEAE 1009

light, sulfide, and turbidity among the lakesduring the maximum development of plates ofphototrophic bacteria. The location of phototro-phic bacteria can be easily detected by a sharpdecrease in water transmissivity (increase inturbidity) at depths in which sulfide appears,generally also in coincidence with temperaturegradients (thermocline in holomictic stratifiedlakes or chemocline in meromictic lakes). Thebasin III of Lake Banyoles (Banyoles III) andLake Vilar (Fig. 1D and F) belong to the mero-mictic crenogenic type (12, 13). Undergroundwater rich in sulfate (10 to 17 mM S042-) entersthrough the bottom. Sulfide is produced in theanaerobic sediment mostly by sulfate-reducingbacteria and diffuses to the monimolimnion,which starts at the 5- to 7-m depth in Lake Vilarand at the 15- to 20-m depth in Banyoles III,depending on the time of the year. Lake Ban-yoles is oligotrophic, whereas Vilar is eutrophic,both developing algal blooms in the mixolimnionduring the summer. Lake Moncortes is alsocrenogenic meromictic, with the sulfide-contain-ing monimolimnion starting at the 15- to 20-m

0

EII.-0.w

. 0

10 20

0.5 1

40 80

TEMPERATURE (°C)

SULFIDE (mM)

LIGHT (CA. of surface)TURBIDITY (*. transmission)

FIG. 1. Physicochemical characteristics of LakeCis6 on 28 August 1980 (A), Lake Nou on 16 July 1979(B), Lake Negre on 24 August 1979 (C), Lake Ban-yoles III on 15 October 1978 (D), Lake Estanya on 27July 1979 (E), and Lake Vilar on 16 July 1979 (F).Symbols: turbidity (0), light energy (x), temperature(0), and sulfide (A).

depth (data not shown). Lakes Ciso and Nou(Fig. 1A and B, respectively) are also fed byunderground sulfated water, but they becamestratified during summer. Thermal and chemicalgradients were located at 0.5 to 1.5 m in LakeCiso and at 3 to 4 m in Lake Nou. Every yearthere is a fall overturn period in Lake Ciso, butanaerobic conditions are still maintained in thewhole water column. Lake Nou is also mixedduring winter, but only until 4 m, remaininganaerobic below that depth. Lakes Estanya,Negre (Fig. 1E and C, respectively) and Coro-mines'(data not shown) are holomictic aerobic,but develop an anaerobic sulfide-containing hy-polimnion in late summer and autumn, duringthe algal bloom decline.

Species composition. To detect the presence ofpurple and green phototrophic bacteria in thelakes, the in vivo absorption spectra of sampleswere recorded (Fig. 2). In Lakes Cis6, Estanya,and Nou, coexisting populations of Chromatia-ceae and Chlorobiaceae could be detected bymeans of the in vivo absorption spectra of thesamples, but the Chromatiaceae were dominant.The spectra could be divided into two zones.The zone between 350 and 600 nm showedclearly Bchl a (370 to 372 nm) and okenone (515to 520 nm). In the zone between 650 and 900 nmtwo peaks could be detected, corresponding tothe mixed absorption of Bchl c, d, and e (725 to730 nm) and Bchl a (830 to 832 nm).

In Lakes Vilar and Banyoles III, clearly domi-nant populations of brown Chlorobiaceae couldbe observed from the main peaks at 460 to 462,520 to 521, and 715 to 716 nm (Bchl e).

[ E F-|IF \N400 600 800 W00 600 800

WAVELENGTH (nm)FIG. 2. In vivo absorption spectra of samples of

photosynthetic bacteria from Lakes Vilar (A), Cis6(B), Banyoles III (C), Nou (D), Negre (E), and Es-tanya (F). The numbers over the peaks are the wave-lengths at which the maximum absorption was ob-served.

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z *.X*,,2 Vin

430 510 590WAVELENGTH (nm)

FIG. 3. Absorption spectra of purified acetonic ex-tracts of bacteriochlorophylls from samples of LakeCis6 during 1978. 11 February ( ), 22 May (...and 12 August (-- - ).

Lakes Negre and Coromines showed mixedpopulations of green algae (chlorophyll a, 668nm) and green Chlorobiaceae containing Bchl c

or d (420 to 440 nm and 724 nm).Isolation of dominant Chlorobiaceae. The

above-mentioned results were confirmed by theisolation in pure cultures of Chlorobium phaeo-bacteroides UA5001, Chlorobium limicolaUA5002, and C. limicola UA5003. The pigmentcomposition of each strain was analyzed by thin-layer chromatography. Strain UA5001 containedisorenieratene (maximum absorption in petro-leum ether at 426, 452, 484 nm) and Bchl e

(maximum absorption in acetone 438, 462, 604,649 nm). Strain UA5002 had chlorobactene (436,461, 489 nm) and Bchl c (418, 436, 630, 663 nm).Strain UA5003 contained chlorobactene andBchl d (408, 427, 578, 613, 654 nm). At least agreen and a brown Chlorobium could be isolatedfrom all the studied lakes. Thin-layer chroma-tography of the pigments extracted from thefield samples confirmed these results. In sam-

ples of lake Vilar from 16 July 1979, Bchl e

(maximum absorption in acetone at 462 nm)accounted for 86% and Bchl c and Bchl d(maximum absorption at 428 to 436 nm) account-ed for 14% of total Bchls.

Analysis of the ratio R428. The proportionbetween green and brown Chlorobium spp. wasmeasured in samples by following the ratio be-

tween absorbances, of Bchl c plus Bchl d (428 to436 nm), and Bchl e (468 nm) in purified acetonicextracts. To avoid interferences, the carotenoidswere removed previously with petroleum ether(see above). The 4628 ratio was used becausebrown and green Chlorobium spp. cannot bedistinguished on the basis of microscopic analy-sis. No attempt was made to evaluate viablecounting of brown or green colonies in syntheticmedia because it might select one of the groups,and it is not accurate to perform by the agarshake method due to the high dilution factorsnecessary to obtain a few colonies in each tube.Figure 3 shows variations in the absorptionspectra in the 350- to 630-nm band in samplesfrom Lake Cis6 during 1978. Coexisting brownand green Chlorobium spp. can be detected.A correlation analysis was performed to study

the main factors that control the above-men-tioned ratio (Tables 1 and 2); a total of 483samples were analyzed. Since previously we haddescribed competition for light and sulfide be-tween Chromatium minus and Chlorobium spp.in Lake Ciso (1), we included as main factors inthe correlation analysis the average number ofChromatiaceae cells, the average depth at whichthe plate was located (this parameter is a mea-sure of the potential light intensity and qualityreaching the plate), and the average concentra-tion of sulfide. The results show that there is asignificant correlation between R168 and the fol-lowing two factors: (i) the depth at which theChlorobium spp. layer was located (r = -0.618)and (ii) the average concentration of Chromatia-ceae (r = +0.646). The other possible correla-tions among the parameters were not significant.

Although both sets of correlations are low,they are still significant. Generally speaking,correlation coefficients obtained in ecologicalstudies are often poor due to the fact that

TABLE 1. Ratio R468, sulfide concentration, depthof the plate, and concentration of Chromatiaceae in

eight Spanish lakesaChroma-

Lake No. of 428 H2S Depth tiaceaeLake samples R:68 (mM) (m) (cells/ml[x 105])

Coromines 25 3.34 0.5 3 3.46Estanya 4 2.79 0.7 13 4.71Negre 25 2.67 0.2 4 0.00Cis6 200 2.46 1.9 2 4.10Nou 25 2.22 0.9 4 2.28Moncortes 4 1.28 0.3 19 0.00Vilar 150 0.65 3.8 6 1.20Banyoles 50 0.19 0.7 20 0.03

a Average values for the water column occupied bythe phototrophic bacteria were used for calculations.

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COMPETITION FOR LIGHT AMONG CHLOROBIACEAE 1011

TABLE 2. Correlation analysis between the ratioRU', sulfide concentration, depth of the plate, andconcentration of Chromatiaceae in eight Spanish

lakesa

Variable Rj H2S Depth Chroma-

R4468 1 -0.393b -0.618c +0.646cH2S 1 0.298b +0.115bDepth 1 -0.430dChromatiaceae 1

a Data are from Table 1.b Not significant.c 0.05 > P > 0.025.d 0.1 > P > 0.050.

ecosystems are more complicated than simplelaboratory experiments and many variables thatare interacting cannot be sufficiently controlledin the statistical analysis. For this reason, weincluded in the study a substantial part of labora-tory experiments to confirm the hypothesis de-duced from field data. One possible explanationfor the low values may be the fact that the R8ratio is affected to some extent by changes in thespecific pigment content of cells and by changesin other environmental factors. On the otherhand, although light quality and intensity wererelated with depth, both parameters were alsodependent on other physicochemical and biolog-ical factors. The reason for using depth as arepresentative parameter was that we had notenough measurements of the actual intensity andquality of light.

Light shading bgY Chromatium sp. Seasonalchanges of the R S in Lake Cis6 during 1978 inrelation to the Chromatium sp. and Chlorobiumspp. numbers are shown in Fig. 4. It is clear thatthe dynamics of both bacterial populations werecomplementary. When Chromatium sp. startedto grow, Chlorobium spp. started to decrease,and when the Chromatium sp. bloom declined

A 02 iL0 A L

e0 \\

0 1^ ._____.-.

(after August), Chlorobium spp. increasedagain. Furthermore, as soon as the number ofChlorobium spp. decreased due to the lightshading effect of the plate of Chromatium sp.located near the surface, selection of the greenChlorobium spp. was apparent from the increasein the RIM. The onset of this increase wascoincident with the middle of the Chromatiumsp. growth phase in June and July. Under thissituation, light energy and quality available forChlorobium spp. were controlled by Chroma-tium sp., which acted as a biological light filter.Figure 5 shows the changes in the spectraldistribution of light into the plate of Chromatiumsp. and the possible selective effect on thepopulation of Chlorobium spp. The combinedabsorption of both the lake water that absorbsstrongly in the infrared and UV and that ofChromatium sp. with three main absorptionpeaks at 370, 510 to 530, and 830 nm resulted intwo main transmission peaks at 450 and 650 nm.Light transmitted at 650 nm cannot be absorbedby Chlorobium spp., but radiation at 450 nmcoincides with the maximum absorption of C.limicola at 445 nm, whereas C. phaeobacte-roides is strongly shadowed in the region be-tween 500 and 550 nm.To confirm whether this spectral difference

would result in the positive selection of C.limicola in mixed cultures together with C.phaeobacteroides, laboratory experiments weredone with a chemical light filter that reproducesthe combined transmission spectrum of Chro-matium sp. cells and the water column (Fig. 6).The results were compared with a control ex-periment without a filter. The specific growthrate in both mixed cultures (control and filterexperiment) was adjusted at about 0.013 h-1 byattenuating adequately the incident light in thecontrol as determined in previous experiments.The specific growth rate on the basis of theabsorbancy at 428 nm (Bchl c and Bchl d) and at468 nm (Bchl e) of the acetonic extracts was also

FIG. 4. Dynamics of Chromatium sp. (0), Chlorobium spp. (@), and R428 (x) during 1978 in Lake Cis6. Totalnumbers were integrated throughout the water column.

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z

0

-:<0

A

B .7

<0

.Cf

350 550 750WAVELENGTH (nm)

FIG. 5. Shading and light filtering effect of Chro-matium sp. on the population of Chlorobium spp. on28 August 1980. (A) In vivo absorption spectrum of theChromatium sp. layer. (B) Spectral distribution oflight into the plate. (C) Absorption spectra of Chloro-bium phaeobacteroides (-) and Chlorobium limi-cola (..) cultures isolated from below the plate ofChromatium sp.

determined. The results for the control experi-ment were, respectively, 0.014 and 0.011 h-1,whereas for the experiment with the filter theresults were 0.018 and 0.006 h-1, respectively.Thus, the growth rate based on absorbancy at428 nm increased when cultures were incubatedwith the filter, whereas the growth rate based onabsorbancy at 468 nm decreased, and after 120 hof incubation the R4628 changed from 1 to 2.Based on the fact that under the experimentaldesign the green C. limicola received more lightthan did the brown, one might expect an in-crease in the specific content of Bchl e in thebrown C. phaeobacteroides. As a consequence,the observed changes in the R288 ratio wereunderestimations of the actual changes in theirrelative numbers, pointing to a selection of greenChlorobium against brown.Light shading by algae and effect of depth.

From the analysis of the lakes it seemed obviousthat brown Chlorobium spp. were dominant indeep layers, especially in meromictic lakes de-veloping a population of algae in the mixolim-

nion, such as Lakes Vilar and Banyoles III.Light quality and light energy in these lakes arecontrolled by the abundance of algae and thethickness of the water column absorbing lightand modifying its spectral distribution. Figure 7shows the spectral distribution of light at differ-ent depths in Lakes Vilar and Banyoles III.Banyoles III is a typical oligotrophic lake and, asin other similar lakes, maximum light transmis-sion lies between 450 and 650 nm with a maxi-mum at 540 nm. In Lake Vilar, the spectraldistribution was similar, but a marked influenceof the algae living in the mixolimnion was evi-dent from the strong absorption between 400 and500 nm. When comparing the spectral distribu-tion of light at different depths in both lakes withthe absorption spectra of C. limicola and C.phaeobacteroides, it was apparent that the maxi-mum transmission at 540 nm approximately co-incided with the maximum absorption of thebrown Chlorobiaceae (maximum peaks at 470and 520 nm), but not with the green (maximumat 440-450 nm).Photopigment synthesis. Since Chlorobium

spp. populations are mainly light limited (qualityand quantity) at the depths where they arefound, we studied in the laboratory the synthesisof the light-harvesting pigments under differentlight intensities. Three Chlorobium strains were

4WJ~

WAVELENGTH (nm)

40 80 120TIME (hJ

FIG. 6. Selection experiment in mixed cultures ofChlorobium phaeobacteroides UA5001 and Chloro-bium limicola UA5002. (A) Absorption characteristicsof the chemical light filter (----) in relation to theabsorption spectra of cells from UA5001 ( ) andUA5002 ( ). (B) Variation of the ratio R 21 uponincubation.

B+FILTER

CONTROL

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p

'n 20

-

0

- 0

6

600 400WAVELENGTH (nm)

500 600

FIG. 7. Spectral distribution of light through the water column in Lakes Banyoles III (A) and Vilar (B). Thestippled area indicates the depth at which the plate was located.

used. Two of them were UA5001 and UA5002,and the third, C. vibrioforme 8327, was used forcomparison.

Figure 8 shows the effect of light intensity onthe growth and specific carotenoid and Bchlcontent in the studied bacteria. Growth wasdependent on the light intensity; at approximate-ly 1,500 lux, light saturation was observed. At 45lux, no significant growth was observed duringthe course of the experiment.An inverse relationship of both sets of pig-

ments with light intensities between 250 and9,000 lux was observed, indicating great adapt-ability. Nevertheless, a direct relationship exist-ed below 250 lux. Below these light intensitiesand with fhuorescent light, Chlorobium spp. didnot grow, and probably pigments were progres-sively degraded upon incubation, thus showing adecrease in the specific carotenoid and Bchlcontent. The green Chlorobium strains (C. limi-cola UA5002 and C. vibrioforme 8327) exhibiteda specific Bchl content 1.6 to 2.5 times higherthan did the brown species under the sameconditions, whereas maximum specific carot-enoid content was 1.5 to 3.8 times lower. As aresult, the carotenoid/Bchl ratio was about 4times higher in C. phaeobacteroides than in C.limicola, suggesting that carotenoids were moreabundant than Bchl in brown Chlorobium. Fur-thermore the carotenoid/Bchl ratio increased atlower and higher light intensities than that pro-ducing the maximum specific pigment content.

DISCUSSIONIt is a general observation that Chromatiaceae

and Chlorobiaceae, when living together in alake, frequently distribute at different levelswithin the vertical profile (4, 10). Laboratoryevidence explaining this observation is nowavailable. Under saturating light intensities (with

incandescent lamps), generation times of Chro-matium and Chlorobium are quite similar, butbelow 15 to 45 lux the former is unable to grow,whereas the latter can grow (3).The fact that Chromatium needs more light to

1200A

B

W ~~~~~~~~~~D

o o.oI

0

2000 4000 6000 8000

LIGHT INTENSITY ( lux)

FIG. 8. Effect of light intensity on the specific

content of Bchls (A) and carotenoids (B), caroten-

oidlBchl ratio (C), and specific growth rate (D) in

Chiorobium limicola UA5002 (0), Chlorobium vibrio-forme 8327 (x), and Chiorobium phaeobacteroides

UA5001 (0).

B

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grow than Chlorobium could be explained on thefollowing basis: (i) the higher light-trapping effi-ciency in Chlorobium due to both higher specificpigment content and the arrangement of thechlorosomes in the periphery of the cytoplasm(5, 6) and (ii) the significantly lower maintenancerate constant exhibited by Chlorobium (8 timeslower than in Chromatium) (29).The typical vertical distribution in which

Chromatium occupies the uppermost layer,where there is more light energy, and the non-motile Chlorobium remain underneath can beexplained taking into account that (i) light isattenuated as it passes across the water column,(ii) there are different light energy requirementsamong Chromatium and Chlorobium, and (iii)Chromatium organisms are able to find the opti-mal conditions by active movement. In thissituation light energy and light quality availablefor Chlorobium became primarily regulated byChromatium. Evidence supporting this hypothe-sis is based on the fact that biomass of Chloro-bium spp. was inversely correlated with thequantity of pigments of Chromatium sp. (oken-one and Bchl a) during 1978 in Lake Ciso (21a).Under such conditions the spectral distribu-

tion of light transmitted by Chromatium sp.favors the development of green Chlorobiumspp. The laboratory experiments with mixedcultures of brown and green Chlorobium andfilters that combined the absorption propertiesof Chromatium sp. and the water column sup-ported this hypothesis and also the correlationanalysis. Among the Chlorobiaceae, only Bchlsthat absorb in vivo at about 440 to 450 nm areable to trap light transmitted by the layer ofChromatium sp. with a maximum at 450 nm.Therefore, having a strong absorption in thisband, as is the case of green Chlorobium, due toa high specific Bchl content results in a selectiveadvantage against brown Chlorobium underthese specific conditions.

Pfennig (22) suggested that brown Chlorobia-ceae could have a selective advantage over theirgreen counterparts in deep layers of lakes.Truper and Genovese (28) also hypothesizedthat carotenoids of brown Chlorobium couldplay an important role in this advantage ofbrown Chlorobium for growing deeper in lakes.Nevertheless, the experiments of Matheron andBaulaigue with mixed cultures of Chlorobiaceaeincubated in situ at different depths in marinecoastal waters (18) did not support that hypothe-sis. Probably the spectral differences at themaximum incubation depth (30 m) in marinewaters were not sharp enough to create a selec-tive advantage of brown Chlorobiaceae over thegreen C. vibrioforme.Our results are in favor of the above-men-

tioned hypothesis. The literature contains rele-

vant data that also support our observations.Table 3 lists a series of 30 freshwater lakes fromaround the world in which dominant species anddepth of the plate have been indicated by differ-ent authors. Only lakes in which species compo-sition of Chlorobiaceae were provided fromabsorption spectra or isolation in cultures wereincluded. By adding our results and arrangingsuch data in relation to depth at which the platewas located, it can be seen how in lakes thatexhibit plates near the surface (2 to 4 m) greenChlorobiaceae were dominant; lakes between 4and 9 m showed high variability, whereas indeeper layers (9 to 25 m) brown Chlorobiaceaewere dominant.Maximum light transmission in many of the

included lakes is between 450 and 650 nm,

TABLE 3. Relation between dominant species ofChlorobiaceae and the depth in which the plate is

located in 30 freshwater lakes

Cis6SolaiWakPomrCorcMar!RoseNouNegiKisaHarnVilarPlus,PaulSuig(VechSugaVallcMogiMirrKon4EstaiFishFaroCram

Lake Depth(m)

2,r 2;u-ike 2.5yaretskoe 3mines 3y 3.75-1 4

4re 4Lratsu 5itori 5r 6see 6

6.5etsu 8hten 81 9e San Juan 10ilnoye 10or 11onier 11.5nya 13

1313.5

vford 15

KinneretFayettevilleMoncortesBanyoles IIHiruga

Dominantspecies'

Clm, CpbPtcClmCvbClmClmClmClmClmClm, CpbClmCpbAncPtcCpb, ClmCpbCpb, ClmPelCpbCpbPelClm, CpbCpbCpbCpb

18 Cpb18 Cpb19 Cpb, Clm20 Cpb25 Cpb

Source orreference

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2027212724112010

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a Green species: Clm, Chlorobium limicola; Anc,Anchalochloris sp.; Ptc, Prostecochloris sp.; Cvb,Chlorobium vibrioforme. Brown species: Cpb, Chloro-bium phaeobacteroides; Pel, Pelodyction sp.

b H. Biebl, Ph.D. thesis. University of Freiburg,1973.

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COMPETITION FOR LIGHT AMONG CHLOROBIACEAE 1015

showing a maximum peak about 540 nm. Theshape of these spectral distributions is mostlyaffected by the combined absorption by algae orcyanobacteria living in the upper layers and alsoby the optical properties of water. The spectraldistribution of light above the plates in meromic-tic lakes is generally quite similar to the absorp-tion spectrum of brown Chlorobiaceae and re-sults in a selective advantage for itsdevelopment. On the other hand, the high simi-larity between in vivo absorption spectra ofgreen algae and cyanobacteria with those ofgreen Chlorobiaceae prevents the latter fromgrowing actively under dense populations ofthese chlorophyll a-containing organisms due tothe strong shading and light filtering.The advantage of brown Chlorobiaceae is

based on their absorption spectral properties,strongly influenced by the high specific contentof carotenoids, which permit a better utilizationof the light spectrum available in deeper layersin lakes. The response to light intensity in termsof synthesis of light-harvesting pigments indi-cate that C. phaeobacteroides has a caroten-oid/Bchl ratio about 4 times higher than that ofC. limicola, and the specific carotenoid contentis 1.5 to 3.8 times higher. This fact was found notonly in the laboratory, but also in natural popu-lations. During the summer, Lakes Cis6, Vilar,and Banyoles III exhibited maximum caroten-oid/Bchl ratios of 0.77, 1.48, and 1.98, respec-tively, clearly increasing with depth at which theplate was located (E. Montesinos, Ph.D. thesis,Universidad Aut6noma Barcelona, 1982). Thissuggests an increasing light stress, both in quan-tity and quality, depending on the thickness ofthe algal layer over the bacterial plate. One caninterpret these field results in three differentways: (i) the increase in the proportion of carot-enoid-rich brown Chlorobiaceae; (ii) the in-crease in carotenoidlBchl ratio at light intensi-ties below that which produces the maximumspecific pigment content; and (iii) the possibilitythat, depending on the light quality, a differentialbiosynthesis of the light-harvesting pigments(carotenoids and Bchls) exists. Thus, regardlessof which of the three factors is more important,it is clear that carotenoid-rich Chlorobiaceaedominate in deep layers.By combining field data from several lakes

together with laboratory experiments we areable to show that light quality plays an essentialrole as a selective factor controlling the speciescomposition among Chlorobiaceae in naturalhabitats. The most streaking feature of our ob-servations is that, although light quality is affect-ed by the chemical and physical properties ofwater, the main effect is due to phototrophicpopulations themselves (both algae and bacteria)as a result of biological light filtering.

ACKNOWLEDGMENTS

We thank Hans Van Gemerden and Carlos Pedr6s-Ali6 forhelpful discussions during the preparation of the manuscript.

E. M. was the recipient of a doctoral scholarship from theSpanish Ministry of Education and Science.

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