APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2142-21500099-2240/87/092142-09$02.00/0Copyright © 1987, American Society for Microbiology

Diel Vertical Movements of the Cyanobacterium Oscillatoriaterebriformis in a Sulfide-Rich Hot Spring Microbial Matt

LAURIE L. RICHARDSONt* AND RICHARD W. CASTENHOLZ

Department of Biology, University of Oregon, Eugene, Oregon 97403

Received 13 February 1987/Accepted 16 June 1987

Oscillatoria terebriformis, a thermophilic cyanobacterium, carried out a diel vertical movement pattern inHunter's Hot Springs, Oreg. Throughout most daylight hours, populations of 0. terebriformis covered thesurface of microbial mats in the hot spring outflows below an upper temperature limit of 54°C. Upon darknesstrichomes moved downward by gliding motility into the substrate to a depth of 0.5 to 1.0 mm, where thepopulation remained until dawn. At dawn the population rapidly returned to the top of the mats. Field studieswith microelectrodes showed that the dense population of 0. terebriformis moved each night across an

oxygen-sulfide interface, entering a microenvironment which was anaerobic and reducing, a dramatic contrastto the daytime environment at the mat surface where oxygenic photosynthesis resulted in supersaturated 02.

Laboratory experiments on motility with the use of sulfide gradients produced in agar revealed a negativeresponse to sulfide at concentrations similar to those found in the natural mats. The motility response may helpexplain the presence of 0. terebriformis below the mat surface at night. The movement back to the surface atdawn appears to be due to a combination of phototaxis, photokinesis, and the onset of oxygenic photosynthesiswhich consumes sulfide.

Oscillatoria terebriformis Agardh ex Gomont (Fig. 1)forms large populations at temperatures below 54°C inseveral hot spring outflows in the northwestern UnitedStates (3). At sulfate-rich (>2.6 mM) Hunter's Hot Springs,2 miles (ca. 3.2 km) north of Lakeview, Oreg., the unusualvertical migration pattern of this species was studied. Duringdaylight hours, the population generally formed a dense,dark reddish-brown layer at the surface of a microbial matcommunity which contained as the other dominant mat-forming species the cyanobacterium Synechococcus lividusCopeland and the anoxygenic phototrophic bacteriumChloroflexus aurantiacus Pierson and Castenholz. Duringdarkness, nearly the entire uppermat population of 0.

terebriformis moved, by gliding motility, downward 0.5 to1.0 mm into the microbial mat, remaining within the matthroughout the night. This particular vertical movementpattern is apparently unique when compared with the pat-terns of other cyanobacteria and motile procaryotes found inmicrobial mats (Table 1).

Castenholz (2) has investigated the daytime behavior of 0.terebriformis in Hunter's Hot Springs. The populationspreads out over the mat surface under low to moderate lightintensities and either contracts to form dense clumps ormigrates down into the mat during periods of high lightintensity, such as midday in summer. By this behavior thepopulation apparently maintains an optimal light environ-ment for the support of uninhibited oxygenic photosynthe-sis. 0. terebriformis demonstrates photokinesis (a light-intensity-dependent gliding rate) and positive phototaxis (4).The stimulus and potential benefit of the nightly down-

ward movement into an organically rich anaerobic environ-ment are not obvious. Most cyanobacteria are obligatephototrophs (11, 25) and fail to grow heterotrophically in the

* Corresponding author.t Contribution number 303 from the Center for Great Lakes

Studies.t Present address: Center for Great Lakes Studies, University of

Wisconsin, Milwaukee, WI 53204.

laboratory in darkness. It has been suggested that themetabolism of these cyanobacteria in darkness is limited torespiration of endogenous carbon, involving glycogen deg-radation together with NADP+ reduction via the oxidativepentosephosphate pathway coupled to 02 reduction vianormal electron transport chains. Therefore, the presence ofpopulations of cyanobacteria in environments where little orinfrequent light penetrates has continually perplexedcyanobacteriologists (19). An understanding of the nighttimebehavior of 0. terebriformis may be important in clarifyingsome of these observations.The downward migration may involve a little-known form

of taxis for cyanobacteria. Whereas some benthic cyanobac-teria are phototactic or have photophobic responses, therehave been only a few reports of chemotaxis in cyanobacteria(7, 12, 16).

Steep vertical gradients of 02 and sulfide often exist inmicrobial mats that have high sulfate concentrations, as dothose associated with Hunter's Hot Springs. The downwardmovement of 0. terebriformis is toward a position wheresulfide, as well as anoxia, is present. This is of interest interms of physiology. Many species of cyanobacteria havebeen reported to "tolerate" sulfide, a normally toxic sub-stance (20). Certain species have shown increases in photo-synthetic '4CO2 fixation when they were exposed to lowamounts of sulfide in the light (28). The effects of sulfide oncyanobacteria in darkness, however, are unknown.

In this study we have defined the microenvironment of themicrobial mat at Hunter's Hot Springs, within which thevertical movements occur, with the use of macro- andmicroelectrodes and chemical techniques. The data obtainedhave served as the basis for laboratory work with pure

cultures isolated from the springs. This paper reports theresults of the environmental study, as well as some of thefindings on tactic behavior.

MATERIALS AND METHODSField measurements. Water temperature was measured

with a Yellow Springs (model 42SC) Tele-thermometer.

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______________ II1U\xVV II IFIG. 1. (A) 0. terebriformis in a thermal outflow of Hunter's Hot Springs, Lakeview, Oreg. S, Source (arrow indicates the direction of

flow); OT, 0. terebriformis; SYN, S. lividus; CHL, C. aurantiacus. (B) Bright field Nomarski interference photomicrograph of 0.terebriformis culture OH-80-Ot-D. Bar, 6 ,.m.

Measurements of pH were carried out with a Cole-ParmerDigi-Sense pH meter (model 5985-40). Eh was measured witha Radiometer PHM-29b pH/mV meter with a calomel (K401)and platinum (P101) electrode. For determination of solublesulfide, the Pachmayr colorimetric assay was used (1). Theassay was modified slightly for small-sample analysis. Sam-ples (0.5 ml) obtained by syringe were rapidly injected into 5ml of H20. One milliliter of a solution containing 2 g ofN,N-dimethyl-para-phenylenediamine sulfate in 20%(vol/vol) H2SO4, followed by 0.1 ml of 10% (wt/vol)FeNH4(SO4)2. 12H20 (in acidified aqueous solution), was

added. Samples were refrigerated in darkness, and 20 minbefore spectrophotometric analysis, 3.5 ml of distilled H20was added to each sample. Optical density was determinedat 668 nm against a reagent blank by using a Gilford (model240GH) spectrophotometer with a Beckman DUmonochromator. Measurements were calibrated againststandard solutions of Na2S 9H20. This method can detectH2S at a concentration as low as 10 p.M. Light measurementswere recorded with a Biospherical Instruments (modelQSL-100) averaging quantum meter by using a silicon diodefiltered to eliminate all but 400- to 700-nm light. A LambdaInstruments L1-185 light-measuring instrument with an

LI-200S pyranometer sensor was also used.Microelectrodes. Microprofiles were obtained in situ with

02, pH, and sulfide microelectrodes (22) with the assistance

of N. P. Revsbech and D. Ward. The 02 cathode was 2 to 10p.m in diameter at the tip. The 02 microelectode is notaffected much by sulfide after an initial sulfide conditioning,an important feature for these studies. The pH microelec-trode had a tip approximately 10 p.m in diameter; the sulfideelectrode tip was about 100 pLm. The sulfide probe sensesS2-, the concentration of which is dependent on pH. There-fore, calibration of the electrode is dependent on bothconcentration of total dissolved sulfide and pH, as well astemperature. The sulfide microelectrode was calibratedagainst chemical sulfide analyses of sulfide-containing wa-ters in the field. The three microelectrode tips were gluedtogether to allow simultaneous measurements. The com-bined electrodes had a total diameter of less than 500 p.m,and insertion into the soft mat was controlled with amicromanipulator. Vertical profiles were obtained by takingmeasurements at increasing depths at one point in the mat inincrements of 250 p.m. The response time (90%) ranged fromless than 0.2 s (02 microelectrode) to 2 min (sulfide micro-electrode).

Artificial hot springs. Controlled experiments on verticalmigration were carried out in the laboratory. Intact matmaterial and sediment were transported from Hunter's HotSprings and suspended on elevated stainless steel wirescreens in noncirculating heated (45°C) water baths. Matswere placed on a bed of sediment, with the mat surface 1 cm

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TABLE 1. Position of motile microorganisms in mats

Position in microbial mat in:Mat type and organism (reference) Darkness Low light High light

A

Id4

water

Q terebriformls

mat/sediment

sunrise noon sunset

FIG. 2. Vertical migration of 0. terebriformis. (A) Diagram of24-h migration cycle. The population is represented by the heavyblack line and is shown in relation to the surface of the microbialmat. Migration is more rapid upward than downward. (B) Daytimeposition of 0. terebriformis on the mat surface. This photograph was

taken at 09:25. Under the relatively low light conditions, thepopulation was spread out over the mat surface. (C) Surface of thesame microbial mat before sunrise at 07:00. Little 0. terebriformis isvisible on the surface of the mat. The white cover is Beggiatoa sp.

Water temperature for both panels B and C was ca. 40°C.

NonthermalaOscillatoria margaritifera(M) (Castenholz,unpublished)

Microcoleus chthonoplastes(M) (29)

Microcoleus lyngbyaceus(FW) (21)

Chloroherpeton thalassium(M) (Castenholz,unpublished)

Oscillatoria sp. (M) (14)Beggiatoa sp. (M) (14)Chromatium sp. (M) (14)

Hot springOscillatoria terebriformis

(this publication)Beggiatoa sp. (18)Chromatium sp.

Up Down

Up Down

Up Down

Up Up(if anoxic) (if anoxic)

..Up- b

UpUp

(in part)

Down

UpDownDown

Down

DwDownDown

Up Down

Up Down DownUp Down Down

(Castenholz, unpublished)Oscillatoria princeps Up

(Castenholz, unpublished)Chloroflexus aurantiacus Up

(10) (in part)

Up Up

Down Down

a M, Marine; FW, freshwater.bAppears down because Beggiatoa sp. moves up and covers the Oscil-

latoria sp.'-, No data given for high-light conditions.

below the air-water interface. Spring water, collected fromHunter's Hot Springs, was supplied to the baths by anelevated container which allowed a controllable inflow. Theinflow was countered by evaporation and by a small-scaleoverflow. Increases in salinity due to evaporation of waterwere negligible because of the short time periods involved(less than 3 weeks). Light was supplied by cool whitefluorescent lights at an irradiance of 60 to 75 W/m2. Varyinglight-dark cycles were instigated, with the entire systemsurrounded by black cloth to minimize extraneous light.

Laboratory studies on vertical migration. Instead of moni-toring the behavior of individual trichomes, laboratory stud-ies involved the behavior of 0. terebriformis populationsexposed to gradients of test substances. Soft (0.7%) agarwith D medium (6) buffered with EPPS (N-[2-hydroxyethyl]-piperazine-N'-3-propanesulfonic acid; 1.2 g/liter at pH 8.0)was used as a support. 0. terebriformis can glide through, aswell as on top of, 0.7% agar. Test substances were added tounsolidified agar at 45°C, and 4 ml was poured into 8.5-mlvials, forming a test plug after solidification. An inoculumwas mixed into additional soft agar to form a uniformsuspension of trichomes; 4 ml of this suspension was thenpoured over the plug and allowed to solidify, forming anoverlay. The vials were incubated at 45°C in the light or darkand visually inspected for vertical movement in relation tothe gradient of the test substance.

RESULTS

Diel vertical migration. Figure 2 shows the pattern ofvertical movements of 0. terebriformis and the surface ofthe mat during the day and the night. The undermat isvariable in nature but is generally composed of a gel (perhaps

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FIG. 3. Vertical migration of 0. terebriformis in a laboratory mat. Irradiance was about 60 W/m2 at the water surface (water depth was0.5 cm). The water temperature was maintained at 45°C. Shown is the mat after a 10-h period of darkness (A), after 15 min of light (B), after40 min of light (C), and after 1 h of light (D).

derived from the undermat C. aurantiacus), a well-defined

HOURS layer of the unicellular S. lividus mixed with C. aurantiacus,or soft silty sediment. Sections of mat were cut out during

is 21 24 3 6 9 12 the night and preserved in warm agar (1.5%) with 3%Formalin. The sections were allowed to solidify, and crosssections were made which were examined with a dissecting

+170 | | | | / microscope. It could be seen that during the night 0.terebriformis moved downward to form a fairly distinct layer

+ go] ~ | ||0te.5 to >1.0 mm below the mat surface (not shown).90| | | | | | | \ /The vertical pattern is apparently unrelated to any endog-

Eh | X | | | | E | \ / enous rhythm. Laboratory experiments with artificial hotmv o i 8 ! | § a | Ysprings inoculated with native, intact mat demonstrated that

for 3 days under relatively low light intensity (ca. 60 WIm2),-90 _ g f . 0. terebriformis remained on the surface of the mat. Simi-

e mentlarly, with continuous darkness the population maintained a

- 1 70 g _ysedi sent position within the mat. The overriding controlling factor inthe vertical migration pattern was apparently the presence or

50] __ absence of light. When subjected to an artificial light-dark°0 1 -- cycle, the population migrated downward into the mat40 ater during darkness; when present within the mat, the popula-40

_ l tion would move upward at any time with the stimulus of

8- __ ] light at an intensity of 60 W/m2 (Fig. 3). An ongoing,water complete migration cycle of 3 h (1 h of light, upward

pH migration; 2 h of dark, downward migration) was maintained7 _ _ _ | for 2 weeks, after which the experiment was halted. In the

.14 field during the night, the population of 0. terebriformis also

ui .12ax mM .10 -1 .08 FIG. 4. Daily (24-h) cycle of Eh, pH, temperature, and sulfide in

sn 06] |- sed > | the microbial mat. Sediment from a depth of 2 to 3 mm in the matU') .06 was analyzed. Water immediately overlying the mat was sampled.

.04 Redox potential values were corrected for both pH and 02

.02 concentration.

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pH 6 7 8 9 6 7 8 9H2S

FIG. 5. Microprofiles of 02, pH, and sulfide just before, during, and after sunset in May. The dark arrows represent the vertical positionof 0. terebriformis in the mat, whereas the open arrows indicate the stationary layer of S. lividus. Water temperatures varied between 41.5and 42°C. Sundown occurred at 19:53. Shown are microprofiles at 19:10 (A) (irradiance was less than 50 W/m2), 19:25 (B) (25 W/m2), 19:50(C) (20 W/m2; at this time the population was moving down into the mat), 20:20 (D) (in full darkness), and 21:45 (E) (with most of thepopulation within the mat). These microprofiles depict the exact location of the oxygen-sulfide interface in the microbial mat. The absolutevalues of the sulfide concentrations may be slightly shifted because of a lack of calibration against sulfide standards in the field (calibrationwas carried out against sulfide concentrations in water samples obtained at the same time). The shapes of the profiles are accurate. In thisfigure H2S refers to total sulfide (H2S, HS-, and S2-). Organism abbreviations are defined in the legend to Fig. 1.

migrated from within the mat to the mat surface after a brightbeam of light was directed to the mat for 15 to 30 min with ahigh-power flashlight.

Eh, pH, and sulfide 24-h cycles. Figure 4 illustrates datafrom a single experiment in which Eh, temperature, pH, andsulfide were measured every 3 h over a 24-h period (9 and 10May) with conventional (macro) electrodes and water chem-istry techniques. Additional experiments showed a similarpattern (data not shown). Temperature at the point studiedvaried over a 10°C range, from 43 to 53°C. Although thesource remained constant at 79.5°C, the cooler night aircaused the fluctuation in temperature downstream (water atthe experimental site was 0.75 to 1.0 cm deep, with avelocity of ca. 5 cm/s). The pH varied from 7.2 to 8.0 in thesurface waters and from 7.1 to 7.6 in the sediments at a depthof approximately 2 mm. This fluctuation can be attributed tophotosynthetic activity during the light hours and follows thepredicted pattern of variation; i.e., highest pH values oc-curred during midday when photosynthetic activity wasprobably maximal. The widest variations were observed inEh values and sulfide concentrations. Eh was measured bothin the surface waters above the mat and in the sediment at adepth of 2 mm. Eh in the sediment varied by 135 mV, from-40 to -175 mV. There was a steady decline in sedimentredox values for the first 6 h of darkness. This can beattributed to the accumulation of biogenic sulfide (17). Dur-ing darkness, 02 is presumably consumed by microorga-nisms, including C. aurantiacus and many other species offacultatively aerobic bacteria, and consumed abiotically viachemical reduction by sulfide. During darkness 02 is presentin the continuously flowing air-exposed surface waters (thesaturating 02 concentration is approximately 0.1 to 0.2 mMat 50 and 43°C, respectively, for an elevation of 1,472 m).

After dawn the redox potential of the sediments remained

fairly constant at approximately -80 mV for 6 to 8 h andthen gradually rose to -45 mV as 02 produced by thephotosynthetically active surface layers diffused downward.Sediment Eh values continued to rise until darkness. In thewater immediately overlying the surface of the mat, Ehvalues followed a different pattern. Here values ranged from-10 to +210 mV, a difference of 220 mV. The variation wascharacterized by a very sharp drop immediately after sun-rise, followed by a rapid steady increase throughout lateafternoon and the first half of the dark period. The extremelyrapid decline in redox values after dawn may have been dueto physical perturbation of the mat by the upward migrationof 0. terebriformis. Measurements revealed bursts of sulfidein the water immediately overlying the mat during the dawnperiod accompanying the decrease in Eh values (data notshown).

Sulfide, 02, and pH microprofiles. While the above dataillustrate the general cyclical fluctuations in the environmentof 0. terebriformis, they do not reveal the precise micro-environment within the mat during darkness. The populationmigrates downward to a position 0.5 to 1.0 mm below themat surface, but with the assays used, it was impossible todetermine the microenvironment at this depth. Figure 5depicts a series of microprofiles recorded shortly before,during, and after sunset. Throughout this period the popu-lation of 0. terebriformis migrated downward into the mat.At 19:10 a thick layer of 0. terebriformis was present on themat surface. The decrease in photosynthetic 02 productionwith diminishing light is evident in the 02 and sulfide profiles.The O2-sulfide overlap was minimal, with little or no stablecoexistence of the two compounds. 02 was not presentbelow 0.8 mm. At 19:25 sulfide buildup began while 02 wasfurther depleted. 0. terebriformis was still present on thesurface of the mat. By 19:50 the surface population began to

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7 8 9 6 7 8 9

FIG. 6. Microprofiles of 02. pH, and sulfide during dawn and early morning. These mneasurements were recorded at the same location as

those of the experiment shown in Fig. 5 but were taken on the following morning. The water temperature varied from 40 to 41.5°C. Arrowrepresentations are the same as for Fig. 5. Sunrise occurred at 06:44. Shown are microprofiles at 05:50 (A) (irradiance was 8.0 W/m2; at thistime, 0. terebriformis was within the mat), 06:40 (B) (irradiance was 50 W/m2), 07:33 (C) (with sunlight striking the mat surface; irradiancewas 300 W/m2, and 0. terebriformis was now present on the mat surface), and 08:04 (D) (light was approximately 350 W/m2). As in Fig. 7,H2S refers to all sulfide species. Organism abbreviations are defined in the legend to Fig. 1.

visibly thin, as trichomes disappeared into the substrate. Atthis time the 02-sulfide interface was located 0.2 to 0.3 mmbelow the surface of the mat. By 21:45, after 1 h of fulldarkness, almost the entire population of 0. terebriformiswas below the mat surface. At a depth of 0.5 to 1.0 mm,

which was the location of the layer of trichomes, sulfide waspresent at concentrations of approximately 0.14 to 0.34 mM.02 was absent below 0.25 mm. Thus, 0. terebriformis movesduring darkness to a microenvironment which is both anaer-

obic and reducing.

A C F G

FIG. 7. Vertical migration of 0. terebriformis in artificial gradi-

ents. This photograph was taken after 17 h of incubation at 450C. (A)

Dye safranin placed in the test plug (bottom half) to show the general

rate of diffusion of test substances. No 0. terebriformis was placed

in the overlay. (B) Control; no test substance was added to the plug

(dark incubation). 0. terebriformis is evenly distributed in the vial.

(C) Test plug containing 7 mM glucose (dark). (D) Test plug

containing 7 mM fructose (dark). In vials B, C, and D, 0.

terebriformis was evenly distributed after the incubation period. (E)Test plug containing 0.7 mM sulfide (dark); (F) 7 mM fructose plus0.7 mM sulfide (dark); (G) 7 mM fructose, 0.7 mM sulfide, 5 F.MDCMU (light). In vial G both the overlay, and the test plugcontained DCMU, an inhibitor of Photosystem 11. In all vials withsulfide as a test substance, trichomes were observed to form a layerabove the test plug.

Figure 6 illustrates microprofiles recorded during the dawnand early-morning period of 8 May. At 05:50 the O2-sulfideinterface was between 0.2 and 0.5 mm deep, above the layerof 0. terebriformis. Sulfide increased steadily with depth andwas about 0.5 mM at a depth of 1.3 mm. 02 was present onlyin the top 0.5 mm. At 06:40, just before sunrise, only smallnumbers of 0. terebriformis were present on the mat sur-

face. With a light intensity of 50 W/m2 (recorded at the matsurface), some oxygenic photosynthesis was taking place,which could be seen because of the presence of 02 down toa depth of 1 mm. Sulfide concentration was decreasing bydepth during this time. At 07:33 a large peak of 02 was

present at the mat surface, produced presumably by thephotosynthesis of both 0. terebriformis, now present on themat surface, and S. lividus, immediately underlying 0.

terebriformis. The highest concentration of 02 coincidedwith the positions of these two species of cyanobacteria.

Laboratory experiments on vertical migration. The tactic

FIG. 8. Response of 0. terebriformis to sulfide in darkness. (A)Plate 6.5 h after exposure to an Na2S granule placed at X. (B)Control plate after 6.5 h without sulfide exposure. In both cases

plates were inoculated in the center.

pH 6mM

mat .surface

EE

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nature of the downward movement was investigated bysetting up artificial gradients of test substances in soft (0.7%)agar through which 0. terebriformis could move. Experi-mental conditions were designed to simulate the gradients of02 and sulfide found in the natural mat, although the verticaldistance was expanded. Figure 7 shows the results of one ofthese experiments. It was determined that sulfide, at aninitial concentration of 0.7 mM in the test plug (see thelegend to Fig. 7), inhibited motility of the trichomes andresulted in formation of a layer of 0. terebriformis similar tothat seen in the microbial mat. This response occurred bothin the dark and in the light, but in the light only when DCMU[3-(3,4-dichlorophenyl)-1,1-dimethylurea] was present.Without DCMU in the light, the effect did not occur (data notshown). Seven millimolar glucose, fructose (Fig. 7), acetate,and lactate (data not shown) did not have an effect similar tothat of sulfide; i.e., none of these other substances elicitedformation of a dense layer of trichomes.An additional set of experiments was carried out in which

a granule of Na2S 9H20 was inserted into the agar to oneside of a culture plate 45 min after 0. terebriformis had beenheavily inoculated in the center. The plates were thenincubated in the dark or light at 45°C for several hours andinspected and photographed periodically. After 6 h of expo-sure to sulfide (and polysulfide by-products) in the dark, 0.

terebriformis had not only accumulated along a sharp line ofdemarcation (Fig. 8) but had also retreated somewhat from aposition held 3 h earlier (data not shown). In continuous lightthe results were less conspicuous. It is possible that anegative sulfide taxis or a step-up sulfide phobic response isinvolved.

DISCUSSION

The field studies at Hunter's Hot Springs demonstratedthat as a result of the diel vertical movement pattern, 0.

terebriformis is exposed to large-scale fluctuations in 02,

pH, Eh, sulfide, and light and regularly alternates between an

aerobic, light environment and an anaerobic, dark, reducingenvironment. Table 2 summarizes a range of environmentalfactors to which 0. terebriformis is likely to be exposed ona daily basis. Most of these factors are extremely importantin terms of physiology.

During daylight hours 0. terebriformis is exposed to 3 to5 x supersaturated 02 (up to 0.7 mM), a value in starkcontrast to the nightly environment of anoxia. The range ofsulfide in the environment of 0. terebriformis is extremealso, from 0 (day) to 0.8 mM (night). Free sulfide overapproximately 100 to 200 ,uM is toxic to most aerobicorganisms, inhibiting electron transport in both respirationand oxygenic photosynthesis (8). At the pH values found inthe mat at night (6.0 to 7.0), sulfide should have an evenmore toxic effect, since relatively more is in the mostpenetrable H2S form (9). Field samples of 0. terebriformishave been routinely exposed to sulfide from 0.3 to 1.0 mM,with little toxic effect on photosynthesis (5).As mentioned previously, the nighttime vertical migration

pattern of 0. terebriformis is unusual (Table 1). 0. terebri-formis is the only species reported to migrate downward intoa mat during darkness. In contrast, some filamentouscyanobacteria migrate up to microbial mat surfaces in com-

plete darkness, probably exhibiting aerotaxis (21, 29). Itappears that the marine Oscillatoria mats described byJ0rgensen (14) are covered at night by upwardly migratingBeggiatoa sp. but that the Oscillatoria sp. probably does notactually migrate downward. In any case, this marine Oscil-

TABLE 2. Range of conditions to which 0. terebriformis wasexposed owing to the diel vertical migration

Environmental Organism Timefactor Minimum Maximum position in of daymata

02 0 -0.5--1.0 Night0.7 mM Surface Day

Sulfide 0.8 mM -1.0 Night0 Surface Day

Eh -170 mV -1.0 Night+ 200 mV Surface Day

pH 6.3 -1.0 Night8.8 Surface Day

aPosition in mat is expressed as on the surface or in millimeters below thesurface of the mat.

latoria sp. is also exposed to anaerobic, reducing conditionsat night. A similar nighttime event occurs in a cooler,downstream part of Hunter's Hot Springs, where Beggiatoasp. overrides Oscillatoria princeps, a species that appearsnot to move downward at night (unpublished observations).The daytime positioning of motile mat organisms, includ-

ing 0. terebriformis, appears to be one of obtaining anoptimal light intensity for photosynthesis or avoiding toohigh an intensity or both.

Periodic exposure to anaerobic, reducing conditions is notunusual for cyanobacteria in the natural environment. Illu-minated aquatic environments which fluctuate between aer-obic and anaerobic conditions are often dominated bycyanobacteria, whereas in stable anaerobic, photic zones,dominance is usually assumed by photosynthetic purple andgreen bacteria (19). The presence of cyanobacteria in envi-ronments which regularly fluctuate between oxic and anoxicconditions has, for the most part, been viewed as the resultof tolerance of these conditions, not preference (19). Fluc-tuations in oxygen and sulfide may select against obligatelyaerobic (eucaryotic) or anaerobic photoautotrophs, thusindirectly selecting for cyanobacteria. There are many re-ports of the presence of filamentous cyanobacteria in anaer-obic or reducing environments, as well as in zones ofrelatively low 02 concentration. For example, populations ofArthrospira sp. form in the aerobic-to-anaerobic transitionzone of lakes (24). The genus Oscillatoria is apparently oneof the most common of the cyanobacterial inhabitants ofsuch environments. 0. splendida (27) and 0. margaritifera(Castenholz, unpublished observation) have been found inlow Eh, sulfide-containing sediments. Other Oscillatoriaspecies (e.g., 0. utermoehliana and 0. prolifica) are com-monly found in the 02-depleted hypolimnia of lakes (13, 24).Two reports suggest that the development of blooms ofcertain Oscillatoria species is dependent on a lowered redoxpotential. Leventer and Eren (15) claimed that 0. chalybeablooms are correlated with a lowered partial pressure of 02accompanied by an increase in sulfide at increasing depth inlakes. Tash (26) attributed the presence of 0. redekei in thelower strata of some lakes to reducing conditions and sulfide.

Various cyanobacteria encounter free sulfide togetherwith anaerobic conditions in the light and have differentresponses involving either sulfide tolerance, sulfide utiliza-tion, or both (9). For 0. terebriformis, tolerance at leastwould be expected in view of the regular exposure of thisspecies to high sulfide levels during darkness.

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VERTICAL MOVEMENTS OF 0. TEREBRIFORMIS 2149

While the ability to tolerate low Eh, anoxia, and sulfidemay serve as a selective advantage for dominance, this failsto account for the fact that cyanobacteria often dominateaquatic sediments or thick mat environments which, alongwith alternating aerobic-anaerobic conditions, are character-ized by dim light or prolonged darkness. As 0. terebriformisactively migrates during darkness to a position where anaer-obic, reducing conditions exist, maintaining this position forat least 9 to 15 h every 24-h period, a potential benefit to theorganism is suggested. It is also likely that many trichomesremain in the undermat for an additional 24 h if the day isovercast and relatively dark. It has been determined (23) thatthis species is a facultatively anaerobic, slow-growingchemoheterotroph and that prolonged survival in darknessunder aerobic conditions is not possible. In view of theseresults, the observed dominance of other cyanobacteria indimly illuminated, anaerobic habitats could also be based onsomething more than tolerance.

It seems that the disappearance of most of the 0.terebriformis population from the mat surface at night is aresult of random gliding in all directions by trichomes withinthe mat. During daylight hours, gliding motility has a direc-tional component controlled by light gradients and intensi-ties. When these directional cues are absent, gliding move-ments may be random. Within 1 to 2 h, a narrow piling-up orlayering of trichomes occurs a short distance below the matsurface, whether in the field or laboratory. This may be dueto the reversible inhibition of motility by sulfide present atdepth. Gliding motility of 0. terebriformis is known to occurat rates of up to about 0.3 mm/min in aerobic darkness, a rateeasily sufficient to account for the subsurface layering. Thisrate is also more than sufficient for the complete randompenetration of trichomes to the bottom of the soft agar plugsused in the laboratory experiments (Fig. 7) or the formationof a layer on the surface of sulfide plugs.

In the natural environment at Hunter's Hot Springs, thelayering effect in darkness is overridden and the populationascends to the mat surface when light is shown on the mat(either at dawn or with artificial light at any time during thenight). The exact mode of the light effect is uncertain.Resumption of oxygenic photosynthesis may release glidingmotility from the inhibition of sulfide, perhaps directlythrough the oxidation of sulfide. Light directly increases therate of gliding motility in 0. terebriformis (photokinesis).Possibly both of these factors, plus positive phototaxis,combine to bring the population rapidly to the surface. Thetime involved for upward movement of the population ismuch less than the 1 to 2 h required for the downward, morerandom movement after dark.The physiological advantages for 0. terebriformis of an

anaerobic, reducing environment at night, in contrast to theaerobic mat surface, are the subjects of an accompanyingreport (23).

ACKNOWLEDGMENTS

We thank Niels Peter Revsbech and David M. Ward for theirassistance and expertise in the microelectrode studies at Hunter'sHot Springs.

This work was supported by a grant to R.W.C. from the NationalScience Foundation.

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