APPLiED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1977, p. 433-452 Vol. 34,No. 4 Copyright X) 1977 AmericanSocietyforMicrobiology Printedin U.S.A. Structure, Growth, and Decomposition of Laminated Algal- Bacterial Mats in Alkaline Hot Springs W. N. DOEMEL' AND THOMAS D. BROCK2* BiologyDepartment, Wabash College, Crawfordsville,Indiana 47933,1 andDepartmentofBacteriology, University ofWisconsin, Madison, Wisconsin 537062 Received forpublication 12 April 1977 Laminated mats of unique character in siliceous alkaline hot springs of Yellowstone Park are formed predominantly by two organisms, a unicellular blue-green alga, Synechococcus lividus, and a filamentous, gliding, photosyn- thetic bacterium, Chloroflexus aurantiacus. The mats can be divided approxi- mately into two major zones: an upper, aerobic zone in which sufficient light penetratesfornetphotosynthesis, andalower, anaerobiczone, wherephotosyn- thesis does not occur anddecomposition isthe dominantprocess. Growth ofthe mat was followed by marking the mat surface with silicon carbide particles. The motileChloroflexus migratesvertically at night, dueto positive aerotaxis, responding to reduced 02 levels induced by dark respiration. The growth rates ofmats were estimated at about 50 ,um/day. Observations ofa single mat at Octopus Spring showedthatdespite the rapid growth rate, thethickness ofthe mat remained essentially constant, and silicon carbide layers placed on the surface gradually moved tothe bottom ofthe mat, showing thatdecomposition was taking place. There was a rapid initial rate of decomposition, with an apparent half-time ofabout 1 month, followedby a slowerperiod ofdecompooi- tionwithahalf-timeofabout12months. Withinayear,completedecomposition of a mat of about 2-cm thickness can occur. Also, the region in which decompositionoccursisstrictlyanaerobic, showingthatcompletedecomposition oforganic matter from these organisms can occur in the absence of02. Widespreadinterestinthepaleomicrobiology temperatures around 40to450C. More detailed ofPrecambrianstromatoliteshasfocusedatten- studies on these structures are reported by tiononlivinglaminated algalmatsthatmight Walter et al. (37). The present study deals be similar to those which formed the Precam- withadifferenttypeofmatwhichoccursexten- brian deposits (35). Extensive work has been sively in hot springs ofneutral to alkaline pH done over the past several decades on stroma- at temperatures of 55 to 650C. This mat is toliticblue-greenalgalmatsinmarineenviron- composed oftwo quite different photosynthetic ments, and a few studies on freshwater algal organisms, a unicellular blue-green alga (Sy- reefs have also been carried out. Early work nechococcus lividus) and a gliding, filamen- onthealgalmatsofYellowstoneNationalPark tous, photosynthetic bacterium (Chloroflexus by Weed (38) focused geological attention on aurantiacus). These two organisms, and asso- these extensive and interesting structures, but ciatedheterotrophicbacteria,formwell-defined little microbiological and ecological work was laminated mats of considerable thickness, in done until the mid-1960s, whenthis laboratory which the photosynthetic bacterium is respon- began a long-term investigation ofthe geomi- sible for the structural integrity. These mats crobiology ofgeothermalhabitats. The Yellow- are of special interest because they occur at stone mats are ofspecial interest because they temperatures too high for the development of are siliceous and hence may provide a model grazing animals (or any other eucaryotic orga- (oranalog) forthe depositional environment of nisms) and so provide some insight into how Precambrian iron formations (34). Indeed, mats might have developed in the Precam- manyofthemicroorganismsoftheYellowstone brian, when metazoans were absent. AsAwra- mats resemble morphologically the fascinatinig mik(2)hasshown, stromatolitediversityshows microbiota ofthe Gunflint (3). Inabriefstudy, a marked decrease in the Late Precambrian, Walter et al. (36) reported on living conophy- at a time when metazoan life first appeared. ton-likestructures formedbyablue-greenalga However, decomposition processes in the Yel- (Phormidium sp.) that grew in hot springs at lowstonematsoccurreadilydespitetheabsence 433 434 DOEMEL AND BROCK APPL. ENVIRON. MICROBIOL. of metazoans, so a complete procaryotic food m from Mushroom Spring (4). This spring first chain leadingto mineralization oforganicmat- started to flow as a geyser during the summer of ter can occur. 1969 and then subsided to a spring. During the summer of1973, the spring had a source tempera- STUDY AREAS tureofabout85°C, agoodflow, andasingleeffluent channel withagradient from 80°C to ambienttem- The locations of Octopus Spring and Ravine perature. By 30 May 1974, the source temperature SpringhavebeengivenbyMadiganandBrock (24). had decreased to 75°C and the minimal flow rate OctopusSpring (PoolA) isatypicalalkaline spring from the source no longer supported an extensive (pH 8.5 to 8.7). It is located about 0.15 km SSE of mat. InJune 1975, the springno longerflowed and GreatFountainGeyserandtheWhiteCreekValley. the algal mat had dried. Sulfide Spring (source: An extensive microbial mat, about 2 to 4 cm deep, 86°C, pH6.6;sulfidelevel, 1.3mg/liter)isoneofthe exists in a shallow extension ofthis pool (Fig. 1). two springs located about 0.2 km ENE ofthe Fire- This backwater region is separated from the main hole Lake parkingarea, 50 m southofthe Firehole poolby anincomplete barrierofsiliceoussinter (5). Lake LoopRoad. Although many hot springs have short life spans, Octopus Spring seems to be relatively stable, since MATERIALS AND METHODS itcanbereadilyidentifiedfromPeale's1883descrip- tion (25) as his no. 10 (p. 165), with temperature Measurement of light intensity. Measurements andflow conditions similarto those now existing. of light intensity in the field were made with a Springs 74-6 (source: 80°C, pH 7.7), 74-7 (source: Gossen Pilot light meterequippedwith an incident 68°C, pH 9.2), and 74-3 (source: 78°C, pH 8.0to 8.2) lightattachment. Measurementsofthereductionof arenearRavineSpring. BuffaloPoolisanintercon- lightbymicrobialcoresweremadewithaKippand nected group ofthree springs (source: 91°C, pH 9.3) Zonen solarimeter connected to a Keithley model located on the east bank ofWhite Creek upstream 601 electrometer (Keithley Instruments, Inc.). The from pool 74-3. Toadstool Spring is located about 5 solarimeterwasmodifiedbyreplacingtheprotective FIG. 1. Octopus Spring mat. The source is in the background and the mat is in the foreground, in the backwater behindthesinterdike. VOL. 34, 1977 ALGAL-BACTERIAL MATS IN ALKALINE HOT SPRINGS 435 globe with a cardboard mask having a 5.8-mm- synthetic biomass. Although in vivo absorption diameter hole. Light from a 12-V, 60-W ribbon spectra differ, in solvent extracts bacteriochloro- filament lamp (Zeiss microscope illuminator) was phyll c and chlorophyll a have similar maxima, focused with alens sothatacircle coveredthehole between 662 and 668 nm. To distinguish these two in the mask. To calculate the radiation intensity, chlorophylls, an in vivo procedure (27) was used the calibration value supplied by Kipp and Zonen whichinvolveseliminatingmostofthelightscatter- was used (1 gcal/cm2 per min = 8.8 mV). ingcausedbythecellsbysuspendingmaterialina Measurement ofphotosynthesis. The procedures saturated sucrose solution. The refractive index of for measuring photosynthesis have been described this solution approximates that ofthe cells, so the elsewhere (9, 24). To distinguish photosynthesis by in vivo peaks ofchlorophyll a, 680 nm, and bacte- Chloroflexus from photosynthesis by blue-green al- riochlorophyllc, 740nm, canberesolved. Sincesome gae, 0.1 mlof5 x 10-4 M3-(3,4-dichlorophenyl)-1,1- scattering still remains, thusraising the base line, dimethylurea (DCMU) was added to appropriate the high base lines were corrected by subtracting vials (4). the absorbance at 640 and 700 nm, respectively. Thin sections ofalgal cores. Cores (3.5-mm ID) The concentration of both chlorophylls was also were prepared for thin sectioning by dehydration determined in methanolic extracts if two distinct throughanacetoneseriesandtheninfiltrationwith peaks could be discerned (4). In some of the later DurcupanA/M(Polyscience, Inc.,Warrington, Pa.). work, a thin-layer chromatographic procedure (23) Sections 1 um thick were prepared with a Porter- was used to separate and quantify the two chloro- Blumultramicrotome, placedontoclean slides, and phylls. dried at 70 to 80°C for 15 to 30 min. After cooling, Protein was determined bythe Lowry method as the sections were rinsed with three changes of modified by Brock and Brock (8). Before protein distilled water and then dried. Oneto two drops of was extracted from the cores in agar, the samples Richardson's strain (0.5% azure II, 0.5% methylene were boiled for 30 min in 0.5 N perchloric acid, blue, 0.5%borax)wasplacedontoeach section, and which solubilized the agar and precipitated the the slide was warmed at 60 to 65°C for 5 min. The protein. excess stain wasremoved, and theslide was rinsed Oxygen measurements. Dissolved oxygen in the indistilled water. Afterdrying, adropofresinwas water immediately above the surface of the mat added, followedbyacover slip, andthe slideswere was measured by modifying the standard azide dried at 55to 60°C for48h. modification of the Winkler method for dissolved Measurement of mat growth. The mat was oxygen(1). Serumbottles(60-mlVitro400;Wheaton marked periodically with 15.5-cm circles of120-grit GlassCo.)withsleevedrubberserumstopperswere silicon carbide(BuehlerLtd.,Evanston, Ill.),placed flushed with nitrogen gas having less than 0.05% on the surface with a metal template. About 1 oxygen (Matheson Gas Products Co.) for 5 min. teaspoon ofsilicon carbide sprinkled onto the mat Afterflushing andremoval oftheneedles sothat a gave a thickness of 0.3 to 0.7 mm. The center of slightpositivepressureremained, thestopperswere each circle was marked with an iron nail. These sealed with a thin film of silicone sealant (Dow nails appeared not to oxidize in the spring and Chemical Co.). A30-mldisposablesyringewitha2- were intact after 7 years. Two to three cores were inch needle was then flushed with nitrogen and collected from each ofthese stations with a brass used to withdraw 30 ml ofgas from the vials. This cork borer (8.4 or 9.4 mm in diameter). The cores syringe then was slowly filled with water from the were immediately placed into 30-mm petri dishes spring, and the water was injected into the vial; containing2%moltenagar, whichthenwasallowed again a positive pressure was maintained. To each to harden. The agar stabilized the core during vial, 0.2 ml of alkaline potassium iodide solution transport and storage and also enabled the core to was injected, followed by 0.2 ml of manganous be sectioned. The agar disk around the core was sulfate solution. The vials were vigorously mixed removed from the petri dish and trimmed so that for several minutes; the floc was allowed to settle; the core was encased in a rectangle of agar. This and0.2mlofconcentratedsulfuricacidwasinjected agar block was then halved longitudinally with a into each vial. The iodine freed was then titrated razorblade. Oneofthehalveswasplacedonaglass with a 0.0025 N thiosulfate solution, with a soluble slide so that the surface of the core was exposed. starchsolutionaddedtowardtheendofthetitration With a Zeiss Photomicroscope I equipped with a toincrease sensitivity. Optivar, using either a x2.5 or x6.3 objective and anoptical micrometer, the heightofmatabove the RESULTS silicon carbide was estimated. The magnification was adjusted to minimize measuring errors, and at Morphology ofSynechococcus-Chloroflexus least 10 measurements of the height above the Mats. Although there are several classes of silicon carbide were made oneach core. microbial mats in alkaline thermal springs, After the height ofthe mat above the carborun- only the microbial mats containingS. lividus, dum was measured, this portion of the core was ablue-green alga (cyanobacterium), andChlo- sectioned from both halves ofthe core with a razor blade, and the protein was measured as described roflexus, a filamentous, photosynthetic bacte- below. rium, are considered here. A typical Synecho- Measurementofbiomass. Chlorophylla andbac- coccus-Chloroflexus microbial mat is in a tide teriochlorophyll c were used as measures ofphoto- pool of Octopus Spring (Fig. 1) and in the 436 DOEMEL AND BROCK APPL. ENVIRON. MICROBIOL. stream flowing from this pool. Although the 1.1-,m-diameter filaments that autofluoresced surface ofthis mat appeared at first glance to andwere similartothefilamentousblue-green be smooth to slightly roughwithno distinctive alga Pseudanabaena (S. Golubic, personal structure, a closer examination revealed the communication). Below about 3 to 5 mm, most presence of several distinctive types of struc- ofthefilamentous bacteriaappeared moribund tures. (i) On the mat in the pool and the (Fig. 8), andunicellularrodswereconcentrated stream, clusters of 1- to 2-mm conical projec- in bands having sulfide-forming activity, as tions, called nodules, were presentattempera- revealed by the ferrous ammonium sulfate tures up to about 650C (Fig. 2). Often adjacent technique (see Materials and Methods), sug- nodes were interconnected by short, raised gesting that at least some are sulfate-reducing ridges, producing a mat consisting of a fine bacteria. Often in the lower regions, refractile network ofnodes and ridges (Fig. 3). (ii) Also spherical bodies resembling myxobacterial present on these mats, but less frequently, cystswereobservedthatneitherautofluoresced were 2- to 10-mm circular structures, called nor fluoresced when stained with acridine or- colonies (Fig. 4). These colonies usually were ange. Also, in a number ofsamples, chains of orange with yellow-green centers, and in the spherical cells were seen that were similar to orange region there sometimes were several an organism described by Geitler (19) as the concentric ridges and/or nodules. (iii) On the blue-green algaIsocystis. However, these cells mats in flowing water there were yet other never autofluoresced and hence either are not distinct structures, called streamers (Fig. 5 blue-green algae or are not viable. showsstreamersfromToadstool Spring). These Bauld and Brock (4) demonstrated that bac- streamers extended several centimeters from teriochlorophyll c distribution correlated with their attachment to the mat and appeared to the distribution ofthe bacterial filaments and be more abundant where the flow was more isolated anumberofstrains ofthe filamentous turbulent. photosynthetic bacterium Chloroflexus. Since Thetypical laminated mat, similartoastro- microscopy of isolated nodes, colonies, and matolite (43), was beneath these surface struc- streamers revealed that all ofthese structures tures (Fig. 6). To better understand the distri- were composedpredominantly ofbacterialfila- butionofmicroorganismswithinthelaminated ments with some Synechococcus, the composi- mat, microscopy was done on verticle sections tion of these structures was further investi- (prepared with a razor blade) of fresh cores gated with [14C]bicarbonate incorporation, embedded in 2% agar. Because ofthe opacity chlorophyll analysis, and autoradiography. of such sections, visualization was done with Colonies, nodules, and streamers were sam- vertical fluorescence illumination. The Syne- pled from mats at Octopus Spring and Toad- chococcus could be observed directly with this stool Geyser. Colonies were removed with a microscopic technique because ofthe autofluo- cork borer having a larger diameter than that rescence of chlorophyll a. The sections were of the colony. With a dissecting microscope, thenstainedwitha0.1%solutionofthefluores- the cores were separated in the field into sev- cent dye acridine orange; with this stain, bac- eralserialthinsections, approximately0.5mm teria fluoresce either red or green. Also, sam- thick. Eachofthesesectionswasgentlyhomog- ples were removed from the various lamina- enized in5 ml ofwater, andthephotosynthetic tions and were examined directly with phase activityandchlorophyllcontentofthehomoge- andphase-fluorescenceillumination. Thesemi- nate was measured. Although the chlorophyll croscopicobservationsaresummarizedinTable concentrations and proportions were variable 1. in different cores, the colonies were enriched Inthe upperlayer, 0.2 to 1 mm in thickness, in bacteriochlorophyll c in the upper layers. Synechococcus andChloroflexus predominated In contrast, the regions surrounding the colo- (Fig. 7). Often there was a second layer, 0.1 to nieswerehighinchlorophylla. Inthecolonies, 0.6 mm thick, immediately beneath this upper a high percentage of incorporation of [14C]bi- layer which was similar except that it was a carbonate was insensitive to DCMU. dark blue-green and the autofluorescence of Similarexperiments were donewithnodules the Synechococcus was much more intense. and streamers. Both were removed from the Beneath this region,Synechococcus wasrarely microbial mat by aspiration with a Pasteur observed, even as empty cells, but there was pipette and rubber bulb. In nodules, bacterio- an abundance of Chloroflexus in this next chlorophyll c was present in considerably layer, 0.8to 1.4mmthick. Althoughthemajor- higher amounts than in the surface layers of ity offilaments in this region did not autoflu- surrounding mat. Microscopy suggested that oresceandalthoughotherorganismswereusu- nodules are composed ofa dense mass ofChlo- ally absent, in some thin sections there were roflexus filaments arranged in aconical shape, FIG. 2. Nodules on the Octopus Spring mat. Thesurface ofthe microbial mat is often covered with1- to 2-mm conicalprojections. Diameterofcore, about3.5 mm. FIG. 3. Surface ofa core similar to that inFig. 2. The nodules extend1 to2 mm above the surface and areoften connected byridgesformingafine lattice. FIG. 4. OrangecoloniesontheOctopusSpringmat.Phototakendirectlythrough the water. Thecenterof thecolony isoftengreen, shown here byasmalldark dot.Distanceonthephotograph about20cm. FIG. 5. Streamers. Filamentous streamers (St) in the effluentofToadstool Spring. The lightcircles are silicon carbide (C), 15 cm in diameter, and serve as markers. The streamers are present only in flowing water. Thecurrentflowsfrom left to right. FIG. 6. Macrophotograph ofa complete core, showing laminations. This is onefrom the Octopus Spring mat. Note the surface nodules (N), the upper dark layer (D), and the laminae (L) below this layer. Often there are bandsofsinter (Si) in thecore (coarse whitezones). 437 438 DOEMEL AND BROCK APPL. ENVIRON. MICROBIOL. TABLE 1. Topography andcompositionofa matatOctopusSpringa Coresection Phase-contrastandfluorescencemicroscopyofmacer- Coresection Directmicroscopyofwholecores, (mm) ationsofcoresections (mm) usingacridine orangefluores- cencebyincidentlight 0-0.2 Synechococcus >Chloroflexus 0-1.2 Synechococcus, filaments,'Is- ocystis" 0.55-1.6 Highly granulated Chloroflexus > rods, noSy- 1.2-2.5 Filaments anddebris nechococcus, refractile spheres, some algal filaments 1.6-2.1 Moribund Synechococcus, refractile Chloro- 2.5-3.2 Amorphous debris flexus and short rods 2.9-3.65 Chloroflexus and "Isocystis," some algal fila- 3.2-4.2 FewSynechococcus,withlarge ments numbers ofrods 5-7.5 Moribund filaments 4.2-6.5 Amorphous material 8.7-10.2 Rods, few moribund filaments, no Synechococ- 6.5-7.5 Bacterial rods and moribund cus, sulfate reduction filaments, some Synecho- coccus 10.2-11.7 Gelatinous matrix, primarily unicellular rods, 7.5-7.8 Dense rods moribtnd filaments 11.7-12.55 Unicellular rods, few moribund filaments 7.8-8.9 Dense rods and moribund fil- aments 12.55-15.2 Unicellular rods, sulfate reduction 8.9-11 Denserods 15.2-15.4 Unicellular rods (0.4 ,um), no filaments, pine 11-17 Amorphous material pollen - a Thisisacompositeofaseriesofcoresincubatedin0.4%ferrousammoniumsulfateagar (2%)todetect regions ofsulfide production, and then split vertically with a razor blade and examined microscopically. Regions ofsulfate reduction are indicated in column two. The designation"bacterial filaments" includes all filamentous organisms lacking algal chlorophyll a (as revealed by absence ofred autofluorescence). They were 0.5 to 1.5 ,im in diameter and were ofindeterminate length; they were probablyChloroflexus. Algalfilamentsshowedredautofluorescenceandwere1.1 Aumindiameter;theyresembledPseudanabaena. Thebacterialrodswere0.7 Amindiameterand4.0 ,uminlengthandwereprobablynotphotosynthetic. In addition to direc' visualization by phase-contrast and fluorescence microscopy ofmacerated samples, the verticaldistributionoforganismswerevisualizedwithoutdisturbancebyadding100 ,ugofacridineorange permltotheflatsurface ofsplit cores andobservingwith incident lightby fluorescence microscopy. with the Synechococcus concentrated in pock- then removal ofthe Synechococcus should not ets of filaments. In streamers, chlorophylls significantly alter the structure of the mat. were extracted and isolated with thin-layer Preliminary experiments demonstrated that chromatography (23). About 40% of the total when amicrobial matwas darkened, within24 chlorophyll absorbing at 665 nm was bacter- h the mat turned orange in color and this iochlorophyll c, the remainder being chloro- orange layer was enriched in Chloroflexus. phyll a. Results with [14C]bicarbonate incorpo- Also, as shown by Madigan and Brock (24), ration support the observation ofhigh Chloro- Chloroflexus is able to photosynthesize at con- flexus concentrations in the streamers. These siderably lower light intensities thanSynecho- observations with colonies, nodules, and coccus. This observation implied that at low streamers demonstrate that a large proportion light intensities, Synechococcus would not be ofthe population in these structures is photo- abletomaintainapopulationandChloroflexus synthetic bacteria. Since bacterial filaments wouldpredominate. To testthis, an areaofthe are abundant whereas other bacteria are few, mat at 56°C in the stream flowing from Toad- and since Chloroflexus has been isolated from stool Geyser was covered by two layers of a these structures, this is indirect evidence sup- dense nylon cloth held on a frame, which re- porting the structural role ofChloroflexus. duced the incident light by 98%. At the same Ifthe mat is held together by Chloroflexus, time, a circle of 120-mesh silicon carbide was FIG. 7. Organisms ofthe upperlayerofthe mat, as seen byphase-contrast microscopy. Thefilamentous cells are Chloroflexus, andthecurved rod-shapedcellsareSynechococcus. Bar = 10 ,um. FIG. 8. Photomicrograph byphasecontrastofa smearfrom a lowerregion ofthe mat. The Chloroflexus filaments are moribund. Mostly the material is aformless debris. Though not obvious in the micrograph, bacterial rods wereoftenpresent. Same magnification asFig. 7. 4 tO"A 439 440 DOEMEL AND BROCK APPL. ENVIRON. MICROBIOL. placedonthematasamarkerandsubstratum. prepared from resin-embedded corings and Within 4 h small nodules had formed on the after staining were examined with a light mi- surface of the mat and on the surface of the croscope (see Materials and Methods). Obser- silicon carbide, and within 24 h a number of vations of cores from Octopus Spring are re- pinkish-orange nodules were present (Fig. 9). ported here. Two patterns oforientation were MicroscopyshowedthatSynechococcus wasab- observed. In some samples, in the uppermost sentfromthenodulesandinternodularregions, region, the unicellular blue-green alga was yetthenodulesweresimilartonormalnodules. present but showed no preferred orientation, Uptake by homogenates of these nodules of and the photosynthetic bacterial filaments [14C]bicarbonate incubated with and without were oriented horizontally (Fig. 10). In the DCMU had equivalent radioactivity, suggest- lowerregions, mostofthe filamentousbacteria ingthatthealgaewerenotcontributingsignif- were oriented vertically and Synechococcus icantly to the primary production of these was not apparent (Fig. 11). In some regions, nodes. Further, autoradiograms prepared of vertically oriented filaments extended into the this material indicated that all ofthe incorpo- upper region (Fig. 12). ration of['4C]bicarbonate wasbybacterial fila- ments (Table 2). Together these observations TABLE 2. Photosynthesis by nodules underlow add direct evidence to support the notion that light, as seen byautoradiographya Chloroflexus isthestructural componentofthe Chloroflexus Synechococcus Condition mat and furthermore that it accounts for the (grains/25 Am3) (grains/25 Am3) growth ofthe mat. Light. 23.3 0 The apparent involvement of Chloroflexus Lightwith DCMU 15.3 0 ... in the structure and growth ofthe mat and in Dark. 4.6 0 the development ofthe differentiated nodules, a Nodules on the surface ofa mat under a 98% streamers, andcolonies impliesthattheremay light reduction cover were harvested by aspiration be a nonrandom orientation of the filaments withaPasteurpipette,homogenized, andincubated within the mat. To determine whether the with ['4C]bicarbonate for4h. Autoradiograms were orientation of filaments within the microbial prepared, and grains over Chloroflexus and Syne- mat is nonrandom, 1-,um thin sections were chococcus werecounted. FIG. 9. Pinkish-orange nodulesofChloroflexus, whichdevelopedin24 hon topofasiliconcarbidecircle laiddown in Toadstool Geyser. A neutraldensityfilteroverthe mathadreducedthelight intensity to98% offullsunlight. Diameterofcircleonphotograph about85 mm. FIG. 10. Photomicrograph ofa thin section ofthe upper layer ofOctopus Spring from resin-embedded material. The surface ofthe mat is towardthe topofthepage. The cells arepredominantly Synechococcus. Nopreferred orientation is observed. Bar = 10 ,um. FIG. 11. Photomicrograph ofa thin section ofthe lower region ofthe same mat as Fig. 10, showing verticalorientation ofChloroflexusfilaments. NoSynechococcus ispresentat this depth.Bar = 10 ,um. FIG. 12. Photomicrograph ofa thin section showing vertical filaments extending into the upper region through azone ofdenseSynechococcus. Bar = 10 gm. FIG. 13. Photomicrograph of nodular mat, showing alternating regions with horizontal Chloroflexus filaments atrightangles. The top ofthe mat is in the upperright-handcorner. Bar = 10 gm. FIG. 14. Verticalsectionofacorefrom ToadstoolGeyser, showingasiliconcarbide (C) layer. Thesilicon carbide hadbeenplacedonthesurfaceofthemat29daysbeforeandisnowatadepth of4 mm intothemat. The silicon carbide layeris about 0.2 mm thick. 441 442 DOEMEL AND BROCK APPL. ENVIRON. MICROBIOL. Adifferentpatternwasobservedinaportion teria, insome experiments thewhite lightwas ofthe mat having abundant nodes. Again, in passed through an infrared (IR) filter (Tiffen the upper regions of the mat, about 0.4 mm no. 88A) before being passed through the mat. thick, the blue-green alga was present andthe (This filter passes no radiation below 730 nm.) filamentous bacteria were horizontally posi- Although the reduction of IR radiation was tioned. Belowthisregion, thealgawasmissing greaterinsome cores, the difference was small andthebacterialfilamentswereagainhorizon- so that the reduction ofboth tungsten and IR tally positioned, but in different directions. In radiation was approximately the same. Thus, the section shown in Fig. 13, in the upper inthese compactmats, lightattenuation is not regions most filaments are in cross-section. absorption only by pigments, but also by scat- Immediately below this, there is a region 0.15 ter. Indeed, because there was no difference in mm thick in which most filaments are in tan- attenuation by tungsten and IR radiation, it gential section. Below this there is another seemslikelythatscatteringcausesmoreatten- region, 0.23 mm thick, in which the filaments uation than does pigment absorption. are again in cross-section. These regions of On a bright cloudless day, light intensities alternating stacks of filaments do not extend mayreachabout 1.5gcal/cm2permin (equiva- through the entire core. In one portion ofthe lent to about 8,400 lx) at noon. Since the thin core, the filaments arestillhorizontalthrough- layer ofwater overlying the mats, 1 to 5 cm at out, but there is no obvious stacking of fila- the Octopus Spring mat, does not significantly ments. In all sections and cores examined, the reduce the light intensity, the light energy algae were restricted to the upper 0.3 to 0.4 passing through the green layer and reaching mm ofthe mat. the orange layer can be calculated (Table 3B). Response of the mat components to light. About 2,500 to 3,500 lx passes through the The depth of light penetration into the mat green layer, which is probably sufficient to was estimated directly by use of an artificial support autotrophic growth, but only 180 to light source and a solarimeter (see Materials 450 lx passes throughboth layers, about 1 mm and Methods). The top green layer and an thick. Because these are mid-day values, most underlying orange layer of similar thickness ofthe time the light intensity is much lower. were used. These sections were placed onglass Thus, below the green and the top orange slides, and the reduction of light energy by layers, the mat is essentially dark, implying each of the sections was determined. The re- that the light penetrates only about 1 to 2 mm sults of a typical core are shown in Table 3A. into the mat. The green layer reduced the radiation by 94%, Although some light does reach the orange and the orange layer reduced it by about 91%. layerofthemat,Synechococcus isrestrictedto Tocheckforattenuationbyphotosyntheticbac- the green layer (10), and even cell ghosts of TABLE 3. Reductionofradiantenergy by thegreenandorange layersofa microbial mata Lightintensity (gcal/cm2) (A) Lightsource Percent reduc- Percentreduc- Lightalone Withgreenlayer tion Lightalone Withorangelayer tion White.............. 1.0 0.056 94 1.0 0.097 91 Infrared............ 0.76 0.025 97 0.89 0.080 91 Calculated lightintensity(lx)atthebottomof: (B) Location Greenlayer Orangelayer Mat (50C)......................................... 3,440 450 Mat (490)......................................... 2,580 230 Mat (54C)......................................... 2,580 460 Outflow (59-65°C) ............... 4,620 420 ................... Outflow (50-54°C) ............... 2,580 180 ................... Mat (580)......................................... 2,580 360 (5000). a (A) Samples from Octopus Spring The values for (B) were calculated by assuming a light in- tensity of84,000 lx (maximum intensity at mid-day). The lightintensitypassing through the green layer (0.2to0.5mmthick)wasdeterminedbysubtractingtheradiantenergyattenuatedbythegreenlayerfrom thetotalenergy. Thelightintensityatthebottomwasdeterminedbymultiplyingthelightpassingthrough the green layer by the percentage reduction by the orange layer and subtracting this from the light passing through the green layer (total distance = 0.4 to 1 mm).