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BACTERIOLOGICAL REVIEWS, Dec. 1969, p. 476-504 Vol. 33, No. 4Copyright @ 1969 American Society for Microbiology Printed Ln U.S.A.

Thermophilic Blue-Green Algae and theThermal Environment

RICHARD W. CASTENHOLZDepartment of Biology, University of Oregon, Eugene, Oregon 97403

INTRODUCTION.................................................. 476DISTRIBUTION OF THERMAL WATERS.................................... 481SOLUTES OF THERMAL WATERS.......................................... 482DISTRIBUTION OF SPECD DS............................................... 486Upper and Lower TemperatureLLmts.......................................... 486Clas tion and Geographical Distribution..................................... 487Problems of Survival and Transport............................................ 488

STUDIES OF NATURAL POPULATIONS.................................... 489Mat Formation and Stability.................................................. 489Movements of Filaments and Mats............................................. 491Measurements of Photosynthesis and Growth.................................... 491

CULTIVATION OF THERMOPHILIC CYANOPHYTES ....................... 493Medium and Nutrition ................................................... 493Isolation and Maintenance................................................... 494Rates of Growth, Photosynthesis, and Respiration in Culture....................... 495

RESPONSES TO TEMPERATURE AND LIGHT INTENSITY.................. 497Optimal Temperature and Light Intensity....................................... 497Effects of Light and Temperature on Pigmentation............................... 498Growth and Survival at Suboptimal Temperatures................................ 499

LITERATURE CITED........................................................ 500

INTRODUCTIONThe blue-green algae (cyanophyta) are con-

sidered to be thermophilic (in this review) whenpart or all of their optimal growth temperaturerange is above 45 C. Different definitions are usedfor thermophily in bacteria, fungi, and animals.A maximal growth rate at temperatures over 45 Cis mainly a characteristic of procaryotic or-ganisms. Only a few species of eucaryotic protistsor animals tolerate temperatures above this(Table 1). In the range between 50 and 60 C, thereare a few fungi and the eucaryotic alga of acidwaters, Cyanidium caldarium. Photosyntheticblue-green algae are known to grow at constanttemperatures as high as 73 to 74 C (29), andnonphotosynthetic bacteria as high as 95 C (26,Table 1). Even many species of blue-green algaeof nonthermal habitats have higher temperatureoptima than the eucaryotic algae of the samewaters (79). Because of this, blue-green algae maybe enriched for by incubating samples in light andnonselective mineral medium at 35 C (11).

Blue-green algae are becoming more con-spicuous in this age of increasing environmentalpollution. In nutrient-enriched waters, blooms ofplanktonic blue-green algae are more frequent,denser, and longer lasting (80). Similarly, thermalpollution from the water coolant of power plants(nuclear and conventional) has, as one of its

most striking effects, the promotion of blue-greenalgal growth (164). It is probable that habitatssuitable for thermophilic organisms are going toincrease substantially.Most thermal habitats are aquatic, and the

source of heat is telluric for nearly all of these.Nevertheless, insolation can raise the temperaturesufficiently in a few situations and the self-heatingof organic materials (thermogenesis) may bringlocalized temperature to the point of ignition.Hot springs and their drainways provide themost abundant aquatic habitats for thermophilicblue-green algae. It will be these natural environ-ments and these blue-green algae that are dis-cussed through the remainder of this paper.However, the other (rarer) thermal habitats

require a brief description, even though little isknown of their organisms in situ. Temperatures inexcess of 50 C may be attained in a few trulyaquatic situations solely from insolation. Themonimolimnion (bottom waters) of a few smallmeromictic saline ponds and lakes may be heated(to >50 C) during the seasons of high lightintensity and retain fairly high temperaturesthroughout the year, since circulation of the bot-tom water is completely lacking (15, 95). Con-siderably higher temperatures (90 to 95 C) werereached in artificial solar ponds at the NegevInstitute in Israel (J. Schechter, personal com-

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TABLE 1. Organisms that live in hot springs attemperatures above 45 Ca

Organism Temp

Filamentous and unicellular bacteria[mainly heterotrophic ? (26, 29, 32) ] ...... 95 C

Acidophilic thiobacilli (34) ......... about 60 CPhotosynthetic (purple sulfur) bac-

teria (unpublished data) ........... 57 to 60 CPhotosynthetic blue-green algae (31, 38,

146) ............. .............. 74 CCyanidium [(acidophilic) (16, 34)]. .. 56 to 57 CFungi [(?) in hot springs (58)] ............ 60 CDiatoms (120, 169; R. P. Sheridan, personalcommunication) ......................... 50 C

Green algae (120, 139, 169) ............... 48 CCiliata (45) ............................ 50 CRotifera (45) ............................ 45 COstracoda [crustacea (45; Castenholz,

unpublished data)] ......... ........ 49 to 50 CAcarina [arachnida (45, 46)] ........ 50 to 51 CDiptera [larvae (28, 45, 46, 175) ] ............ 50 CColeoptera [larvae (45)] .................. 45 C

a Approximate highest constant temperaturesat which they occur or at which growth has beendemonstrated are indicated. Selected referenceson temperature limits are given for each group oforganisms. Except for Cyanidium and certainbacteria and fungi, all of the organisms occurmainly in waters ofpH above 6.

munication). In these, meromixis was establishedby artificially increasing the salt concentrationtowards the bottom. Small freshwater or salinepools in warm desert or tropical island environ-ments sometimes exceed 40 C (B. Whitton,personal communication), although air tempera-ture is also important in such circumstances. Theorigin of heat in the deep, hot brines of the RedSea is telluric, and only bacterial life has beenreported to date (174). Blue-green algae are theprincipal inhabitants of alternately moist and drycliffs in most mountainous regions of the world.The conspicuous dark streaks of blue-green algaemay be heated by sun and air to temperatures ofover 40 C (109), but it is not known thatgrowth occurs during such periods.Hot springs and most solar-heated aquatic

environments are composed of mineral waters,whereas the environment in self-heating piles ofvegetation is richly organic. Thermophilic fungi,sporeforming bacteria, and actinomycetes appearto be the main agents of thermogenesis in ac-cumulations of hay, compost, peat, and manure.A few of the fungi are active between 50 and60 C, whereas some of the bacteria are active toabout 70 C (5, 58, 77). Although self-heating vege-tation may be one of the main habitats for thegrowth of thermophilic spore-forming bacteria,

isolations of these organisms can easily be madefrom hot springs (134) and a variety of non-thermal habitats such as soil, sand, air, seawater,and snow (5).Although the source waters of hot springs are

usually quite low in dissolved organic compounds,there are obviously a number of heterotrophicbacteria associated with the photosynthetic blue-green algae on which they probably depend forcarbon-energy sources. Some of these are fila-mentous flexibacteria; others are nonsporeform-ing filaments or rods of other types (42). How-ever, in many hot springs in Yellowstone (26)and Oregon (unpublished data) there are bacteriagrowing above 90 C where no endogenous photo-synthetic organisms are present to supportheterotrophic growth. Either the rapid flow ofspring gyater (containing a minute quantity oforganic matter) is sufficient to sustain growth orthese bacteria are autotrophs of some type.

Blue-green algae are particularly concentratedin hot-spring waters with a pH of over 6 wherethey form conspicuous and often unialgal matlikecovers over submerged substrates. Since there isin many hot springs a surface effluent with athermal gradient ranging from supraoptimal toambient air temperature, specific differences ingrowth temperature optima may result in distinctspecies bands covering different portions of thegradient (Fig. 1). Since the component organismsdiffer in their amounts of chlorophyll-a, bilipro-teins, and carotenoid pigments, these bands maybe quite different in color, but ranging from a darkbrown to a yellow or rich green or blue-green.The orange or flesh color of many hot spring matsis often caused by compact masses of hetero-trophic, carotenoid-containing, filamentous bac-teria. Although these generally form gelatinouslayers (sometimes a few centimeters thick) under-neath a top cover of photosynthetic blue-greenalgae, they are sometimes exposed over wideareas (Fig. 1, 2). In some springs, pink to redlayers of photosynthetic purple sulfur bacteriaoccur directly under the algal cover.Because of the shallowness and the clarity of

most thermal waters, and the exposure of manyhot springs to high light intensities, various typesof "sun adaptation" have occurred in manythermophilic organisms. Laboratory and fieldstudies of this have added an interesting dimen-sion to studies of adaptation to high temperature.In addition, the organisms of many hot springshave adapted to high salinity or to high concentra-tions of certain ions. Being partly of magmaticorigin, most thermal waters differ considerablyin their chemistry from surface waters of lakesand streams (Table 2; 127, 183). Most alkalinehot springs contain between 1,000 and 2,000

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mg of total dissolved solids (TDS) per liter; somehave salinities higher than that of seawater. Eventhose of lower salinity may be notably enrichedin S (reduced), As, F, Mn, Fe, Al, Li, Si, or in

several other elements (Table 2). Although thegross chemistry of a large number of hot springsis known and there is some information on thenutrition of thermophilic cyanophytes, few

Fir. 1. Hunter's Hot Springs, Lakeview, Ore.: a portion of the main thermal stream. The arrow indicates thedirection offlow. The distance between the no. I and no. 3 markers is approximately 0.5 m. (1) Green-coloredSynechococcus cover, >54 C. (2) Dark brown 0. terebriformis cover, <54 C. (3) Pink-orange exposed undermaroffilamentous bacteria, < about 45 C, plus several cyanophytes. The water depth rangesfrom afew millimeters toabout 5 cm in midchannel. 30 March 1965.

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FIG. 2. Hunter's Hot Springs: a closer view of a mat, with a portion cut away. (1) Green-colored top cover ofSynechococcus. (2) Brown-colored 0. terebriformis top cover in various contraction patterns and overriding theSynechococcus. (3) The undermat of pink-orange filamentous bacteria. Total mat thickness is about 2 cm. Thearrow indicates the many separable layers of the undermat. The 5-cm scale marker is on the sandy substrate.Slow-moving water here was 1 cm or less deep over the top cover and about 53 C. 29 May 1967.

attempts have been made to correlate field andlaboratory data.One of the most interesting ecological features

of most hot springs is the great constancy oftemperature and chemistry of the water at thesource, generally unequaled by other aquaticsystems. In practice, this means that a thermo-philic alga has its optimal range of temperatureavailable in the thermal gradient at any season.Similarly, any chemical limitations that existexert their influence year round. This is quiteunlike the situation in typical streams and lakeswhere great seasonal fluctuations of temperatureand nutrient content may occur. In addition, therelatively few species of each trophic level greatlysimplify studies of species interactions and ofproductivity.

It is generally assumed that blue-green algaeevolved in the early Precambrian and were re-sponsible for the first significant increase in at-mospheric oxygen. Microfossils of cyanophyte-like filaments have recently been found in early,middle, and late Precambrian strata (171).Fossils of the late Precambrian (about 1 billion

years ago) resemble living members of the blue-green algae (151). Most of the fossil-rich Pre-cambrian material occurs as chert or as stromato-lites which could have originated in aquaticthermal environments similar to those of today.In particular, the description by Schopf (151) ofthe probable conditions of deposition of the latePrecambrian cyanophyte-rich material soundsvery similar to the manner in which blue-greenalgae are embedded by siliceous sinter in con-temporary hot springs of high or moderatesalinity. The question of whether the adaptationsof modem thermophiles to high temperatureoccurred in Precambrian or more recent times isunanswerable at present (see reference 5).The physical-chemical bases of the stability of

compounds and cellular structures at elevatedtemperature are still poorly understood. Most ofthe work has been with the thermophilic spore-forming bacteria, primarily Bacillus stearother-mophilus (e.g., 3, 12, 18, 20,25, 29, 34, 48, 50, 56,72, 86). It may be fallacious to extrapolate theseresults in toto to the blue-green algae which arephotosynthetic and from a different chemical

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environment. It has been suggested (29) that thephotosynthetic apparatus itself is the mosttemperature-sensitive component of the blue-green algae with the highest temperature toler-ances (i.e., 73 to 74 C). On the basis of very littleinformation, thermophilic blue-green algae appearto have the same functionally defined and struc-turally defined systems as other blue-green algae(8, 9, 69, 121, 132). Among thermophilic types,only Synechococcus lividus and Mastigocladuslaminosus have been described at the ultrastruc-tural level (69, 132), and even the gross aspects ofphotosynthesis, respiration, and other metabolicprocesses at high temperature are undescribedin most species (52, 133).The isolation of thermophilic variants of meso-

philic bacteria capable of growth at 55 C has beenconsistently successful with several spore-formingtypes (5). The ability to grow at 55 C has alsobeen genetically transformed to nonthermophilicbacteria by mesophilic forms, indicating that theoriginal thermolability may have resided in asingle system or component (21, 136). In view ofthis, it seems possible at least that all cell systemsof mesophilic procaryotes such as blue-greenalgae may be capable of operating at temperaturesof about 55 C with only minor genetic alterations.Perhaps 55 to 60 C represents a borderline, abovewhich cells are required to use very different andperhaps more cumbersome methods of assuringthermostability, methods which may prevent theoperation of metabolic systems at lower tem-peratures (22).

DISTRIBUTION OF THERMAL WATERSThe water of hot springs may discharge at

temperatures somewhat higher than the commonboiling temperature, although superheated watersare uncommon except in volcanic regions (17).Superheated steam issuing from vents may haveconsiderably higher temperatures (2, 17). A springmay discharge into a basin, thereby forming apool or lake [limnotherm (179)]. Some of thistype have no surface effluents. The pool itself(with or without an overflow) may be of a uni-form temperature if the water is well mixed by abottom source of gas or water. If less turbulent,water along the edges may cool slightly. Otherpools have stream outlets. Whether thermalstreams originate from pools or directly from theearth [rheotherm (179) ], they will display an entiretemperature gradient from source temperature toclose to ambient atmospheric temperature unlesstruncated by the flow entering another body ofwater or disappearing into the ground. Althoughmany springs have quite constant temperaturesat their sources over many years, others varyconsiderably in the course of years, days, or hours

(3, 17, 176). Flow rate may be less constant,particularly in geyser areas where the well-knownsurges of water cause great changes in drainwayflow and temperature over short periods of time.

Springs in which the water discharges at atemperature of over 45 C occur in every continentexcept Antarctica (Fig. 3). Most of these springareas have been mapped, listed, and referencedby Waring (181). Concentrated thermal activityis usually associated with Tertiary or Quaternaryvolcanism, where magma may still lie closer tothe surface (Fig. 3). Many groups of springs,however, are associated solely with the deeppenetration of water in fault zones. The greatestconcentrations of thermal springs occur in theYellowstone Plateau of North America, theNorth Island of New Zealand, Iceland, andJapan. Large numbers of hot springs are alsowidely scattered over most of the United Stateswest of the Great Plains, the line of the Andes,Italy, Algeria-Tunisia, Greece, Turkey, portionsof central Africa, India, central Asia, Indonesia,Melanesia, the Philippines, and Kamchatka (Fig.3). Australia has few hot springs and the wholeof northern Europe above 52° lat is devoid ofthermal waters above 40 C. A few hot springs lieon the Greenland coast 300 to 400 miles to thewest of Iceland, but all of North America eastof the Rocky Mountains lacks thermal water,except for the few springs in Virginia, NorthCarolina, and Arkansas (181).Although there is a great variety of chemical

types, there seem to be thermal waters on everycontinent that are essentially identical to someon almost every other continent. Any area ofintense thermal and volcanic activity usuallyhas the greatest variety of spring types. The majorareas of Yellowstone, Iceland, and New Zealandhave probably been active continuously since theearly Pleistocene. It is likely that throughout thebiological history of the earth thermal waterhas existed, although not in the same areas.Terrestrial volcanic belts are not connected byunderground water systems (13), although thishas been suggested.

Exposure to extremely high light intensity isalmost universally characteristic of hot springenvironments. Most of the extensive thermalareas are devoid of higher vegetation, or nearlyso. In addition, some of the best known areasare at high elevation or in regions of little over-cast. Since the water of most thermal pools isclear and the effluent streams are usually shallow,there is little radiation extinction. As a result, alarge number of thermophiles must adapt to highlight-level situations or else physically avoid thelight. In Yellowstone, (elevation 6,000 to 8,000ft) exposures to 0.6 cal per cm2 per min of radia-

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FIG. 3. Global distribution of thermal areas with springs discharging at temperatures over 45 C (0) and ofcontemporary or Recent volcanism (A). The regions with particularly high densities ofhot springs are indicated bysolid circles and arrows. Most of the information has been obtained from Waring (181). Capital letters refer toselected hot-spring areas where floristic studies of the component blue-green algae have been made. Major refer-ences are: A, Oregon (145); B, Yellowstone Park (59, 139); C, Iceland (23, 27, 54, 139, 153); D, France (73, 139);E, Hungary, Czechoslovakia, and Yugoslavia (57, 115, 116, 139); F, Greece and Israel (14, 62, 139); G, India(173); H, Indonesia (91); I, New Zealand (139); and J, Japan (71, 187). The reference to Nash (139) is includedfor several regions because of his comprehensive review of historical colections (before 1923) from European andNorth American hot springs.

tion in the visible range (400 to 700 nm) arecommon at midday in summer, whereas similarintensities occur in the hot-spring areas of easternOregon (altitude 1,500 to 4,800 ft). Such highintensities, with spectral distributions similar toovercast or sun-plus-sky radiation, have beenapproached in the laboratory with quartz iodinevapor lamps and appropriate liquid filters (110).

SOLUTES OF THERMAL WATERSThe solute concentration of hot springs may

vary greatly, even within a local area (2, 17, 61,181, 183). Thermal water with less than 500 mgof TDS per liter is uncommon. The medianconcentration for the earth's thermal springs isprobably only slightly less than 2,000 mg ofTDS per liter. This is considerably higher thanthe typical value (less than 150 mg/liter) forsurface waters of lakes and streams (127; Table2). Hot subsurface waters are rich in solutesbecause of their greater leaching and carryingcapacity. It is generally assumed that the mineralcontent of a hot spring remains quite constant.However, erratic or seasonal changes may occur,

probably varying with the increase or decreaseof precipitation in the watershed. Thus, theratios of certain elements and the total salinitymay change considerably in a short period (176).Nevertheless, the great majority of thermalsprings appear to have remarkably constantchemical composition and temperature at thepoint of emergence.

Springs of somewhat lower temperatures(<80 C), in which the waters are wholly ofrecent meteoric origin, generally have lowersalinities, often between 200 and 400 mg/liter(183; Table 2). Highly saline hot springs (>20,000mg of TDS per liter) occur in widely scatteredregions, associated with deep sediments orvolcanism [e.g., Utah, Israel, Turkey, Greece,Japan, Philippines, Indonesia (181, 183; Table2)]. In a highly saline spring on Reykjanes (SWIceland), with a salinity of over 45,000 mg ofTDS per liter, no algae were found (T. D. Brock,personal communication). The pH of this springis below 7, and the water may contain poisonousconcentrations of some elements (183). Blue-green algae are well known in highly saline lakes,

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and many also thrive in certain saline hot springs.The Tiberias Hot Springs of Israel, containingover 30,000 mg of TDS per liter with a pH from6.2 to 7.0 (183), have an abundant blue-greenflora which includes about 35 species, accordingto Dor (62). In Greece, hot springs with over15,000 mg of NaCl per liter also have severalblue-green species (14).

It is difficult to estimate to what extent thewater of thermal springs is of magmatic origin. Ingeneral, very hot waters with deep subterraneansources are partly derived from magmatic steam,although an ultimate meteoric source probablyaccounts for the major portion. Magmatic con-tributions are more significant in regions ofTertiary or Quaternary volcanism, where themagma is still relatively close to the surface.The water of a few springs, both thermal andnonthermal, is of a connate origin (i.e., trappedin sediment at the time of its deposition). Sincethe origins of thermal waters differ, so do therelative concentrations of numerous ions; thereis no simple method of classification.Somewhat condensing a general classification

of subsurface waters used by White, Hem, andWaring (183), we may list the common types ofthermal waters (based on origin and chemistry)as: (i) volcanic-Na, Cl-HCO3; (ii) volcanic-acid,S04; (iii) calcareous, travertine-depositing; (iv)meteoric-low salinity; (v) brine-Na-Ca, Cl.Examples of the chemical compositions of thesegeneral categories are given in Table 2.

(i) Volcanic-Na, Cl-HCO3 springs are neutralto highly alkaline waters which occur near Ter-tiary or more recent volcanic activity and arepartly derived from magmatic steam. The princi-pal solutes are sodium, chloride, and bicarbonate(or carbonate), and silicate (Table 2). The chlo-ride may be largely magmatic. Salinities aregenerally between 1,000 and 3,000 mg/liter ingeyser areas (i.e., high temperatures near theearth's surface), but may be higher elsewhere(183). Almost all the hot springs of the Yellow-stone Plateau (>3,000) have salinities in thisrange (2). They usually also contain over 200 mgof silica per liter, in which case opaline (siliceous)sinter may be deposited. The sulfate concentrationmay be high but it varies with the spring (Table2). This category of spring comprises the bulk ofthe alkaline hot springs of the earth.

(ii) Volcanic-acid S04 waters are closelyassociated with volcanic activity and surfacefumaroles (solfataras). Sulfuric acid, resulting inpH values between 1 and 4, originated mainlyfrom the oxidation of the abundant sulfides inthe subterranean water. Hydrochloric acid (mag-matic chloride) may contribute to the acidity insome waters (Table 2). Silica is generally quite

high ( > 120 mg/liter). Most volcanic acid springs(e.g., North America, Japan, Indonesia, NewZealand, Italy, Iceland) are inhabited by thephotoautotrophic eucaryote, Cyanidium calda-rium in the thermal range below 56 to 60 C (34,54). Highly saline acid springs are also common,but I am not aware of any reports of photo-autotrophs in such waters (Table 2). Salinities offrom 35,000 to 100,000 mg/liter are known inwestern parts of the Pacific circle of contemporaryvolcanism (183).

(iii) Calcareous, travertine-depositing springsare widely scattered, and are probably in contactwith limestone deposits (183). They are generallynear neutrality and have higher concentrationsof Ca, Mg, and HCO3 (relative to sodium andchloride) than neutral or alkaline springs of thevolcanic type (Table 2). Hydrogen sulfide may behigh in some springs of this type. They precipi-tate large amounts of calcite or aragonite trav-ertine. This material is also deposited by thecommon volcanic-Na, Cl-HCO3 springs, but notin such large amounts. The water is mostly, if notentirely, of meteoric origin. All travertine-deposit-ing springs are supersaturated with CO2. As aresult of the decrease in pressure upon surfacing,CO2 is evolved and CaCO3 is precipitated.

(iv) Meteoric, low-salinity (<500 mg of TDSper liter) thermal waters are characterized bytheir association with diastrophism rather thanvolcanism. Nitrogen is usually the major gas.Sodium or Ca is the dominant cation and Cl isusually low compared to HCO3 or even S04(Table 2). Temperatures are often lower thanthose of volcanic springs, although the circulationdepth may reach thousands of feet (183).

(v) Thermal brines (Na-Ca, Cl), resemblingoil-field brines, are not common but occur inmany geographical regions. Salinites range frombelow to well above that of sea water, and thepH is usually between 6 and 7 (Table 2). Thesewaters are thought to be connate (183). Highconcentrations of methane are common.Hot acid sulfur springs and acid brines provide

such restrictive conditions that blue-green algaeare either absent or very rare. However, mosthot springs, including the common neutral-alkaline types of the volcanic areas, are greatlyenriched with many ions rare in the surface watersof lakes and streams (Table 2). Some of theseelements are micronutrients, but are present atconcentrations that are probably toxic to manymicroorganisms. Some are lost from solutionsoon after surfacing, but this may depend on thetemperature, pH, and oxygen tension. Uzamasa(176) reported the following concentrations asextremes from a variety of volcanic hot springs

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in Japan (mg/liter): Al, 1,000; As, 5.1; Co, 2.19;Cu, 68; F, 16; Fe, 1,000; Pb, 2.6; Ni, 9.38; Mn,278; and Zn, 2.0. The greatest enrichment of themetallic ions is in the more acid waters (Table 2).The mean copper level was about 0.098 mg/literin the acid springs of Hokkaido, whereas concen-trations between 0.020 and 0.030 mg/liter oc-cuffed in the neutral-alkaline springs (176).Manganese is also higher in the acid springs, butwaters with a pH of up to 8.0 may still containlarge quantities. The mean for Mn-containingsprings in Japan is 2.3 mg/liter, an extremelyhigh level compared to lake or stream water.Dark oxides of Mn and Fe are characteristicdeposits in the drainways of many Na, Cl-HCO3springs. Aluminum and Fe are commonly atconcentrations over 10 mg/liter in volcanic-acidwaters, and extremes of over 1,000 and 10,000mg/liter, respectively, have been reported (183;Table 2). Arsenic may be high in acid or alkalinesprings, and values over 2.0 mg/liter are commonin Yellowstone (2) and in other alkaline hotsprings (183). In 174 neutral and alkaline springsin Japan, the mean concentration was 0.28 mg/liter. About twice as much occurred in 16 acidsprings (176). Fluoride is generally higher inmildly alkaline waters. For Japanese springs,the concentration was generally between 1.0and 2.0 mg/liter. Yellowstone springs are notedfor high fluoride concentrations, commonlybetween 15.0 and 20.0 mg/liter (2; Table 2).Bromide concentrations of over 1.0 mg/literare most common in more saline springs of eitherthe volcanic or connate types (Table 2). Sulfidesmay be common in springs of diverse types.

Tolerances of blue-green algae to most of theabove solutes have not been established (seereference 94). The high tolerances of some blue-green algae are evident from their growth in hotsprings containing large amounts of some of theseelements. Some of the obvious differences in floraand productivity among hot springs may resultin part from the different concentrations of"minor" elements. Perhaps the frequently con-spicuous absence of eucaryotic algae below about25 to 30 C in drainways of many hot springs(particularly in volcanic types) is explicable bytheir relatively high sensitivity to certain ions.

Practically all analyses of thermal springs havebeen made by geochemists or hydrologists.Consequently, analyses of biologically importantelements have been incomplete. The elementsknown to be micronutrients for blue-green algae(e.g., Cu, Co, Zn, Mn, Fe, B, Mo) are oftenomitted from the analyses unless their presencein large quantities is suspected. Except possiblyfor Mo and Co, these elements are usually more

abundant than in nonthermal surface waters(176)The macronutrients which are most often

limiting in lake waters are inorganic phosphateand combined nitrogen. Thermal waters, al-though of comparatively high salinity, did notacquire their salts in the same evaporative manneras surface waters of closed basins which areusually enriched in both, but particularly in P04.From the relatively few analyses available, itappears that NO3-N may be very low or lackingentirely in many hot spring sources (30, 52, 183).However, even low levels may not limit microbialgrowth if the flow rate is sufficiently high. Springswhich do not have detectable amounts of NO3at their sources may acquire higher levels down-stream, either from surface drainage or as a resultof N-fixation. In Hunter's Hot Springs of easternOregon, NO3 was undetectable at one source,but ranged from 0.042 to 0.142 mg of N03-Nper liter 20 m downstream (52). Brock also foundan increase in nitrate and ammonia in a down-stream direction (30). NO3 ion was found in 15%of the Japanese springs, presumably at the sources(176). In those, the mean level was about 0.2mg/liter. Much higher values may occur in moresaline thermal waters (183). Among the blue-green algae there is increasing evidence thatN-fixation is restricted to heterocyst-containingspecies, possibly to the heterocyst itself (76), butapparently exceptions may occur (186). Amongblue-green algae which grow above 54 C, onlyM. laminosus has heterocysts, and N-fixationhas been demonstrated in axenic cultures andcell-free preparations (78, 152). Steward has alsoshown in situ N-fixation in hot spring drainwaysdominated by Mastigocladus (168).Ammonium-N is generally more abundant

than NO3 or NO2, at least at the spring source,in both alkaline and acid types, but particularlyin the latter (183; Table 2). Ammonium ion wasfound in less than one-half of 860 hot springsanalyzed in Japan; in these it averaged about 1.6mg/liter-considerably higher than NO3 (176).The depressed upper temperature boundary forSynechococcus in some springs might be attributa-ble to source waters very low in all forms ofcombined nitrogen, although such correlationshave not been made.

Inorganic phosphate analyses of thermal watersources often show values much higher thanthose found in surface fresh waters (30, 127, 170,183; Table 2). Nevertheless, P04 was detectablein only 37% of the Japanese springs; in these itaveraged about 6.5 mg/liter-a very high valuewhen compared to surface waters (176). Mush-room Spring (Yellowstone), Brock's main re-

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search spring, contained 2.7 mg of P04/liter-also high (30). In some springs, phosphate valuesmay increase downstream if drainage or seepagefrom the shore occurs. In Hunter's Hot Springs, aconcentration of <0.040 mg of P04-P/liter atone source increased to over 0.100 mg of P04-P/liter (i.e., 0.300 mg of PG4 ion/liter) 20 mdownstream (52).

It is probably safe to state that most hot springs

TABLE 3. Blue green algae of thermal springs"

Order: Chroococcales Family: ChroococcaceaeSynechococcus lividus Copeland var. (31,

38, 146) ........................... 74 CA, B, (I?), J

"Type IV" (146) ......... ........... 72 C"Type III" (146) .......... .......... 67 C"Type II" (146) .......... ........... 62 C"Type I" (146) ...................... 58 C

Synechocystis elongatus Naeg. var. (14,91) ............................ (66-70C)

A, B, E, F, G, H, (I?), J

S. minervae Copeland var. (14, 145) ..... (60 C)A, B, F, (I?), J

S. aquatilis Sauvageau (62) ........... (45-50 C)(B?), E, F, G, H, J

Aphanocapsa thermalis (Kutz.) Brugger(14, 145)........................ (55+ C)

A, B, D, E, F, G, I, J

Order: Chamaesiphonales Family: PleurocapsaceaePleurocapsa [? minor Hansg. (145)]... (52-54 C)A, G, (H?), J

Order: Oscillatoriales Family: OscillatoriaceaeOscillatoria terebriformis Ag. "thermal-

red" (53) ......................... 53 CA, (J?) (B, D, E, F, G, and I-other

forms ?)0. animalis Ag. (73) ......... .......... (?55 C)A, D, E, F, G, J

0. amphibia Ag. (62) ........ ........... (57 C)B, D, E, F, G, J

0. geminata Menegh. (62, 145) .......... (55 C)A, B, D, E, F, G, H, I, J

o okenii Ag. (116)................... (60+ C)(B?), E, F, H, J

0. tenuis Ag. (unpublished data) ...... 45-47 CB, C, D, E, F, G, H, I, J

Spirulina sp. [incl. S. labyrinthiformisGom. (unpublished data)] ...... (55-60 C)

A, B, F, H, JPhormidium laminosum (Ag.)Gom.

(54) .......................... 57-(60) C[? incl. P. tenue (menegh.) Gom. and

P. valderianum (Delp.) Gom.]A, B, C, D, E, F, G, H, I, J

TABLE 3.-Continued

P. purpurascens (Kutz.) Gom. (un-published data) ........ ......... 46-47 C

B, C, D, I, JSymploca thermalis Kutz.

var. (unpublished data) .45-47 C or (?>50 C)(A?), C, D, F, J

Order: Nostocales Family: RivulariaceaeCalothrix sp.[incl. C. thermalis

(Schwabe) Hansg. (145)]...... (52-54 C)A, B, C, E, J

Order: Stigonematales Family: StigonemataceaeMastigocladus laminosus (Ag.) Cohn

(54, 154) ........................ 63-64 CSecond type (54) .......... .......... 57 C

A, B, C, D, E, F, G, H, I, J

a Selected list of species that are common in atleast some thermal areas. The upper limit withoutparentheses refers to the maximum constant tem-perature at which growth has been demonstrated(in the field or in culture); the upper limit withparentheses is based on certain observations ofoccurrence only. The references apply mainly totemperature limits. The distribution of specieswith respect to some of the thermal areas of theworld is indicated by the letters in capitals. Thelocations of these are shown in Fig. 3, and relevantreferences are given there.

have the high nutrient characteristics of eutrophicwaters, but these are often countered by poi-sonous concentrations of certain elements, lowpH, or extremely high salinity. It is probablethat many thermal streams cause eutrophicationin the bodies of water into which they drain.Harrington and Wright (96) have measured aconsiderably higher standing crop and primaryproductivity in the Firehole River below thethermal tributaries from the Upper Geyser Basin(Yellowstone) than above. Similarly, the chemis-try or temperature of hot spring effluents appar-ently causes increases in insect biomass in theGibbon River of Yellowstone (178). If poisonousconcentrations of some elements were present inthe thermal streams, these would be diluted tosubtoxic levels in major freshwater streams.The dissolved organic compounds present in

thermal streams have seldom been measured.Brock (30) found 2.4 mg of organic carbon/literboth in the source pool and at the foot of thedrainway of his main research spring. The con-centration of organic solutes and their identitiesshould be of considerable interest, since littleis known about the natural energy or carbonsources for bacteria that grow at very high tem-peratures [75 to 95 C (26)]. Brock and Freeze

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(42) have recently described a new aerobic,nonsporulating bacterium (Thermus aquaticus)isolated by high temperature (70 to 75 C) enrich-ment from many hot springs. This may representone of the important heterotrophs of hot-springmats.The undermat of carotenoid-containing fila-

mentous bacteria (presumably heterotrophs)must also depend on organic solutes either fromthe spring source or from excretions or lysatesof the photoautotrophs of the upper layers. Insome springs (e.g., Kah-nee-ta, Oregon) thicktop mats of orange filaments (about 1.5 /Amwide) predominate in summer over large areasat temperatures ranging from about 55 to 40 C.The specific energy and carbon sources for growthare unknown, but chlorophyll-a is lacking.

DISTRIBUTION OF SPECIES

Upper and Lower Temperature LimitsThe maximum constant temperature that will

sustain growth under natural conditions is stillunresolved for most cyanophytes, but 73 to 74 Cmay be the upper limit for the highest tempera-ture type [Synechococcus (Table 3)]. Brock (31)has demonstrated photoincorporation of 14C-carbonate in Synechecococcus populations attemperatures as high as 73 C in a Yellowstonehot spring. In complex hot-spring medium, S.lividus has a generation time of less than 24 hr at70 C (146). There are numerous reports of blue-green algae occurring at higher temperatures(see 27, 52, 59, 145, 179, 187). Copeland (59)listed five species above 75 C in Yellowstone,one as high as 85 C. Mann and Schlicting (131),more recently, reported similarly high tempera-ture ranges in Yellowstone. Brock (29, 32) andI (52) have examined some of the same springsand have concluded that the organisms inquestion are probably flexibacteria or othernonchlorophyllous filamentous procaryotes, notphotosynthetic cyanophytes. Although fluores-cence microscopy may not be capable of resolvingvery small quantities of pigment, Brock did notfind any indication of chlorophyll-a in the mate-rial collected (32). Organisms of these highertemperatures are sometimes brightly colored,presumably because of carotenoid pigments. Sofar, there has been no demonstration of photo-synthesis or growth of photoautotrophs over73 C. Most of the reports of higher tolerances(particularly numerous in the older literature)are based simply on presence; and the tempera-ture, even if measured at the precise site of thespecimen, was probably taken with a bulb ther-mometer in water, which may often stratify tothe extent of a 10-degree difference in a vertical

centimeter. On the other hand, Setchell (158)and Nash (139) reported blue-green algae inYellowstone at temperatures no higher thanabout 73 and 75 C, respectively, which agreesessentially with the observations by Brock andmyself. Kempner (113) measured 32p incorpora-tion into nucleic acids in a few adjacent springsin Yellowstone and found none above 73 C; heconcluded that this temperature represented theupper limit of life. Probably he was working withwaters that contained Synechococcus but notthe bacteria which grow at considerably highertemperatures ( >90 C) in nearby springs (26, 29).Some of the discrepancies referred to might

be resolved if the duration of exposure to thetemperature in question were known. Some ofthe reports of blue-green algae above probablemaximum constant growth temperatures mayrepresent short-term exposures to the highertemperature. For example, M. laminosus wasfound in Iceland in a particular spring that variedfrom about 60 to 70 C approximately every 20min (54). What is thought to be the same organ-ism was isolated from the spring, cultured, andcloned. In the laboratory, its maximum constantgrowth temperature was 63 to 64 C. However,it could withstand about 9 hr of exposure to 70 Cwithout noticeable mortality (54).

In North American springs, 74 C is about themaximum constant temperature at which oneor more species of Synechococcus occurs (Table3). Where these species do not occur or wherechemical conditions may be inhibitory, the uppertemperature boundary for photoautotrophs islower. In hot springs of the Cascade Range inOregon, 64 to 68 C is approximately the upperlimit; unicellular Synechococcus is usually theonly cyanophyte above 58 C. Farther to the eastin Oregon, and in Yellowstone, 73 to 74 C is theapparent boundary in alkaline springs. In acid-sulfur springs below pH 4 in Yellowstone, nocyanophytes occur; C. caldarium is the onlyphotoautotroph over 45 C and it extends to about57 C (16, 34). In Iceland, the photosyntheticupper limit in alkaline springs is about 63 C.This is a reflection of the absence of all species ofSynechococcus and the presence of M. laminosuswhich tolerates this temperature in many alkalinesprings throughout the world (Table 3; 54, 154).Similar reasons for depressed upper-temperatureborders probably apply to New Zealand (139)and many other thermal areas lacking high-temperature races of Synechococcus.The lower temperature limits for growth are

very poorly defined. Most of the thermophiliccyanophytes which have been examined in thisrespect grow poorly or not at all below 30 to 35 C.Certain races of Synechococcus will not grow

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100

<60

<o0

25 30 35 40 45 50 55 60 65 700C

FIG. 4. Growth and photosynthetic rates of blue-green algae at different temperatures, as percentage ofthe maximum rate. Symbols: 0, A. nidulans (117)growth rate with 0.5% C02 and 450 ft-c light (saturat-ing)-maximum rate was about 11 doublings per day;elongated circle, M. laminosus (105)-growth rate withline air and about 1,000 ft-c-maximum rate was lessthan 1.5 doublings per day; 0, 0. terebriformis-growth rate with air and saturating light intensities-maximum rate was about 4.8 doublings per day; 0,

S. lividus (145)-average growth rates of three clones,probably representing one race, no aeration, and about1,000 ft-c-maximum rate about 8.3 doublings perday; M, S. lividus (66)-growth rate with 2% CO2and 400 ft-c (below 45 C) and 1,500 ft-c at 45 to 55 C-maximum rate was 6.6 doublings per day; vertical barsrepresent photosynthetic rates (14C02 incorporation)ofa field population of Synechococcus collected at 58.5C (31).

below 55 to 50 C; others ceased growth between50 and 45 C, some between 40 and 35 and othersbelow 30 C (146). High-temperature clones ofM. laminosus from Iceland, however, are ableto grow over the range of 62 to 29 C and perhapslower (54). There are great differences amongcyanophytes, and unlike the upper temperaturelimit the real lower boundary is much harder todefine in field populations, since growth ratestaper off gradually below the optimum (Fig. 4).Upper temperature limits, on the other hand, areoften a few degrees or less above the optimum.The lower limit is flexible, depending on previousconditioning, light intensity, and other factors.Field populations taper off gradually in somecases, but competition with species better adaptedto lower temperatures may result in an abruptlower boundary in field population, which isoften several degrees higher than the real mini-mum temperature for growth.

Classification and Geographical DistributionIndividual species of thermophiles appear

generally to have very wide geographical distribu-tions. However, it is difficult to make categorical

statements about this, primarily because thespecific identification of blue-green algae is souncertain at present and few thermal areas havebeen examined exhaustively. Nevertheless, Ihave chosen several of the species of blue-greenalgae that are most frequently reported from hotsprings above 45 C. These are included in Table3 together with some general information on theirglobal distribution. On the world map (Fig. 3)the few hot-spring areas where reasonably com-plete floristic studies have been made are spottedwith capital letters, and references are includedin the figure legend. The reader should be warnedthat the distribution patterns implied by theinformation provided must be considered tenta-tive. My choice of binomials used in Table 3has been difficult because of conflicting systemsof classification. Gross morphology has beenused as the basis of all systems, yet blue-greenalgae present relatively few morphological charac-ters to the observer under the light microscope.After examining the classical compilation ofGeitler (90), one has the impression that differentnames were frequently given to specimens thatare only variants of the same genome. Drouet(64, 65) has simplified the matter with respectto the unicellular types and the oscillatoriaceaeby reducing large numbers of species to synonomyand by considering many morphological typesas ecophenes (nongenetic variants) of a fewspecies with high degrees of plasticity. However,Drouet's decisions were not based on observedvariations in clonal cultures, and, unless the sim-plicity which he implies has a genetic basis, the newsystem has no advantage over the old. The readershould be warned that the use of the Drouetsystem exclusively might lead to considerableconfusion. For example, the organism that I callS. lividus Copeland (old system) is apparentlythe same organism that Stockner (169) calledSchizothrix calcicola (Ag.) Gom. We are bothreferring to the unicellular rodlike organismthat divides in a single plane transverse to thelong axis and which grows at high temperaturesin hot springs of Oregon (146) and Yellowstone(29). In addition, the filamentous blue-green algawhich is primarily responsible for buildingcalcareous stromatolites in marine waters ofBermuda is called S. calcicola (159, 160). Drouetconsiders all of these and many other morpho-logical forms as ecophenes of S. calcicola, nor-mally a filamentous nonthermophilic organism(63, 64). This apparent problem cannot be re-solved at present because neither Drouet nor Iknows the degree of genetic homology betweenthe obligate thermophile of the hot springs andthe marine type. However, information of agenetic type, based on deoxyribonucleic acid

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hybridization studies, base composition, andother characteristics may soon revolutionize theclassification of blue-green algae (60, 68, 130). Atpresent, to avoid the confusion of organisms byoversimplification, it is important that the morenarrowly descriptive names be used, with Drouet'sname in addition, or vice versa.

Besides being the most ubiquitous and cosmo-politan thermophile, M. laminosus (Ag.) Cohnand its several "forms" is one of the easiestspecies of blue-green algae to recognize becauseof its unusually high degree of morphologicalcomplexity (27, 85, 154). Some of the morpho-logical forms may simply be developmentalphases or temperature-controlled manifestationsof the same organism. Preliminary experimentshave indicated, however, that there are at leasttwo genetic "races" with unique morphologiesand temperature tolerances (54). Although theliterature has not been exhaustively searched, itis evident that M. laminosus has been reportedin thermal waters (pH 5 to 9+) from almost allparts of the earth (154), with the possible excep-tion of some midoceanic islands [e.g., the Azores(37)] or recently deglaciated areas [e.g., westcoast of Greenland (27)]. In regions of many hotsprings, such as Yellowstone Park, some springsare dominated by Mastigocladus, whereas others(superficially similar chemically) are almost com-pletely dominated by Synechococcus. Throughoutthe world a "Mastigocladus Spring" appears tobe a recognizable entity. No one, however, hasfixed the reasons for this discrimination byhabitat. From the little that is known aboutMastigocladus, other thermophiles, and hotspring chemistry, it is tempting to suggest thatspring sources deficient in combined nitrogenwill allow the N-fixing Mastigocladus to dominatefrom about 60 to about 50 C, since neitherSynechococcus nor other thermophiles of thistemperature range are apparently capable ofnitrogen fixation (168). On the other hand,nitrate- or ammonium-rich springs may allowother thermophiles to compete successfullyagainst Mastigocladus, perhaps through morerapid growth rates. There are also reports thatMastigocladus is less tolerant of hydrogen sulfidethan other species in hot springs (see reference154). Other chemical constituents could also beinvolved in the exclusion of Mastigocladus fromsome springs.Most remote thermal areas have not been

investigated with regard to the species present.Phormidium laminosum is alleged to be a commoncompanion of the cosmopolitan Mastigocladus.However, specific differentiations in the narrow-trichome (<3 Am) members of the genus Phor-midium are so uncertain that one cannot be

confident that the same species occurs in all theselocalities.

Iceland probably has only about six or sevenblue-green algae capable of growing above 45 C,whereas hot waters of Europe, Asia, and NorthAmerica generally support at least twice thisnumber of species (54). Iceland has been activethermally throughout the last glaciation; buthot waters were probably covered by the ice untilabout 9,000 years ago. Relative youth and therarity of dispersal from other thermal areas areprobably major factors in accounting for theimpoverished condition of the Iceland flora (54).The range in chemistry of thermal waters inIceland is probably great enough to support anyof the earth's thermophilic blue-green species(17, 174, 181; Table 2). The species in Icelandall appear to be cosmopolitan.Some thermophilic species and varieties are

broadly endemic, but are absent from a fewgeographical regions (see Fig. 3). For example,the narrowly elongate species of Synechococcus(e.g., S. elongatus) which inhabit waters to about70 C in North America, Japan, and the easternMediterranean are absent from parts of westernEurope, Iceland, and possibly New Zealand. S.lividus, as such, has only been reported fromNorth America and Japan (71, 187). Narrowerdistribution patterns, such as that of Oscillatoriaterebriformis, probably occur, but may representthe distribution of acceptable habitats ratherthan actual momentary stages of geographicalspread. 0. terebriformis apparently occurs onlyin restricted hot-spring regions of western NorthAmerica. The same red-brown thermophilictype may also occur in Japan (155), but doubt-fully in other continents, although a number ofdifferent genetic entities fit the broad description(Agardh ex Gomont) bearing this binomial."Thermal-red" 0. terebriformis seems to occurin almost all alkaline springs of western andeastern Oregon, northern California, and Nevada(53). However, it has not been found by me orBrock in the diverse springs of YellowstoneNational Park, which are merely 600 miles fromabundant populations in Oregon. The 0. terebri-formis referred to by Stockner (169) in tepidsprings at Mt. Rainier is nonthermophilic, green,and with different cell dimensions in culture(unpublished data).

Problems of Survival and TransportThe capacity of thermophilic cyanophytes to

survive outside the normal habitat has been littlestudied. None of the extreme thermophiles pro-duce typical resting spores. Certain species, suchas M. laminosus and P. laminosum, are verytolerant of freezing and drying, whereas S. lividus

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is less so and 0. terebriformis is very labile. Thetransport of thermophiles over long distanceswould probably require drying or freezing, orboth, during the journey if the cells were locatedon the outside of the carrier. Proctor et al. (149)demonstrated that freshwater algae, other cypto-gams, and small invertebrates are transportedinternally by various water birds, but few blue-green algae were found (148).

In hot springs of equatorial or temperatelatitudes, a dormancy period is not required forwinter survival. In Yellowstone and Oregon hotsprings, midday light intensities in winter oftenexceed 0.2 cal per cm2 per min [400 to 700 nm(88)], and blue-green algal populations similarto those of summer are maintained in the steep-ened thermal gradient (39, 41, 52, 53, 145, 169).In Iceland, however, the 2 months of near totaldarkness in winter implies a dormant phase forthe cyanophytes, since they are probably obligatephotoautotrophs. The hot-spring mats disinte-grate and wash out (54, 153, 175). The stress ofprolonged darkness may also limit the numberof species to some extent, although species unableto withstand these conditions probably wouldnot easily survive transport to Iceland initially(54). 0. terebriformis ("thermal-red") of NorthAmerica may be such a labile species.One of the interesting questions is whether,

in the dispersal of thermophilic blue-green algae,the carrier must transport the inoculum fromone hot spring to another or whether sources ofinoculum are available elsewhere. Thermophilicspore-forming bacteria (e.g., B. stearothermoph-ilus) can be isolated from common soils aswell as from hot springs (31). Viable cells ofobligate blue-green algal thermophiles (at leastextreme types) may not be found far from thermalareas, but no one has really looked. I have occa-sionally grown blue-green algae out of samplesof common soil in the Willamette Valley (Ore-gon), with incubation at 45 C, but I have neverhad positive results at 55 C.

STUDIES OF NATURAL POPULATIONSMat Formation and Stability

In the thermal range (above 45 C) of nonacidhot springs, there occur gelatinous or calcareousmats of various colors, sometimes several centi-meters in thickness (Fig. 1, 2). The topmost layeris generally composed of photosynthetic blue-green algae. A variety of mat structures has beendescribed by Schwabe (155, 156), Brock (33),Stockner (170), and a few others. One of thestriking aspects of these mats is the variety ofbright green, brown, red, orange, and yellowcolors. These large mats accumulate without

apparent deterioration at high temperatures.These mats are largely composed of living micro-organisms, held together by abundant extra-cellular gelatinous materials. The cyanophytesusually occupy only the first few millimeters orless, and orange, yellow, red, or flesh-coloreafilamentous bacteria make up the rest. Althoughunicellular bacteria are also present, and becomeabundant in organic enrichments (31), carote-noid-containing flexibacteria may be the principalheterotrophs in many hot springs. The glidingmovements of these bacteria (and flexionalmovements of some) together with slight simi-larities in carotenoid pigments (1, 84) indicatethat they may be nonchlorophyllous homologuesof blue-green algae. The leakage or lytic productsof the primary producers are probably the mainenergy sources available to the bacteria, althoughthe source waters of some hot springs may containsome organic solutes derived ultimately fromsurface waters. The bacterial undermat is essen-tially the closed end of energy flow. This materialdoes not readily decompose except when washeddownstream into nonthermal waters.

It was suggested earlier by Schwabe (157) andby Brock (30) that the organic accumulation inthermal waters is due primarily to the generalabsence of herbivores above 43 to 48 C. Thismay be difficult to demonstrate directly, but whatwould appear to be the effect of animal grazingis quite apparent below certain temperatures innumerous hot springs. In many cases (Oregon,Iceland, and Yellowstone) the mat is corrosivelydissected by burrowing ephydrid (diptera) larvaewhich may reach large numbers below above45 C, although specific predators such as doli-chopodid flies are also present at some springs(28, 175). In some Oregon hot springs, a thermo-philic ostracod forms very dense populationsup to about 47 to 48 C without any apparentcompetitors or predators present. In such situa-tions, there is an abrupt change both in the algalspecies and total mass below the maximal tem-perature of these animals. There is generally nomat accumulation at all. Instead, there may beleathery nodules composed of the cyanophytesPleurocapsa sp. and Calothrix sp. These nodulesare able to withstand the apparent grazing pres-sure in flowing waters from about 47 to 35 C.Brock et al. (28) have demonstrated by directmeans, using "IC-labeled blue-green algae andbacteria, that ephydrid larvae and adults feedon the microorganisms of hot springs. I havefound that the thermophilic ostracods of Hunter'sHot Springs (Oregon) can rapidly decimate apopulation of 0. terebriformis. In these springs,the lower temperature edge of 0. terebriformismats (46 to 48 C) is probably determined by the

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feeding range of this herbivore rather than by agreatly decreased growth rate of the algae at thattemperature (see Fig. 4).The grazers of the mats at lower temperatures

probably aid in decomposition by ingestingmicroorganisms and depositing large amountsof partly changed fecal matter which may thenbe further decomposed by the microorganisms oflower temperatures. In areas worked over bydipteran larvae, ostracods, amphipods, and otheranimals, large amounts of characteristicallyflocculent fecal matter with associated micro-organisms may accumulate in pockets of quietwater. This is probably a reliable indicator ofheavy grazing activity.

In most alkaline hot springs, the blue-greenalgal cells are concentrated in the uppermost fewmillimeters of the mat, although filamentousbacteria may also be abundant there. Brock(33, 41) has demonstrated by 14CO2 incorporationthat photosynthesis is limited to the upper portionof the mat. For example, in a mat 1 to 2 cmthick essentially all activity was restricted to thetop 2 to 5 mm. Chlorophyll measurements madeby spectrophotometry or fluorescence microscopycorresponded with the incorporation results. Inthe top 5 mm of a typical core from 57 C in aYellowstone spring, unicellular Synechococcuswas essentially the only photoautotroph (41).This upper portion was subdivided into fourseparate layers for autoradiographic analysesafter photosynthetic incubation. In the uppermost1.2 to 1.3 mm, 95% of the cells were labeled,whereas only 35% (of fewer cells) in the lowestsublayer were labeled. Furthermore, the topcells averaged 4.8 silver g per cell and the lowestonly 1.3 per cell, indicating the obvious lightlimitations at depth. 0. terebriformis ("thermalred") is ubiquitous in Oregon springs belowabout 54 C (Fig. 1, 2). It forms deep red-brownmats 2 mm in thickness and so dense that com-monly only about 1% of the incident light istransmitted (53). Since these mats are motile andtend to override immotile Synechococcus surfacecovers, the latter become severely shaded. Even-tually the Synechococcus may disappear (53). InNorth America, the photosynthetic layer aboveabout 60 C is dominated by narrow-cell formsof Synechococcus resembling S. lividus Copeland.In Yellowstone, Oregon, and other hot springsthere are great variations in photosyntheticcovers below this temperature. The photosyn-thetic top layer may consist of densely bound orunbound populations of unicells (e.g., S. lividus,S. minervae), compact masses of gel-bound orfree filaments (Phormidium sp., Oscillatoria sp.),or tufts of branching filaments (e.g., M. lamino-sus). There are also situations (e.g., Kah-nee-ta,

Oregon) where a gel-like translucent orange layera few millimeters thick (composed of filamentous"bacteria") lies over a dark green layer of Syne-chococcus.Under nearly all types of photosynthetic covers

in nonacid springs lies the orange-to-flesh colored,heterotrophic undermat. In an early statement,Brock (30) suggested that the many layers oftypical hot-spring undermats represented theaccumulation of several years. Later, he realizedthat accumulation is considerably more rapidand that the distinct layering cannot be explainedeasily (39). Stockner (170), studying mainly tepidcalcareous springs at Mt. Rainier, came to asimilar realization.Brock has examined the manner in which mats

are formed (33). He replaced a portion of thehot-spring drainway (about 55 C) with a woodenchannel 6 inches wide and 7 ft long. An orangecover of undermat formed first and was about 2mm thick in 3 weeks. This was followed by a topcover of green Synechococcus which first appearedas small patches, eventually making a completecover in an additional 4 weeks. In hot springs ofeastern Oregon, I found that the blue-green algawas the first apparent colonizer on natural andplexiglass substrates. It is possible that in somestretches of thermal streams organic solutes areat such low concentrations that bacterial growthdepends on intimate contact with photoauto-trophs. During optimal periods, a new coveringof Synechococcus will coat plates (15 by 15 cm)in a few days, and a typical orange undermat willbe visible a few days later. The orange undermatincreases in thickness by growth and not byimmigration.

In eastern Oregon hot springs, new mat mate-rial on denuded substrates and submerged plexi-glass plates increased in thickness at a rate of1 to 2 mm per week. Commonly, this corre-sponded to values of about 1 to 2 g of organicmatter (ash-free dry weight) per m2 per day and5 to 10 mg of chlorophyll-a per m2 per day.These are not true growth rates since they excludelosses from decomposition, grazing, and washoutand include the possible addition of cells recruitedfrom upstream. Cells of S. lividus are almostbacterial in size (5 by 1.5 Mum) and settle out veryslowly even in unshaken flasks. The ability of adislodged cell to successfully recolonize will surelydiminish with increased flow rate or steepness ofthe thermal gradient. In a few cases, however,whole masses of detached mat from upstreambecame lodged on new substrate and obviouslyare added to the yield. The cause of the verydistinct layering of most mats is not understood(Fig. 2). It occurs in mats of flowing streams andnonflowing thermal pools. If the specific organ-

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isms of the various layers of undermat wereknown, the reasons for layering might be moreobvious. The steepness of the vertical oxygengradient in the mat should also have an importantinfluence on the type and growth rate of these.In the Oregon springs, there is often a vividpink-red layer of Chromatium (thiorhodaceae)immediately under 0. terebriformis at tempera-tures below about 57 to 60 C. If the Chromatiumwere active photosynthetically, the local condi-tions should be nearly anaerobic and at greaterdepths in the orange undermat also, at leastabove the temperature of extensive burrowingby dipteran larvae (about 45 C). Species ofSaprospira isolated from hot springs by Lewin(126) showed aerobic tendencies, but it is notknown whether these were important componentsof undermats. Similarly, the nonsporulatingbacterium T. aquaticus, isolated from Yellowstonesprings by incubation at 70 to 75 C, is an obligateaerobe (44).

Movements of Filaments and MatsIn most hot springs in Oregon [and apparently

in Japan also (155)], top layers of highly motile0. terebriformis are common (Fig. 1, 2, 53). 0.terebriformis forms dense mats, commonly over2 mm in thickness, which consist of free-movingbut intricately interwoven trichomes with littleor no gel binding them. The integrity of the matis maintained by the intricacy of the trichomeentanglement, and the position of the whole matin flowing water is probably maintained by thepenetration of free trichome ends into the sub-strate. Almost all other cyanophyte covers in hotsprings are held together and kept in place bythe gelatinous material formed by the blue-greenalgae, the filamentous bacteria, or both. 0.terebriformis mats, where found, dominate thethermal gradient from about 54 C down to about48 to 35 C, the lower edge apparently dependingon the abundance of the herbivorous ostracoddescribed earlier.During midday periods of high light intensity,

0. terebriformis mats may contract to only asmall fraction of their area under low light ordarkness, exposing covers of Synechococcus orthe orange undermat of filamentous bacteria.Mat-edge retraction rates may be as rapid as 100cm/hr. Trichomes may migrate downward intosome substrates en masse (53). With light reduc-tion they will rapidly return to the surface andspread out by gliding. Mass aggregations andcontractions were described in Oscillatoria (51,53), and a somewhat similar phenomenon in anonthermophilic Anabaena (180). Contractionof the mat edge in 0. terebriformis in responseto supraoptimal temperatures has also been

noted (53). In addition, the upper edge of theOscillatoria mat will advance upstream (by massgliding of trichomes), keeping pace with the 54 Cpoint when the temperature drops because ofwind or decreasing air temperature. Most of themovements of Oscillatoria described are probablyadaptive in that they enable the cells to maintainthemselves close to optimal light and temperatureconditions which change diurnally and seasonally.The mat of moving trichomes also allows littlecompetition within its optimal temperaturerange, since it is always able to form the topmostlayer. The common, more sedentary cyanophytecovers of hot springs have no adequate way tocope with diurnal fluctuations in temperatureand light except by physiological tolerance. Inmany cases, these types are not able to colonizeor grow rapidly enough in an upstream directionto keep pace with the steepening of the tempera-ture gradient with on-coming cold weather. As aconsequence, many thermal mats are left belowthe growth temperature range in winter when theybleach, disintegrate, and wash downstream (53).Over wintering is probably by vegetative ormetabolically inactive cells along the upstreamedges of the springs.

Measurements of Photosynthesis andGrowth

In 1966, Thomas D. and M. Louise Brockdetermined the standing crop of algae and othermaterial along a thermal gradient in alkaline hotsprings in Yellowstone and Iceland (35) by meas-urements of chlorophyll, ribonucleic acid, andprotein (36). In the Yellowstone drainway allthree materials were produced at maximal levelsat a temperature between 51 and 56 C. This wasalso the region in the gradient with the greatestmat accumulation as determined visually. Com-mon values for chlorophyll were 700 to 800 mg/m2 (35). In the single Icelandic spring examined,all three materials were maximal at a temperatureof about 48 C. It has already been mentionedthat the species may be different in the tworegions, and that the upper temperature limit ofphotoautotrophs in Iceland is only about 63 C.In both cases, however, the standing-crop maximawere above the temperature range of herbivoresor any other animals in the springs. Positions ofmaximal mat accumulation do not necessarilyfix the locations of the maximal photosyntheticor growth rates of blue-green algae or the hetero-trophs. Since the first paper, the Brocks havepublished several additional reports, most ofwhich are concerned with natural rates of photo-synthesis in Yellowstone hot springs (30-33, 38-41). Their principal experimental stream wasMushroom Spring in the White Creek drainage

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of the Lower Geyser Basin, a few hundred metersfrom Great Fountain Geyser. The temperaturefor the maximal crop of chlorophyll was againbetween 50 and 60 C (30). The incorporation of'4CO2 per unit area was also highest in the generalarea of maximal algal standing crop, as expected.Incorporation studies were performed with matcores set upright in horizontally positioned vials.These cores were generally incubated withNaH'4C03 in spring water at original tempera-tures for 1 hr. Although objections might beraised about possible stress conditions in the vial(170), the time is short, and there has been noindication yet that thermophilic blue-green algaehave a requirement of high-water velocity whichhas been attributed to some algae of streams(184).Brock (30) also plotted the photosynthetic

rates on a per chlorophyll basis to determinephotosynthetic efficiency. The greatest efficiencyoccurred at temperatures between 40 and 50 Cinstead of in the region of the greatest chlorophyll.It is often useful to express photosynthesis onsome basis other than area, but in this case, theuse of pigment as a basis must be distinguishedfrom the same expression in laboratory studies(see reference 167). Since intact cores of the algalmat were used in the experiments, all photo-synthetic cells were compacted in the upper fewmillimeters. Nevertheless, even in this shortvertical distance there is probably a steep dropin light intensity along with an increase in individ-ual cell chlorophyll (see references 44, 137). Thephotosynthetic rate at the top may have beenlight saturated (or even inhibited) at the sametime that cells at greater depth were severelylimited by dim light. Thus, the expression ofphotosynthetic rate per unit of chlorophyll inthe case of the field experiments differs in meaningfrom the same expression in laboratory studieswhere it would be assumed that all of the cellsare in suspension, equally exposed to light, andcontain the same amounts of chlorophyll. Inthe field, it should be expected that photosyn-thetic efficiency (of intact mats) will be less inregions of thickest algal mat (highest chlorophyllvalues) because of the greater degree of lightextinction.The photosynthetic characteristics of natural

algal mats have been illustrated in several studiesby the Brocks. In one experiment, Brock showedthat the photosynthetic rate per unit of chloro-phyll was dependent on light intensity throughoutthe course of a single day in summer (30). Thiscan be best explained by assuming that lightattenuates greatly within the algal layer, leavingmany cells photosynthetically unsaturated; thepercentage of cells saturated would depend on

the external light intensity. In a later study, theBrocks found that there was seldom any indica-tion of photosynthetic inhibition of algal matseven by the highest light intensities [about 0.56cal per cm2 per min, corrected for 400 to 700 nmby a factor of 0.4 (see reference 172)] unless thepopulations had been adapted to reduced lightintensities previously (41). Photosynthetic lightsaturation, however, occurred in some cases atvalues of about 0.3 cal per cm2 per min (myestimate for station I, 71 C, corrected for 400 to700 nm) which was about 50% of full middaysummer insolation. Brock (in Yellowstone) andI (in Oregon) have noted that mats of Synecho-coccus at the higher temperatures (i.e., 68 to 74 C)appear thinner than at lower temperatures. Thus,it is probable that a greater percentage of thecells in these thinner mats become light saturated,resulting in a relatively high photosyntheticefficiency expression. An increase in apparentefficiency near the upper temperature limit wasindeed found by Brock in one study (30). How-ever, the Synechococcus mats at these high tem-peratures may be thinner because of poorerphotosynthetic and growth rates for individuallight-saturated cells than at somewhat lowertemperatures. If this is true, the higher efficiencyexpression would be rather misleading in termsof physiology.

Additional information has come from twoexperiments in Oregon hot springs (unpublisheddata). In these springs, Synechococcus cells canbe easily removed by suction from the mat surfacesince they are not bound in place by mucilage asin many Yellowstone springs. Thus, dilute cellsuspensions with a minimal degree of self-shadingcan be prepared for photosynthetic studies,although the cells used may still have quite mixedlight histories. Using methods of 'IC incorpora-tion and analysis similar to those of Brock, Imeasured the photosynthetic rates of replicatesuspensions of Synechococcus for 30 min in twoOregon springs under different light intensities,simultaneously. In both experiments, the cellswere collected from a temperature of 60 C andincubated at the same temperature. The resultsshowed that photosynthetic saturation for thesecells occurred at about 0.07 to 0.08 cal per cm2per min (400 to 700 nm), approximately 1,500 to2,000 ft-c. Intensities of about 0.2 cal per cm2per min were still in the saturation range. How-ever, an intensity of about 0.4 depressed photo-synthesis to about 50% of the maximal rate.Although the conditions and the species strains

used might be different in Oregon and Yellow-stone, comparisons are of interest. Brock's finding(41) of photosynthetic saturation at about 0.3cal per cm2 per min in the thinner mat at 71 C

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would indicate that mat thickness is still a factorat intensities lower than this, if we accept theevidence that indicates individual cells are satu-rated at intensities as low as 0.07 cal per cm2 permin. Thicker mats at lower temperatures shouldprobably not show photosynthetic saturationeven under the brightest natural light.The Brocks (38) measured steady-state growth

rates of natural populations of Synechococcus bynovel methods. On a hard-bottom drainway ofsiliceous sinter in shallow thermal streams, thecomplete darkening of a Synechococcus matapparently stops growth completely, as wouldbe expected in the case of obligate photoauto-trophs. In the dark, washout of cells appears tohave continued at a normal rate until eventuallythe sinter was bare. The loss rate was exponential.The time when the population was 50% of theoriginal size (as determined by cell count) wasequated with the generation time required tomaintain the observed steady-state populationunder photic conditions. The estimates of genera-tion time were 40 hr at the 70 C station and 22 hrat the 72 C station. The rate of increase in cellnumbers can generally be equated safely with therate of increase in mass over a 24-hr period, in thecase of unicellular organisms. Assuming thatmass increase is possible only during the 15-hrlight period, I have shortened the Brock valuesfor generation times to 25 and 13.7 hr, respec-tively. This means that during the light period therate would be 1.0 and 1.7 mass doublings per day.These rates are very close to the maximal rate(about 2.0 mass doublings per day) obtained for ahigh temperature strain of Synechococcus in mylaboratory at 65 C, under continuous light and avariety of intensities, gas mixtures, and agitationrates (J. Meeks, unpublished data). Maximalrates at 70 C are consistently somewhat lowerand even more comparable with the Brock fieldvalues. The field method described should be veryuseful in springs or streams where flow rates areconstant and where unicellular algae predominate.Difficulties would arise if components of the algalmat were facultative dark heterotrophs, if dark-ness affected the tenacity of cell attachment to thesubstrate, or if washout was erratic and involvedchunks or tufts of mat rather than individual cells.

Stockner (170) has estimated primary produc-tion by measuring diurnal changes in dissolvedoxygen and also increases in organic matter ondenuded substrates in the drainways of tepid(37 C) springs at Mt. Rainier, Wash. He found acorrelation between blue-green algal productionand monthly light intensity-light durationmeans (gram calories per square centimeter perday) throughout most of the year, even summer.This may be expected, however, with or without

self-shading, since the number of productive hourschanges with day length, even though photo-synthesis may be saturated during many of thedaylight hours.

CULTIVATION OF THERMOPHILICCYANOPHYTES

There is little information on the cultivationof thermophilic blue-green algae. The followingsection will summarize primarily the methodswhich have been used in my laboratory. Mostmedia were originally designed for nonthermo-philic cyanophytes (7, 108) and later modifiedand used with thermophiles (67, 69, 105). Mediaand methods for unicellular types, including thesemithermophilic Anacystis nidulans, have beendeveloped by Van Baalen (177) and Allen (10). Amore detailed description of the culture tech-niques and of the culture collection of thermo-philic blue-green algae at the University ofOregon will be published in SchweizerischeZeitschrift fur Hydrologie during 1970 (55).

Medium and NutritionMany of the predominant photosynthetic blue-

green algae of hot springs grow vigorously inmineral medium. Species from hot springs ofmany types and a variety of geographical regionsare grown on defined medium D, with minormodifications (Table 4). Complex media withappropriate hot-spring waters have also beenused extensively for some species (Table 4; 146).In all cases, medium D has worked as well orbetter. The only organic compound in themedium is the metal chelator, nitrilotriaceticacid (NTA). Although NTA may be used as aC and N source by Escherichia coli (83), it isprobably not metabolized by cyanophytes. Themedium contains all of the nutrients known asrequirements for blue-green algae (92, 104, 142).Nitrate is probably the sole source of combinednitrogen in the unmodified medium, althoughNTA may conceivably provide nitrogen to someorganisms. M. laminosus and Calothrix sp. arethe only thermophilic blue-green algae for whichnitrogen fixation has been reported (78, 152, 168).Auxotrophy is known in relatively few cyano-phytes (104) and not at all in thermophilic species.Although photoassimilation of organic com-pounds, such as acetate, may be common incyanophytes (101, 143), only a few blue-greenalgae will grow heterotrophically in the dark (4,75, 104, 114, 122, 162). Among photosyntheticthermophiles, only the eucaryotic alga C. calda-rium is known to be a heterotroph (6, 16).The salinity of the great majority of neutral-

alkaline hot springs is much higher than that of

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TABLE 4. Composition of media and solutions

Medium DaDouble distilled water.............. 1,000 mlNitrilotriacetic acid..................... 0.1 gMiconutrient solution................... 0.5 mlFeC13 solutions......................... 1.0 mlCaSO4-2 H20........................... 0.06 gMgSO4.7 H20.......................... g0.10NaCl...................... 0.008 gKNO3.................................. 0.103 gNaNO3................................. 0.689 gNa2HPO4....................... 0.111 g

Micronutrient SolutionDistilled water ..................... 1,000 mlH2SO4 (concd).......................... 0.5 mlMnSO4 H20........... 2.28 gZnSO4 7H20 ... ........ 0.50 gH3B0 O.................................. 0.50 gCuSOc45H20........... 0.025 gNa2MoO4-2H20........... 0.025 gCoCl2 6H20.0...........O 045 g

Complex hot spring water mediumHot spring water ...... ..... 1,000 mlSoil extractc........................... 50 mlEthylenediaminetetraacetate-Fe(13% Fe)........... 0.005 g

MgSO4- 7H20 ... ........ 0.100 gKNO3.................................. 0.260 gK2HPPO4................................ 0.100 g

a Designed by R. P. Sheridan, medium D isprepared as a 10-fold concentrated stock andstored at 4 C unautoclaved. The pH is adjustedafter dilution (double-distilled water) to 8.2 with1 M NaOH. The final pH is 7.5 to 7.6 after the auto-claved medium has cooled and cleared completely.

b FeCla solution consists of 1,000 ml of distilledwater and 0.2905 g of FeCl3.

c Approximately 400 g of brown loam is auto-claved in 1,000 ml of water for 40 min. Mixedslurry is filtered through triple-layered Whatmanno. 1 paper while warm. Clear amber liquid results.

most surface waters. Nevertheless, some essentialnutrients such as combined nitrogen or phosphatemay be very low. However, none of the thermo-philes in culture seems to be sensitive to highlevels of these elements, unlike obligate oligo-trophs (see reference 82). Nevertheless, there aresome very common thermophilic blue-greens thathave not responded to my isolation techniques.These include the varieties of S. minervae andspecies of Spirulina.Medium D, with the addition of 0.1% tryptone

and 0.1% yeast extract, was also used successfully(46 to 79 C) for the culture of the nonsporulatingbacterium T. aquaticus, obtained from hot springsand hot tap water (42). Sporulating, thermophilicbacteria (e.g., B. stearothermophilus) from hotsprings and many other sources have been grown

in a variety of complex and defined organicculture media (5), as well as in relatively simplemedium composed of distilled water containing0.08% MgSO4.7 H20, 0.018% CaCl2-2 H20,0.05% KNO3, 0.28% NH4Cl, 4% glycerol, 0.6%sodium glycerophosphate, and 1% glucose mono-hydrate, which was adjusted to pH 6.8 andsterilized by filtration through a membrane filter(47).

Isolation and MaintenanceAlgal material collected from hot springs should

be quite dilute in sample vials containing springwater. Most keep very well, often for severalweeks or months, in the dark at a temperaturebetween 10 and 20 C. A temperature below 5 C isquite detrimental to some. However, even 20 to30 C in darkness will generally not harm samplesfor several days if kept dilute. Therefore, nospecial precautions are required for shipmentexcept padding and darkness.Normal D medium (Table 4) with a pH of 7.5

after autoclaving is used as almost a universalmedium for the isolation and growth of cyano-phytes from hot springs which range in pH from5 to over 9. Most thermophilic blue-green algae,as well as the great majority of mesophilic types,prefer an alkaline environment (104). The pH ofD medium rises as a result of algal growth, as itis essentially unbuffered. Medium D of pH 4 hasbeen used to isolate and cultivate the thermophiliceucaryote Cyanidium caldarium from acid-sulfatehot springs.

Collected material may be handled in severalways in the laboratory. Filamentous types whichare motile or have motile hormogonia (e.g.,Oscillatoria, Phormidium, Lyngbya, Symploca,Calothrix, Mastigocladus) will generally isolatethemselves by gliding away from the inoculumparticle in a few to several hours on D medium orCg-10 medium (177) solidified with 1.0 to 1.5%agar in plates and incubated at an appropriatetemperature. Blocks of agar with single trichomesmay then be picked off for inoculation into liquidor agar medium, in one step establishing cloneswhich may also be axenic. Some thicker tri-chomes (e.g., 0. princeps) penetrate into andglide well through agar medium, and there is aneven greater likelihood in such cases of freeing thetrichomes of bacteria. With some slow-movingspecies the original inoculation on agar mediummay be incubated for a few to several days. Thiswill eventually establish a peripheral mass oftrichomes for subsequent transfer to new agar.A gross inoculation on agar medium may also

be used for nonmotile cyanophytes, but not ifthere is an abundance of motile types in thesample. The almost ubiquitous M. laminosus and

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P. laminosum have often been separated by dilu-tion streaking. Unicellular forms, such as Syne-chococcus sp., may also be isolated in this manner,especially on the Cg-10 agar of Van Baalen (177),and probably on the unicellular medium of Allenas well (10). Mastigocladus, however, is some-what unique among thermophilic types in itsability to come up in agar stabs or in agar shakecultures. In the latter method the inoculum iswashed, blended, and diluted before dispersing inthe unsolidified medium at approximately 45 C.Axenic cultures of Mastigocladus have also beenobtained in this way. For the most commonunicellular thermophiles of North Americansprings, Synechococcus sp., isolations are verysimple for the narrow-form rod types (resemblingS. lividus) which are described as several speciesby Copeland (59). In springs in which Synecho-coccus is present, the bulk inoculation of collectedmaterial into liquid D medium usually results inthe growth of several blue-green algae. However,with time, narrow-form types of Synechococcusalmost always outgrow the others and often forma dense suspension bloom. One or more transfersof suspended cells generally results in an unialgalculture. Clones of these rods (1.2 to 2.2 ,um wide)may be established in different ways. For incuba-tion temperatures of 50 C or less, Synechococcussuspensions may be diluted by streaking on theCg-10 agar medium developed by Van Baalen(177) for single cells of A. nidulans. In my labora-tory this medium is modified only slightly: theglycylglycine buffer is reduced to 0.5 g/liter, 1%agar is used, and the pH is adjusted to 8.2 with 1M NaOH before autoclaving. Colonies fromsingle cells of Synechococcus develop on thismedium but generally not on D agar. Axeniccultures of Synechococcus may be obtained in thisway. Difficulties arise, however, with the hightemperature "races" of Synechococcus whichwill not grow below 50 to 55 C (146). These maybe easily enriched for by incubating the samplein liquid D medium at higher temperatures (e.g.,60 to 70 C). Material collected above 65 C in mosthot springs will contain narrow-form Synecho-coccus as the only viable photosynthetic organism.However, syneresis of agar or silica gel occursand the surface dries rapidly at temperaturesabove 55 C; streak and plating isolation methodswere unsuccessful. A tedious, but sometimeseffective, method of cloning and purification bymanual isolation has been used (124). It involvesthe spotting of minute drops of a dilute cellsuspension in a film of mineral oil spread on aglass plate or slide. After microscopic examina-tions, drops with single cells are sucked up with acapillary pipette and inoculated into liquidmedium. This has not worked well with high-

temperature Synechococcus, however, since cellsmay not generally survive either the pick-up bypipette or the transfer to fresh or conditionedmedium. The picking up of agar blocks withsingle cells that have been sprayed onto thesurface of a plate may prove less damaging. Wehave had some success by inoculating liquid Dmedium with a small amount of a very dilute cellsuspension. In new medium, the cells will settleout and small clonal colonies will appear on theflat bottom, but the flask must not be agitated atall during the incubation. Another method hasbeen to pull a very dilute cell suspension on to aWhatman GF/C glass-fiber filter, then to placethe whole disc in liquid D medium. Since thesefilters become quite fuzzy when submerged, smallcolonies may be removed by picking off a sub-merged tuft with a forceps.Many larger cells or filaments (e.g., Mastigo-

cladus) may be cloned and also purified by aseries of dilution washes when manually carriedthrough a series of depression wells, each withsterile medium. A pulled glass capillary tube,point, or hook may be used under a dissectingmicroscope for the transfers. Agar plates may alsobe conveniently used as a working surface fordragging out and isolating single cells or fila-ments from mixed samples (see reference 125).

Axenic cultures of some cyanophytes may beestablished by treatment with ultraviolet radiation(104). With some of thermophilic types, however,it appears that the several orange-colored bacteriaoften present are less sensitive to ultraviolettreatment than the cyanophyte.

Stock cultures here are usually maintained in125-ml Erlenmeyer flasks with 80 ml of liquid Dmedium or in test tubes with 15 to 20 ml ofmedium. Incubation temperatures vary with thespecies. However, the bulk of the cultures arekept at 45 C in constant-temperature water baths.The flasks are usually stoppered with nonab-sorbent cotton, but at higher temperatures ( >55C) Morton metal closures with "fingers" areused with Bellco (Bellco Glass, Inc., Vineland,N.J.) flasks to slow evaporation. Growth isconsiderably slower in the vessels with closures;in some cases this is preferred. The light intensityat the flask level is kept at about 200 to 500 ft-c(continuous light from Coolwhite fluorescentlamps). The intensity at the culture level in thetubes is often only 50 to 100 ft-c. Both flask andtube cultures, particularly when aged, shouldnever be exposed to high intensities (>600 ft-c)for more than a couple of hours; death of all cellsmay occur. The flask cultures require transferringabout every 4 to 5 weeks, whereas the slowergrowing tube cultures are generally transferredevery 8 to 9 weeks. Agar surfaces are used in flasks

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for maintaining axenic cultures, but agar slants intubes dry too quickly to be used at these tem-peratures. Cotton plugs can best be protectedfrom dust contamination by capping these with a*soft porous tissue (e.g., Kimwipes) held with arubber band. Nonporous caps cause wetting of thecotton plugs at these temperatures. Air-circulatedincubators at 45 C and above result in excessiveevaporation; consequently, water baths are pre-ferred.Many thermophilic cyanophytes will tolerate

freezing or drying for a long time. Slow freezing(5 to 10 min) to -20 C works with several com-mon thermophiles. Holm-Hansen (102) foundthis method somewhat preferable to rapid freez-ing, although the blue-greens used were non-thermophilic types from either polar or temperateregions. All culture strains of the ubiquitous M.iaminosus and P. laminosum tolerated slow freez-ing and storage at -20 C in my laboratory with-out any apparent cell death, although onlyMastigocladus was stored as long as 3 months.During some trials, various strains of S. fividustolerated freezing, and some cells were viableafter 1 to 2 years of storage, but this method hasnot worked in every case. 0. terebriformis, acommon thermophile in Oregon, will not tolerateslow or rapid freezing. Freeze-drying may beeffective with the more labile types, but it has notyet been tried with these thermophiles (seereference 103).Dimethyl sulfoxide, glycerol, or other cryo-

protective agents might also enhance survival ofcells at low temperatures, but they have not beentested with thermophilic blue-green algae.

Dilute cultures and samples of several speciesin liquid medium or spring water have been storedin complete darkness at about 15 C and havesurvived well for over 4 months. Such a simplemethod deserves more intensive trials.M. laminosus will also tolerate dryness at about

25 C (with or without a desiccant) for at least afew months with little loss of viability. Narrow-form Synechococcus sp. survived, at most, a fewdays of drying, and 0. terebriformis does notsurvive at all.

Rates of Growth, Photosynthesis, and Respir-ation in Culture

The remark by Marry (133) that thermal algaegenerally grow slowly was based on the fewstudies available at that time. Since 1961, how-ever, there have been enough additional reports ofthermal growth rates to indicate that such ageneralization is incorrect. A short generationtime (about 2 hr) was known in the case of A.nidulans, which has a maximal growth rate at 41 Cbut tolerated a temperature no higher than 45 C

(117). Dyer and Gafford (66, 67) reported ninedoublings per day for S. lividus at 52 C, which isalmost comparable to A. nidulans, Chlorellapyrenoidosa TX 7-11-05 at 39 C (166), andChlamydomonas mundana at 33 C (129). Similarly,several isolates of S. lividus from eastern Oregonsprings grew at rates ranging from 8 to 10 dou-blings per day from 45 to 55 C (146). Both A.nidulans and S. lividus are small unicellular rodsranging in cell width from about 1.2 to 2.2 Mm.Chlorella and Chlamydomonas are also small, butmost species or strains do not exceed 3 to 4doublings per day (106). Over a fairly wide rangeof phylogenetic groups there appears to be aninverse correlation between cell size and growthrate (see reference 185), but no constant rela-tionship between high growth rates and hightemperature, although rates of over 3.0 doublingsper day appear to be confined to temperaturesover 20 C (106). Brock (29) cites somewhatshorter generation times for higher temperaturespecies of bacteria than for lower temperaturetypes. In thermophilic Synechococcus, the highesttemperature races have reduced growth rates(146). Whether the observed limit is geneticallycontrolled or whether other problems, such asnutrient or CO2 availability, are more manifestat higher temperatures in still unresolved. Currentstudies at 60, 65, and 70 C indicate that themaximal growth rate under a variety of aeratedconditions is only about 2.0 doublings per day(J. Meeks, unpublished data). Estimates ofgrowth rates of Synechococcus near 70 C innatural populations gave comparable values(38; see earlier section on field results).Holton (105) estimated a growth rate of about

1.5 doublings per day for M. laminosus and con-sidered it quite low. It is lower than many blue-green algae but is nevertheless comparable to alarge number of eucaryotes (106). Nonthermo-philic filamentous cyanophytes such as Schizo-thrix calcicola, Anabaena variabilis, Nostocmuscorum, and Tolypothrix tenuis reachedmaximal growth rates (3.0 to 4.0 doublings perday) in culture above 30 C (106). The filamentousbut undifferentiated 0. terebriformis (trichomewidth, 4 to 6 Mum) approached 5 doublings perday under optimal conditions from 45 to 53 C(Fig. 4). 0. princeps, with the great trichomewidth of 20 to 30 + ;Am, has not surpassed 0.5doublings per day in culture at its 40 C optimum(L. Halfen, unpublished data). Even though in-formation on growth rates of the blue-green algaeis still scanty, it seems that the variation amongspecies is as great in thermophiles as in mesophilesand that no clear trends are evident.

Photosynthetic rates in thermophiles, asmeasured by H'4CO3 or 14CO3 incorporation,

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have been measured mainly in field populations.However, neither these nor the few laboratorymeasurements have distinguished thermophiliccyanophytes from the mesophiles or from the bulkof the eucaryotic algae (49, 67, 118, 137, 147, 161).Cyanophytes respire aerobically in the dark,

but sometimes at rates lower than those ofeucaryotic algae (24, 43, 104, 105, 118, 182).In the thermophilic M. laminosus, Holton (105)found a dark endogenous respiratory rate [julitersof 02 uptake per mg (dry weight) per hr] of 8.0for actively growing material at 45 C, as com-pared to 7.5 for active cells of A. nidulans at 39 C(118). Biggins (24) found somewhat lower rates(4 to 6 Mliters of 02 per mg per hr) for A. nidukdnsat 25 C (a suboptimal temperature). The existenceof peculiarities in respiration, oxidative phos-phorylation, and other aspects of "dark metab-olism" in blue-green algae has been suggested andargued recently by several persons (19, 24, 104,107, 123, 144, 165).

RESPONSES TO TEMPERATURE ANDLIGHT INTENSITY

Optimal Temperature and Light IntensityThe optimal conditions for growth may seldom

be described in terms of a single factor. Neverthe-less, this has been attempted, using temperaturealone, nutrient concentration alone, light alone,etc. Most of these factors influence the effect ofone or more of the others. Thus, experimentalprocedure can become very complex when oneattempts to consider several factors simul-taneously. Few workers have done so. The tem-perature optimum for growth or photosynthesisof microalgae may be dramatically or subtlyinfluenced by light intensity, day length, nutrientconcentration and availability, CO2 concentra-tion, pH, constancy of the several factors, andpreconditioning. In a series of field experiments,Brock (31) found that the maximal rate of 14Cphotoincorporation in natural populations ofSynechococcus (mainly) taken from severaldifferent temperature regimes was very close tothe temperature from which they had beencollected (Fig. 4). For example, sample coreswere collected at 58.5 C in the thermal stream.These were equilibrated for about 5 min in asmany as 10 new temperatures (about 23 to 73 C)in glass vials, followed by a 1-hr incubation withthe isotope added. The photosynthetic populationat 58.5 C in this Yellowstone spring consistedalmost entirely of Synechococcus sp. Thus,although genetic adaptation was avoided by theshort time involved, not enough time for physi-ological acclimation was allowed. Maximal 14Cassimilation occurred at the temperature of col-

lection only; rates decreased with temperaturebelow and above 58.5 C (Fig. 4). In the fewthermophilic cyanophytes that have been studiedin the laboratory, maximal growth or photo-synthetic rates occurred over a considerablygreater temperature range (Fig. 4). For thisreason, I suspect that the sharpness of Brock'soptimum temperature peaks is not real and thatthe species involved have the potential to photo-synthesize and grow at maximal rates over aconsiderably wider range, given time for ac-climation. Little is known about the resilience ofthermophilic algae subjected to temperaturechanges. Several strains or races of S. lividuswere able to tolerate abrupt changes within theirgrowth range or below it without apparentdamage (145, 146). Lag periods in the growth ofsome clones usually varied from a few to severalhours, the time being somewhat proportional tothe temperature difference involved. A displace-ment from a suboptimal temperature (e.g., 30 to40 C in clone 53) to a higher temperature in theoptimal range (45 to 55 C) generally resulted in ashorter lag ( <10 hr) than when the direction wasreversed (15 to 72 hr). The ability to withstandconsiderable changes in temperature is also knownin obligate thermophilic bacteria, but rapid dis-placements to higher temperatures are often fatalto facultative thermophiles (see reference 21).To have a wide range in growth temperaturewould have considerable adaptive value in athermal stream.The loss of the ability to grow over the original

full temperature range occurs commonly in somebacteria (74, 141). This is often a loss of low-temperature ability rather than a change in upperlimit. Allen (6), using C. caldarium, and Lowen-stein (128), using Mastigocladus, reported thatthe maintenance of cultures for long periodsbelow the optimal growth temperature resulted ina lowering of the upper tolerance limit and of theoptimum. Allen had mistakenly assumed that theoriginal temperature maximum for Cyanidiumwas well above 55 C. Subsequent studies haveshown that 55 C is very close to the upper limitfor this organism in the laboratory (16) or field(34). In the case of Mastigocladus, the "main-tenance" consisted of cold storage at a tempera-ture of 5 to 8 C, well below the growth minimumof 25 C (128). Holton (105), while "cleaning up"his crude material from Laird Hot Springs, B.C.,which contained M. laminosus, found there was aloss in tolerance to high temperature. In isolationsfrom Iceland hot springs in 1968, I found that byfirst enriching for the high temperature race inculture medium at over 60 C followed by cloningwith agar shake or streak methods at 45 C, nocultures were produced that were sensitive to the

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highest temperatures normally attained by thisorganism [about 62 to 63 C (54)].Peary (145) found no deadaptation in one

clone of S. lividus after 8 months of growth at30 C, a distinctly sub-optimal temperature. Atthat time it responded with the same growth ratesfrom 55 to 30 C as the normal culture which hadbeen maintained at 50 C. There were also nodifferences in lag periods, except at 30 C where the30 C-grown material reached exponential phasefirst. The maintenance of the same clone of 0.terebriformis at 29 to 30 and 44 to 45 C for 3years has resulted in initially better growth ratesat 30 C for the 30 C stocks than for the 45 Cstocks (unpublished data). However, the 30 Cstock was still able to grow well at 50 C. Similarly,Bunning and Herdtle (49) reported higher photo-synthetic rates at 20 to 30 C in 0. geminata thathad been grown in that range than for thosegrown at the optimum of 40 C. The photosyn-thetic rates at 40 C were similar for both cultures.M. laminosus was collected (by T. D. Brock) from58 C water in Iceland in 1966 but was isolated andcultivated in my laboratory at 45 C for 2.5 years.Upon raising the temperature to 62 C, the culturecontinued to grow and appeared similar toothers cultured at 62 C (54). Another strain whichhad a lower initial maximum (about 57 C) re-tained this characteristic. Therefore, although lossof growth potential at the upper or lower end ofthe temperature range may be real in some cases,it is not general.

In some thermophilic blue-greens, the growthtemperature optimum is a broad plateau, but it isusually skewed toward the upper end of the fulltemperature range (Fig. 4). In the case of 0.terebriformis, the maximal growth rate was re-tained to about one degree from the normallethal temperature of 54 C. The curve for Masti-gocladus growth, which is broad and well gradedat both ends, was sharpened at the upper end withCO2 increase (105).

In thermophilic blue-green algae, the growthtemperature optimum or range may be modifiedby other external factors; light intensity has oneof the most profound effects. Most of the curvesin Fig. 4 reflect changes in temperature under thesame light intensity. In 0. terebriformis, however,growth rates were measured after full acclimationat each temperature under a light intensitysaturating for that temperature. An air (CO2)supply sufficient to saturate growth for each lightintensity and temperature was also used. In thisspecies, the growth-saturating light intensityvaried from about 1,200 ft-c (Coolwhite fluo-rescence) at 45 C to about 350 ft-c at 31 C, andinhibitory effects of bright light were felt at about5,000 and 1,500 ft-c, respectively. The maximum

growth rate at 31 C was only about 20% of the45 to 53 C value (Fig. 4). Therefore, althoughacclimation periods and lags differed, maximumsustained growth rates could be attained at about1,200 ft-c for the entire temperature rangementioned. Photosynthetic saturation occurredat about 800 ft-c in one Yellowstone strain ofSynechoccus lividus at 45 C, when the cells werepregrown at that intensity (161). Similar valuesoften apply to Chlorella, but the preceding lightintensity for growth has a great influence on thesaturation intensity for photosynthesis (167).A. nidulans appears to have a similarly highsaturation intensity even when pregrown at muchlower levels (118, 137). In natural hot spring mats,evidence has already been presented that suggeststhat many cells making up the algal layer mightnot become light saturated even at very high mid-day intensities because of self-shading.The high light intensities reaching the algal

surface in many thermal waters should result in"sun-adapted" forms. Typical manifestations of"sun adaptation" are the lack of inhibition bybright light and saturation of photosynthesis andgrowth by a relatively high intensity (see refer-ences 44, 167). Some of Brock's field results indi-cate that there is little or no inhibition of photo-synthesis in the compact mats of Synechococcusat the highest natural light intensities. My resultswith suspensions of Synechococcus cells demon-strate that inhibition at the level of an individualcell does occur, but probably only above about5,000 ft-c [about 0.2 cal per cm2 per min (400 to700 nm)]. With the high intensity values forsaturation and inhibition, S. lividus qualifies wellas a sun-adapted organism. In contrast, someblue-green algae are inhibited by light intensitieslower than 500 ft-c (44).

Effects of Light and Temperatureon Pigmentation

Except for thermophilic organisms that mayavoid bright light by growing at some depth in themicrobial mat or by responding phototacticallyand gliding away from it (53), most thermophilicmicroorganisms are probably able to tolerateexposures by making physiological adjustmentswhich usually involve changes in chlorophyll andcarotenoid pigments.Some cyanophytes, at least, seem to contain

carotenoids which are not efficiently coupled tophotosynthesis (70, 93, 111, 140). Goedheer (93)has evidence to suggest that energy transfer froma-carotene to chlorophyll occurs in photosystemI in blue-green algae and that light energy ab-sorbed by xanthophylls is not transferred tochlorophyll. Some of the carotenoids in most (orall) microorganisms function in protecting living

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cell constituents from photooxidations sensitizedby chlorophyll and other pigments (81, 119). Thishas been demonstrated in photosynthetic bacteria(163), heterotrophic bacteria (135), and eucary-otic photoautotrophs (see references 119, 138).However, the physicochemical nature of themolecular interactions which constitute protec-tion are still poorly understood (81).At this point, there is no reason to suggest that

there is any consistent difference in the pigmentsof thermophilic and nonthermophilic blue-greenalgae (however, see reference 22). Phycoerythrinis absent from S. lividus, M. laminosus, andapparently all blue-green algae which grow attemperatures above 60 C. It is also lacking in themesophiles A. nidulans, Gloeocapsa alpicola, andP. luridwn (44). Phycoerythrin occurs in additionto phycocyanin in thermophilic S. minervae(maximum, 60 C), 0. terebriformis "thermal-red" (maximum, 53 C), and in numerous meso-philes (unpublished data). The identity of some ofthe carotenoids in blue-green algae is still un-certain and there is variation among species(97-100). Myxoxanthophyll, echinenone, and j3-carotene, however, appear to be almost uni-versally present. It has been known for many yearsthat the amount of chlorophyll decreases andthat the carotenoid content increases (at leastrelative to chlorophyll) when blue-green algae aregrown under bright light. Complementary chro-matic adaptations (87, 112) have also beendescribed in blue-green algae, but these areprobably over ridden by high intensity responsesin most hot-spring environments.

Sargent (150) found that high intensity whitelight or light of various broad spectral bandscaused a change in Gloeocapsa montana from darkblue-green to yellow, and that lowering the in-tensity reversed the process. The color changescould be explained solely by a chlorophyll de-crease over a few cell generations. A. nidulans(137) and Phonnidium persicinum (44) respondedsimilarly. In Anacystis, phycocyanin paralleledchlorophyll in its decline with increasing light in-tensity (no phycoerythrin), and in P. persicinumphycoerythrin was even more sensitive to brightlight than chlorophyll (no phycocyanin present).In the field, the greening and yellowing of thermalSynechococcus populations is controlled by theamount of shading, at least in summer (41, 52). InS. lividus (41) and A. nidulans (8) grown at highlight intensities, the decrease in chlorophyll wasapparently accompanied by a decrease in thephotosynthetic thylakoids. In the light intensityrange for saturation, photosynthesis is no longerlimited by the amount of light received but by themaximum rate of the "dark" reactions. A con-tinuing decline in the bulk, light-harvesting pig-

ments as light intensities are increased should beexpected on the grounds that the excess pigmentwould not be required. A decrease in the amountof photochemical machinery during bright-lightexposure and the maintenance of relatively largeamounts of carotenoids at the thylakoids shouldbe of value in reducing photodynamic lesions.At a constant temperature and light intensity it

was characteristic of the chlorophyll and carot-enoid content of cells (pigment per unit of dryweight) to remain fairly constant during theexponential phase of growth in 0. terebriformis,with self-shading kept to a minimum (unpublisheddata). At 45 C, the mean chlorophyll and carot-enoid values for the exponential growth phasevaried inversely with the light intensity. From 200to 4,000 ft-c, there was approximately an eight-fold decrease in the characteristic amounts ofchlorophyll per cell during exponential growth,but the decrease in total carotenoids was onlyabout twofold. The ratio of carotenoid to chloro-phyll (optical density at 472 nm to optical densityat 665 nm, in methanol) was about 0.5 to 0.7when grown in dim light in contrast to about 2.5when grown at 4,000 ft-c and higher. The de-crease in chlorophyll with ascending light in-tensities was accompanied by a large decrease inphycoerythrin. Thus, 0. terebriformis t-r (ther-mal-red) was a deep red-brown color at low lightintensity, due primarily to the large amounts ofphycoerythrin, whereas at very high intensity itwas a pale ochre-yellow with carotenoids pre-dominant. Growth of 0. terebriformis in turbu-lent aerated cultures at high light intensities re-sulted in approximately 100 times more myxo-xanthophyll than at the low intensities, althoughthe other carotenoids were reduced.At optimal temperatures (45 to 53 C), abrupt

upward or downward shifts in light intensity(involving a range of up to 4,000 ft-c) caused nodistress in the cultures. An upward shift to brightlight resulted in an immediate increase in growthrate (dry weight increase). Carotenoid and chloro-phyll pigments adjusted to new levels and newratios in about 12 to 24 hr after the shift. There-after they generally increased at the same rateas total dry weight (unpublished data).

Growth and Survival at SuboptimalTemperaures

When 0. terebriformis cultures were shiftedfrom an optimal to a suboptimal temperature,the initial pigment content appeared to be acritical quantity (unpublished data). If the cultureswere preconditioned by growth at 45 C at a highlight intensity (e.g., >2,000 ft-c) there was no lagin growth when the culture was shifted to 31 C

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at 350, 860, or 1,500 ft-c, but the rate dropped tothat typical of light saturation at 31 C (Fig. 4).However, if cells were preconditioned at a lowlight intensity (350 ft-c) at 45 C and then shiftedto 800 or 1,500 ft-c at 31 C, a lag of 6 to 7 daysoccurred, followed by a gradual attainment ofexponential growth. The preconditioning underdim light at 45 C resulted in low carotenoid-chlorophyll ratios (about 0.5); in bright light,values of 1.0-2.5 were obtained. In the laggingcultures, exponential decreases in chlorophylloccurred for the period of the lag. The carotenoiddrop was not as great proportionately. Only afterhigh pigment ratios were finally attained at thelower temperature did exponential growth begin.A shift to 28 C and 300 ft-c (after growing at 45 Cand 200 ft-c) resulted in an initial increase in dryweight for a period of about 48 hr while chloro-phyll was simultaneously declining. This wasfollowed by a decrease in dry weight until aboutthe sixth day when almost all of the cells hadlysed. An initial increase as described was notuncommon in downward shifts of temperature,even to temperatures below the growth minimum.Cultures grown in bright light (about 5,000 ft-c),however, withstood the shift to 28 C without alag and grew at slow exponential rates for severaldays. Even with properly preconditioned cells,28 C seemed to be about the lowest temperatureat which aerated cultures could be maintained.Cells grown at 45 C and 5,000 ft-c were alsoshifted to 27 and 25 C. These grew for a limitedtime at very low rates (0.2 to 0.4 doublings perday at 27 C), but eventually diminished to zero.The highest growth rates of separate culturesdiffered from each other under identical condi-tions at 27 or 25 C, apparently reflecting differ-ences in initial pigment content.The probable protection provided by carot-

enoids against detrimental lesions was alsodemonstrated in light at much lower temperatureswith 0. terebriformis. At 13 or 18 C, the deathrate may be reduced considerably if cells have aninitially high carotenoid to chlorophyll ratio. At700 ft-c, the lysis of almost the entire cell popula-tion occurred in 24 hr or less at 13 or 18 C whenthe initial pigment ratios were below 1.0. Withhigher ratios (about 2.5), no significant lysisoccurred for about 3 to 4 days, and during thefirst 24 hr there was even a slight increase in dryweight. The death rate in darkness at similartemperatures was independent ofpigment content,and there were large populations of viable cellsafter 2 weeks at temperatures ranging from 10 to25 C.

These results indicate that the most deleteriouseffect of exposure to suboptimal or subminimaltemperatures in the light may be the photooxida-

tion of essential cellular (thylakoidal?) com-ponents which include chlorophyll, the sensitizingpigment. In the absence of syntheses, or with veryslow synthetic rates, these photodynamic lesionswould eventually cause the death of the cell andits subsequent lysis. Even when initially highcarotenoid levels apparently afford completephotoprotection at first, eventually dispropor-tionate changes in pigments will occur at tempera-tures where synthetic rates are too slow to main-tain balances.

In nature, it would seem that 0. terebriformis(and perhaps other blue-green algae, too) wouldlack the resilience to withstand abrupt increasesin light intensity when stranded at suboptimaltemperatures, unless pigment balances werefavorable when the temperature change occurred.Analyses of the pigments of natural populationsof 0. terebriformis at various seasons haveshown, however, that carotenoid-chlorophyllratios are very much lower than for comparablelight intensity regimes in the laboratory (un-published data). Nevertheless, this should beexpected, since this and other species of blue-green algae in the springs occur as dense matswith much self-shading. In the case of the Oscil-latoria mats, the trichomes are so motile that noindividual is likely to maintain an exposed posi-tion for very long. Within an optimal temperaturerange, great increases in light intensity should betolerated even by the most exposed trichomes.However, it is probable that this resilience wouldbe lost if a trichome were washed downstream tolower temperatures or if the thermal gradientsteepened suddenly. Continued growth or sur-vival would probably depend in large part on thepigment content of the cells at the time.

ACKNOWLEDGMMENTSI gratefully acknowledge aid from grant GB-7103 from the

National Science Foundation for some of the work reported here.

LITERATURE CITED1. Aasen, A., and S. L. Jensen. 1966. The carotenoids of Flexi-

bacteria. III. The structures of flexixanthin and deoxy-flexixanthin. Acta Chem. Scand. 20:1970-1988.

2. Allen, E. T., and A. L. Day. 1935. Hot springs of the Yellow-stone National Park. Carnegie Inst. Wash. Publ. 466.

3. Allen, M. B. 1950. The dynamic nature of thermophily. J.Gen. Physiol. 33:205-214.

4. Allen, M. B. 1952. The cultivation of Myxophyceae. Arch.Mikrobiol. 17:34-53.

5. Allen, M. B. 1953. The thermophilic aerobic sporeformingbacteria. Bacteriol. Rev. 17:125-173.

6. Allen, M. B. 1959. Studies with Cyanidium caldarium, ananomalously pigmented chlorophyte. Arch. Mikrobiol.32:270-277.

7. Allen, M. B., and D. I. Amon. 1955. Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen-fixation byAnabaena cylindrica Lemm. Plant Physiol. 30:366-372.

8. Allen, M. M. 1968. Photosynthetic membrane system inAnacystis nidulans. J. Bacteriol. 96:836-841.

500 BACTERIOL. REV.


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br.asm.org/

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nloaded from



9. Allen, M. M. 1968. Ultrastructure of the cell wall and celldivision of unicellular blue-green algae. J. Bacteriol.96:842-852.

10. Allen, M. M. 1968. Simple conditions for growth of unicellu-lar blue-green algae on plates. J. Phycol. 4:1-4.

11. Allen, M. M., and R. Y. Stanier. 1968. Selective isolation ofblue-green algae from water and soil. J. Gen. Microbiol.51:203-209.

12. Amelunxen, R. E. 1966. Crystallization of thermostableglyceraldehyde-3-phosphate dehydrogenase from Bacillusstearothermophilus. Biochim. Biophys. Acta 122:175-181.

13. Amsbury, D. L. 1968. Terrestrial volcanic belts. Science161:298.

14. Anagnostidis, K. 1961. Untersuchungen Uber die Cyano.phyceen einiger Thermen in Griechenland (In Greek,German summary and discussion). Inst. Syst. Bot. Pflan-zengeogr., University of Thessaloniki.

15. Anderson, G. C. 1958. Some limnological features of a

shallow saline meromictic lake. Limnol. Oceanogr. 3:259-270.

16. Ascione, R., W. Southwick, and J. R. Fresco. 1966. Labora-tory culturing of a thermophilic alga at high temperature.Science 153:752-755.

17. Barth, T. F. W. 1950. Volcanic geology, hot springs, andgeysers of Iceland. Carnegie Inst. Wash. Publ. 587.

18. Bassel, A., and L. L. Campbell. 1969. Surface structure ofBacillus stearothermophilus ribosomes. J. Bacteriol. 98:811-815.

19. Batterton, J. C., and C. Van Baalen. 1968. Phosphorusdeficiency and the phosphate uptake in the blue-green alga.4nacystis nidulans. Can. J. Microbiol. 14:341-348.

20. Bauman, A. J., and P. G. Simmonds. 1969. Fatty acids andpolar lipids of extremely thermophilic filamentous bac-terial masses from two Yellowstone hot springs. J. Bac-teriol. 98:528-531.

21. Bausum, H. T., and T. S. Matney. 1965. Boundary betweenbacterial mesophilism and thermophilism. J. Bacteriol.90:50-53.

22. Berns, D. S., and E. Scott. 1966. Protein aggregation in a

thermophilic protein. Phycocyanin from Synechococcuslividus. Biochemistry 5:1528-1533.

23. Biebl, R., and E. Kusel-Fetzmann. 1966. Beobachtungenliber das Vorkommen von Algen an Thermalstandortenauf Island. Osterreich. Bot. Zeit. 113:408-423.

24. Biggins, J. 1969. Respiration in blue-green algae. 3. Bacteriol.99:570-575.

25. Bodman, H., and N. E. Welker. 1969. Isolation of sphero-plast membranes and stability of spheroplasts of Bacillusstearothermophilus. J. Bacteriol. 97:924-935.

26. Bott, T. L., and T. D. Brock. 1969. Bacterial growth ratesat temperatures above 90 C in Yellowstone hot springs.Science 164:1411-1412.

27. Boye Petersen, J. 1923. The fresh-water Cyanophyceae ofIceland, p. 251-324. In The botany of Iceland. Vol. II.

Copenhagen.28. Brock, M. L., R. G. Wiegert, and T. D. Brock. 1969. Feeding

by Paracoenia and Ephydra (Diptera: Ephydridae) on themicroorganisms of hot springs. Ecology 50:192-200.

29. Brock, T. D. 1967. Life at high temperatures. Science 158:1012-1019.

30. Brock, T. D. 1967. Relationship between standing crop andprimary productivity along a hot spring thermal gradient.Ecology 48:566-571.

31. Brock, T. D. 1967. Micro-organisms adapted to high tem-peratures. Nature 214:882-885.

32. Brock, T. D. 1968. Taxonomic confusion concerning certainfilamentous blue-green algae. J. Phycol. 4:178-179.

33. Brock, T. D. Vertical zonation in hot spring algal mats.Phycologia, in press.

34. Brock, T. D. 1969. Microbial growth under extreme condi-tions, p. 15-41. In P. Meadow and S. J. Pirt (ed.), Micro-bial growth, Symposia of the Society for General Micro-biology XIX.

35. Brock, T. D., and M. L. Brock. 1966. Temperature optimafor algal development in Yellowstone and Iceland hotsprings. Nature 209:733-734.

36. Brock, T. D., and M. L. Brock. 1967. The measurement ofchlorophyll, primary productivity, photophosphorylation,and macromolecules in benthic algal mats. Limnol.Oceanogr. 12:600-605.

37. Brock, T. D., and M. L. Brock. 1967. The hot springs of theFurnas Valley, Azores. Inter. Rev. Ges. Hydrobiol. 52:545-558.

38. Brock, T. D., and M. L. Brock. 1968. Measurement of steady-state growth rates of a thermophilic alga directly in nature.J. Bacteriol. 95:811-815.

39. Brock, T. D., and M. L. Brock. 1969. Recovery of a hotspring community from a catastrophe. J. Phycol. 5:75-77.

40. Brock, T. D., and M. L. Brock. 1969. The fate in nature ofphotosynthetically assimilated 14C in a blue-green alga.Limnol. Oceanogr. 14:604-607.

41. Brock, T. D., and M. L. Brock. 1969. Effect of light intensityon photosynthesis by thermal algae adapted to naturaland reduced sunlight. Limnol. Oceanogr. 14:334-341.

42. Brock, T. D., and H. Freeze. 1969. Thermus aquaticus gen.

n. and sp. n., a nonsporulating extreme thermophile. J.Bacteriol. 98:289-297.

43. Brown, A. H., and G. C. Webster. 1953. The influence oflight on the respiration of the blue-green alga Anabaena.Amer. J. Bot. 40:753-758.

44. Brown, T. E., and F. L. Richardson. 1968. The effect ofgrowth environment on the physiology of algae: lightintensity. J. Phycol. 4:38-54.

45. Brues, C. T. 1928. Studies of the fauna of hot springs in thewestern United States and the biology of thermophilousanimals. Proc. Amer. Acad. Arts Sci. 63:139-228.

46. Brues, C. T. 1932. Further studies on the fauna of NorthAmerican hot springs. Proc. Amer. Acad. Arts Sci. 67:186-303.

47. Bubela, B. 1968. Effect of temperature on growth character-istics of Bacillus stearothermophilus. Aust. J. Biol. Sci.21:439-445.

48. Bubela, B., and E. S. Holdsworth. 1966. Protein synthesis inBacillus stearothermophilus. Biochim. Biophys. Acta 123:376-389.

49. Bunning, E., and H. Herdtle. 1946. Physiologische Unter-suchungen an thermophilen Blaualgen. Z. Naturforsch.1:93-99.

50. Card, G. L., C. E. Georgi, and W. E. Militzer. 1969. Phos-pholipids from Bacillus stearothermophilus. J. Bacteriol.97:186-192.

51. Castenholz, R. W. 1967. Aggregation in a thermophilicOscillatoria. Nature 215:1285-1286.

52. Castenholz, R. W. 1967. Environmental requirements ofthermophilic blue-green algae, p. 55-79. In A. F. Bartsch(ed.), Environmental requirements of blue-green algae.Federal Water Pollution Control Administration, Corval-lis, Ore.

53. Castenholz, R. W. 1968. The behavior of Oscillatoria terebri-formis in hot springs. J. Phycol. 4:132-139.

54. Castenholz, R. W. 1969. The thermophilic cyanophytes ofIceland and the upper temperature limit. J. Phycol. 5:350-358.

55. Castenholz, R. W. 1970. Laboratory culture of thermophiliccyanophytes. Schweiz. Z. Hydrol., in press.

56. Chapman, D. 1967. The effect of heat on membranes andmembrane constituents, p. 123-146. In A. H. Rose (ed.),Thermobiology. Academic Press Inc., New York.

57. Claus, G. 1959. Studien uber die Algenvegetation der Ther-malquelle von Bilkkzhk, Nordungarn. Arch. Hydrobiol.55:1-29.

58. Cooney, D. G. and R. Emerson. 1964. Thermophilic fungi.W. H. Freeman, San Francisco.

59. Copeland, J. J. 1936. Yellowstone thermal Myxophyceae.Ann. N.Y. Acad. Sci. 36:1-229.

60. Craig, L. W., C. K Leach, and N. G. Carr. 1969. Studies with

VOL. 33, 1969 501


http://mm

br.asm.org/

Dow

nloaded from


502 CASTENHOLZ

deoxyribonucleic acid from blue-green algae. Arch. Mikro-biol. 65:218-227.

61. Day, A. L., and E. T. Allen. 1925. The volcanic activity andhot springs of Lassen Peak. Carnegie Inst. Wash. Publ.360.

62. Dor, I. 1967. Algues des sources thermales de Tiberiade. SeaFish Res. Sta. Haifa Bull. 48:3-29.

63. Drouet, F. 1963. Ecophenes of Schizothrix calcicola. Proc.Acad. Natur. Sci. Philadelphia 115:261-281.

64. Drouet, F. 1968. Revision of the classification of the Oscilla-toriaceae. Acad. Nat. Sci. Philadelphia Monogr. 15.

65. Drouet, F., and W. A. Daily. 1956. Revision of the coccoidMyxophyceae. Butler Univ. Bot. Stud. 12:1-218.

66. Dyer, D. L., and R. D. Gafford. 1961. Some characteristicsof a thermophilic blue-green alga. Science 134:616-617.

67. Dyer, D. L., and R. D. Gafford. 1963. The use of Synechococ-cus lividus in photogas exchangers, p. 87-107. In Develop-ments in industrial microbiology, vol. 3.

68. Edelman, M., D. Swinton, J. A. Schiff, H. T. Epstein, andB. Zeldin. 1967. Deoxyribonucleic acid of the blue-greenalgae (Cyanophyta). Bacteriol. Rev. 31:315-331.

69. Edwards, M. R., D. S. Berns, W. C. Ghiorse, and S. C. Holt.1968. Ultrastructure of the thermophilic blue-green alga,Synechococcus lividus Copeland. J. Phycol. 4:283-298.

70. Emerson, R., and C. M. Lewis. 1942. The photosyntheticefficiency of phycocyanin in Chroococcus, and the problemof carotenoid participation in photosynthesis. J. Gen.Physiol. 25:579-595.

71. Emoto, Y. 1962. A bibliography of the thermal flora ofJapan. J. Jap. Bot. 37:89-94, 119-124, 129-138.

72. Epstein, I., and N. Grossowicz. 1969. Intracellular proteinbreakdown in a thermophile. J. Bacteriol. 99:418-421.

73. Famin, M. A. 1933. Action de la temperature sur les vigd-taux. Rev. Gen. Botan. 45:574-595, 655-682.

74. Farrell, J., and A. H. Rose. 1967. Temperature effects onmicroorganisms, p. 147-218. In A. H. Rose (ed.), Thermo-biology. Academic Press Inc., New York.

75. Fay, P. 1965. Heterotrophy and nitrogen fixation in Chloro-gloea fritschil. J. Gen. Microbiol. 39:11-20.

76. Fay, P., W. D. P. Stewart, A. E. Walsby, and G. E. Fogg.1968. Is the heterocyst the site of nitrogen fixation in blue-green algae? Nature 220:810-812.

77. Festenstein, G. N., J. Lacey, F. A. Skinner. P. A. Jenkins,and J. Pepys. 1965. Self-heating of hay and grain in Dewarflasks and the development of farmer's lung antigens. J.Gen. Microbiol. 41:389-407.

78. Fogg, G. E. 1951. Studies on nitrogen fixation by blue-greenalgae. II. Nitrogen fixation by Mastigocladus laminosusCohn. J. Exp. Bot. 2:117-120.

79. Fogg, G. E. 1956. The comparative physiology and biochem-istry of the blue-green algae. Bacteriol. Rev. 20:148-165.

80. Fogg, G. E. 1969. The physiology of an algal nuisance. Proc.Roy. Soc. Ser. B Biol. Sci. 173:175-189.

81. Foote, C. S. 1968. Mechanisms of photosensitized oxidation.Science 162:963-969.

82. Forsberg, C. 1965. Nutritional studies of Chara in axenicculture. Physiol. Plant. 18:275-290.

83. Forsberg, C., and G. Lindqvist. 1967. On biological degrada-tion of nitrilotriacetate (NTA). Life Sci. 6:1961-1962.

84. Fox, D. L., and R. A. Lewin. 1963. A preliminary study ofthe carotenoids of some flexibacteria. Can. J. Microbiol.9:753-768.

85. Fremy, P. 1936. Remarques sur la morphologie et la biologie'e l'Hapalosiphon laminosus Hansg. Ann. Protistologie5:175-200.

86. Friedman, S. M. 1968. Protein-synthesizing machinery ofthermophilic bacteria. Bacteriol. Rev. 32:27-38.

87. Fujita, Y., and A. Hattori. 1960. Effect of chromatic lightson phycobilin formation in a blue-green alga Tolypoihrlxtenuis. Plant Cell Physiol. 1:293-303.

88. Gates, D. M. 1961. Winter thermal radiation studies inYellowstone Park. Science 134:32-35.

BACrERIOL. REV.

89. Gates, D. M. 1962. Energy exchange in the biosphere.Harper & Row, New York.

90. Geitler, L. 1932. Cyanophyceae. In L. Rabenhorst's Krypto-gamenflora von Deutschland, Osterreich und der Schweiz,vol. 14.

91. Geitler, L., and F. Ruttner. 1935. Die Cyanophyceen derDeutschen Limnologischen Sunda-Expedition, ihreMorphologie, Systematik und (Okologie. Arch. Hydrobiol.Suppl. 14:308-483; 1936, Suppl. 14:553-715.

92. Gerloff, G. C. 1968. The comparative boson nutrition ofseveral green and blue-green algae. Physiol. Plant. 21:369-377.

93. Goedheer, J. C. 1969. Energy transfer from carotenoids tochlorophyll in blue-green, red and green algae and green-ing bean leaves. Biochim. Biophys. Acta 172:252-265.

94. Goldman, C. R. 1965. Micronutrient limiting factors andtheir detection in natural phytoplankton populations.Mem. Ist. Ital. Idrobiol. 18 (Suppl):121-135.

95. Goldman, C. R., D. T. Mason, and J. E. Hobbie. 1967. TwoAntarctic desert lakes. Limnol. Oceanogr. 12:295-310.

96. Harrington, D., and J. C. Wright. 1966. Primary productionin the Firehold River, Yellowstone National Park, Wyom-ing (Abstr.). Amer. Soc. Limnol. Oceanogr. 29th AnnualMeeting, p. 12.

97. Healey, F. P. 1968. The carotenoids of four blue-green algae.J. Phycol. 4:126-129.

98. Hertzberg, S., and S. L. Jensen. 1966. The carotenoids ofblue-green algae. I. The carotenoids of Oscillatoria rube-scens and an Arthrospira sp. Phytochemistry. 5:557-563.

99. Hertzberg, S., and S. L. Jensen. 1966. The carotenoids ofblue-green algae. II. The carotenoids of Aphanizomenonflos-aquae. Phytochemistry 5:565-570.

100. Hertzberg, S., and S. L. Jensen. 1967. The carotenoids ofblue-green algae. IIL. A comparative study of mutato-chrome and flavacin. Phytochemistry 6:1119-1126.

101. Hoare, D. S., S. L. Hoare, and R. B. Moore. 1967. Thephotoassimilation of organic compounds by autotrophicblue-green algae. J. Gen. Microbiol. 49:351-370.

102. Holm-Hansen, 0. 1963. Viability of blue-green and greenalgae after freezing. Physiol. Plant. 16:530-540.

103. Holm-Hansen, 0. 1964. Viability of lyophilized algae. Can.J. Bot. 42:127-137.

104. Holm-Hansen, 0. 1968. Ecology, physiology, and biochemis-try of blue-green algae. Annu. Rev. Microbiol. 22:47-70.

105. Holton, R. W. 1962. Isolation, growth, and respiration of a

thermophilic blue-green alga. Amer. J. Bot. 49:1-6.106. Hoogenhout, H., and J. Amesz. 1965. Growth rates ofphoto-

synthetic micro-organisms in laboratory cultures. Arch.Mikrobiol. 50:10-25.

107. Horton, A. A. 1968. NADH oxidase in blue-green algae.Biochem. Biophys. Res. Commun. 32:839.

108. Hughes, E. O., P. R. Gorham, and A. Zehnder. 1958. Toxic-ity of a unialgal culture of Microcystis aeruginosa. Can. J.Microbiol. 4:225-236.

109. Jaag, 0. 1945. Untersuchungen uiber die Vegetation undBiologie der Algen des nackten Gesteins in den Alpen, imJura und im schweizerischen Mittelland. Beitr. Krypto-gamenflora Schweiz 9 (3):1-560.

110. Jitts, H. R., C. D. McAllister, K. Stephens, and J. D. H.Strickland. 1964. The cell division rates of some marinephytoplankton as a function of light and temperature. J.Fish. Res. Board Can. 21:139-157.

111. Jones, L. W., and J. Myers. 1964. Enhancement in the blue-green alga, Anacystis nidulans. Plant Physiol. 39:938-946.

112. Jones, L. W., and J. Myers. 1965. Pigment variation inAnacystis nidulans induced by light of selected wavelengths.J. Phycol. 1:7-14.

113. Kempner, E. S. 1963. Upper temperature limit of life. Science142:1318-1319.

114. Kiyohara, T., Y. Fujita, A. Hattori, and A. Watanabe. 1960.Heterotrophic culture of a blue-green alga, Tolypothrixtenuis. J. Gen. Appl. Microbiol. 6:176-182.


http://mm

br.asm.org/

Dow

nloaded from



115. Klas, S., and E. Marnenko. 1959. Mikrovegetacija termalnogvrela Sv. Helena Kod Samobora. Jugo. Akad. 7nanostiUmjetnosti '"Rad" 317:243-290.

116. Kol, E. 1932. 'Ober die Algenvegetation der Hajddszoboszl6erTherme. Arch. Protisten. 76:309-324.

117. Kratz, W. A., and J. Myers. 1955. Nutrition and growth ofseveral blue-green algae. Amer. J. Bot. 42:282-287.

118. Kratz, W. A., and J. Myers. 1955. Photosynthesis and respi-ration of three blue-green algae. Plant Physiol. 30:275-280.

119. Krinsky, N. I. 1966. The role of carotenoid pigments as

protective agents against photosensitized oxidations inchloroplasts, p. 423-430. In T. W. Goodwin (ed.), Bio-chemistry of chloroplasts, vol. I. Academic Press Inc.,New York.

120. Kullberg, R. G. 1968. Algal diversity in several thermal springeffluents. Ecology 49:751-755.

121. Lang, N. J. 1968. The fine structure of blue-green algae.Annu. Rev. Microbiol. 22:15-46.

122. Lazaroff, N. 1966. Photoinduction and photoreversal of theNostocacean developmental cycle. J. Phycol. 2:7-17.

123. Leach, C. K. and H. G. Carr. 1969. Oxidative phosphoryla-tion in an extract of Anabaena varlabills. Biochem. J.112:125-126.

124. Lederberg, J. 1954. A simple method for isolating individualmicrobes. J. Bacteriol. 68:258-259.

125. Lewin, R. A. 1959. The isolation of algae. Rev. Algol. 4:181-197.

126. Lewin, R. A. 1965. Freshwater species of Saprospira. Can. J.Microbiol. 11:135-139.

127. Livingstone, D. A. 1963. Chemical composition of riversand lakes. U.S. Geological Survey Professional Paper440-G.

128. Ldwenstein, A. 1903. Uber die Temperaturgrenzen desLebens bei der Thermalge Mastigocladus laminosus Cohn.Ber. Deut. Bot. Ges. 21:317-323.

129. Maciasr, F. M. and R. W. Eppley. 1963. Development ofEDTA media for the rapid growth of Chlanydomonasmundana. J. Protozool. 10:243-246.

130. Mandel, M. 1969. New approaches to bacterial taxonomy:perspective and prospects. Annu. Rev. Microbiol. 23:239-274.

131. Mann, J. E., and H. E. Schlichting. 1967. Benthic algae ofselected thermal springs in Yellowstone National Park.Trans. Amer. Micros. Soc. 86:2-9.

132. Margenko, E. 1962. Licht- und ElektronmikroskopischeUntersuchungen an der Thermalalge Mastigocladuslaminosus Cohn. Acta Bot. Croatica 21:47-74.

133. Marre, E. 1962. Temperature, p. 541-550. In R. A. Lewin(ed.), Physiology and biochemistry of algae. AcademicPress Inc., New York.

134. Marsh, C. L., and D. H. Larsen. 1953. Characterization ofsome thermophilic bacteria from the hot springs of Yellow-stone National Park. J. Bacteriol. 65:193-197.

135. Mathews, M. M. and W. R. Sistrom. 1959. Function ofcarotenoid pigments in non-photosynthetic bacteria.Nature 184:1892-1893.

136. McDonald, W. C. and T. S. Matney. 1965. Genetic transferof the ability to grow at 55 C in Bacillus subtilis. J. Bac-teriol. 85:218-220.

137. Myers, J., and W. A. Kratz. 1955. Relations between pigmentcontent and photosynthetic characteristics in a blue-greenalga. J. Gen. Physiol. 39:11-22.

138. Nakayama, T. 0. M. 1962. Carotenoids, p. 409-420. InR. A. Lewin (ed.), Physiology and biochemistry of algae.Academic Press Inc., New York.

139. Nash, A. 1938. The Cyanophyceae of the thermal regions ofYellowstone National Park, U.S.A., and of Rotorua andWhakarewarewa, New Zealand, with some ecologicaldata. Ph.D. Thesis, University of Minnesota, Minneapolis.

140. Nultsch, W., and G. Richter. 1963. Aktionsspektrum desphotosynthetischen 14CO-Einbaus von Phormidiumuncinatum. Arch. Mikrobiol. 47:207-213.

141. O'Donovan, G. A., and J. L. Ingraham. 1965. Cold-sensitivemutants of Escherichia coli resulting from increased feedback inhibition. Proc. Nat. Acad. Sci. U.S.A. 54:451-457.

142. O'Kelly, J. C. 1968. Mineral nutrition of algae. Annu. Rev.Plant Physiol. 19:89-112.

143. Pearce, J., and N. G. Carr. 1967. The metabolism of acetateby the blue-green algae, Anabaena varlabilis and Anacystisnidulans. J. Gen. Microbiol. 49:301-313.

144. Pearce, J., C. K. Leach, and N. G. Carr. 1969. The incom-plete tricarboxylic acid cycle in the blue-green alga Ana-baena variabills. J. Gen. Microbiol. 55:371-378.

145. Peary, J. 1964. Ecology and growth studies of thermophilicblue-green algae. Ph.D. Thesis, University of Oregon,Eugene.

146. Peary, J., and R. W. Castenholz. 1964. Temperature strainsof a thermophilic blue-green alga. Nature 202:720-721.

147. Prmt, S. 1956. Zur Physiologie der Mineral-und Thermalwa-sservegetation. Hydrobiologia 8:328-364.

148. Proctor, V. W. 1959. Dispersal of fresh-water algae by migra-tory water birds. Science 130:623-624.

149. Proctor, V. W., C. R. Malone, and V. L. DeVlaming. 1967.Dispersal of aquatic organisms: viability of disseminulesrecovered from the intestinal tract of captive Killdeer.Ecology 48:672-676.

150. Sargent, M. C. 1934. Causes of color change in blue-greenalgae. Proc. Nat. Acad. Sci. U.S.A. 20.251-254.

151. Schopf, J. W. 1968. Microflora of the Bitter Springs Forma-tion, Late Precambrian, Central Australia. J. Paleontol.42:651-688.

152. Schneider, K. C., C. Bradbeer, R. N. Singh, L. C. Wang,P. W. Wilson, and R. H. Burris. 1960. Nitrogen fixationby cell-free preparations from microorganisms. Proc.Natl. Acad. Sci., U.S.A. 46:726-733.

153. Schwabe, G. H. 1936. Beitrage zur Kenntnis isliindischerThermalbiotope. Arch. Hydrobiol. 6 (Suppl.):161-352.

154. Schwabe, G. H. 1960. Uber den thermobionten Kosmopoli-tan Mastigocladus laminosus Cohn. Blau-Algen undLebensraum V. Schweiz. Z. Hydrol. 22:757-792.

155. Schwabe, G. H. 1962. Lagerbildungen bei Hormogonalen,p. 53-60. In Beitrage zur Physiologie und Morphologie derAlgen. Gustav Fischer, Stuttgart.

156. Schwabe, G. H. 1964. Lagerbildungen hormogonaler Blaual-gen in thermalen und anderen extremen Biotopen. Verh.Int. Verein. Limnol. 15:772-781.

157. Schwabe, G. H. 1966. Okologischer Charakter und Systemder Cyanophyten. Verh. Int. Verein. Limnol. 16:1541-1548.

158. Setchell, W. A. 1903. The upper temperature limits of life.Science 17:934-937.

159. Sharp, J. H. 1969. Blue-green algae from Bermuda waters:ecologically selected variations of a single species. J.Phycol. 5:53-57.

160. Sharp, J. H. 1969. Blue-green algae and carbonates-Schizo-thrix calcicola and algal stromatolites from Bermuda.Limnol. Oceanogr. 14:568-578.

161. Sheridan, R. P. 1966. Photochemical and dark reduction ofsulfate and thiosulfate to hydrogen sulfide in Synechococcuslividus. Ph.D. Thesis, University of Oregon, Eugene.

162. Singh, R. N., and H. N. Singh. 1964. Ultra-violet inducedmutants of blue-green algae. I. Anabaena cycadeae Reinke.Arch. Mikrobiol. 48:109-117.

163. Sistrom, W. R., M. Griffiths, and R. Y. Stanier. 1956. Thebiology of a photosynthetic bacterium which lacks coloredcarotenoids. J. Cell. Comp. Physiol. 48:473-515.

164. Sladeckova, A. 1969. Control of slimes and algae in coolingsystems. Verh. Internat. Verein. Limnol. 17: in press.

165. Smith, A. J., J. London, and R. Y. Stanier. 1967. Biochemicalbasis of obligate autotrophy in blue-green algae andthiobacilli. J. Bacteriol. 94:972-983.

166. Sorokin, C. 1960. Kinetic studies of temperature effects onthe cellular level. Biochim. Biophys. Acta 38:197-204.

167. Steeman Nielsen, E., and E. G. Jorgensen. 1968. The adapta-

VOL. 33, 1969 503


http://mm

br.asm.org/

Dow

nloaded from


CASTENHOLZ

tion of plankton algae. I. General part. Physiol. Plant.21:401-413.

168. Stewart, W. D. P. 1968. Nitrogen input into aquatic eco-

systems, p. 53-72. In D. F. Jackson. (ed.), Algae, man, andthe environment. Syracuse University Press, Syracuse, N.Y.

169. Stockner, J. G. 1967. Observations of thermophilic algalcommunities in Mount Rainier and Yellowstone NationalParks. Limnol. Oceanogr. 12:13-17.

170. Stockner, J. G. 1968. Algal growth and primary productivityin a thermal stream. J. Fish. Res. Board Can. 25:2037-2058.

171. Swain, F. M. 1969. Paleomicrobiology. Annu. Rev. Micro-biol. 23:455-472.

172. Szeicz, G. 1966. Field measurements of energy in the 0.4-0.7micron range, p. 41-51. In R. Bainbridge, G. C. Evans,and 0. Rackham (ed.), Light as an ecological factor.John Wiley & Sons, New York.

173. Thomas, J., and E. A. Gonzalves. 1965. Thermal algae ofWestern India. Hydrobiologia 25:(I) 330-340, (II) 340-351; 26:(II) 21-28, (IV) 29-40, (V) 41-54, (VI) 55-65.

174. Trtiper, H. G. 1969. Bacterial sulfate reduction in the RedSea hot brines, in press. In E. T. Degens and D. A. Ross(ed.), Hot brines and recent heavy metal deposits in theRed Sea. Springer-Verlag, New York.

175. Tuxen, S. L. 1944. The hot springs of Iceland, their animalcommunities and their zoogeographical significance, p.1-206. In The zoology of Iceland, vol. I, part 11. EinarMunksgaard, Copenhagen.

176. Uzamasa, Y. 1965. Chemical investigations of hot springs inJapan. Tsukiji Shokan, Tokyo.

BACrERIOL. REV.

177. Van Baalen, C. 1967. Further observations on growth ofsingle cells of coccoid blue-green algae. J. Phycol. 3:154-157.

178. Vincent, E. R. 1967. A comparison of riffle insect populationsin the Gibbon River above and below the geyser basins,Yellowstone National Park. Limnol. Oceanogr. 12:18-26.

179. Vouk, V. 1950. Grundriss zu einer Balneobiologie derThermen. Verlag BirkhaIuser, Basel.

180. Walsby, A. E. 1968. Mucilage secretion and the movementsof blue-green algae. Protoplasma 65:223-238.

181. Waring, G. A. 1965. Thermal springs of the United Statesand other countries of the world-a summary. U.S.Geological Survey Professional Paper 492.

182. Webster, G. C., and A. W. Frenkel. 1952. Some respirationcharacteristics of the blue-green alga, Anabaena. PlantPhysiol. 28:63-69.

183. White, D. E., J. D. Hem, and G. A. Waring. 1963. Chemicalcomposition of sub-surface waters. U.S. GeologicalSurvey Professional Paper 440-F.

184. Whitford, L. A., and G. J. Schumacher. 1964. Effect of acurrent on respiration and mineral uptake in Spirogyraand Oedogoniwn. Ecology 45:168-170.

185. Williams, R. B. 1964. Division rates of salt marsh diatoms inrelation to salinity and cell size. Ecology 45:877-881.

186. Wyatt, J. T., and J. K. Silvey. 1969. Nitrogen fixation byGloeocapsa. Science 165:908-909.

187. Yoneda, Y. 1952. A general consideration of the thermalCyanophyceae of Japan. Memoirs College of Agriculture,Kyoto University, Fisheries Ser. 62:1-20.

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