FEMS Microbiology Reviews 75 (1990) 117-124 Published by Elsevier

FEMSRE 00133

Hyperthermophilic microorganisms

K.O. Steuer, G. Fiala, G. Huber, R. Huber and A. Segerer

Lehrstuhl für Mikrobiologie, Universität Regensburg, Regensburg, F.R.G.

Key words: Hyperthermophiles;

1. INTRODUCTION 7 to 9) rich in NaCl. Also man-made hot environ• ments such as the boiling outflows of geothermal The most extremely thermophilic living beings power plants are suitable environments for hyper• known to date are bacteria growing at tempera• thermophiles [9]. Submarine hydrothermal systems tures between 80 and 110 °C [1-3]. Some of them are slightly acidic to alkaline (pH 5 to 8.5) and are so well adapted to the high temperatures that normally contain high amounts of NaCl and SO4 ~ they do not even grow below 80 ° C [4]. As a rule, due to the presence of sea water (Table 1). Due to non of these hyperthermophilic bacteria are able the low solubility of oxygen at high temperatures to grow at 60 ° C or below. Hyperthermophiles are and the presence of reducing gases, most biotopes occurring mainly within the archaebacterial king• of hyperthermophiles are anaerobic. Within con• dom [5]; some of them are also present within the tinental solfataric fields, oxygen is only present eubacteria [6,7]. Due to their existence within within the upper acidic layer which appears phylogenetically highly divergent groups, the lack ochre-coloured due to the existence of ferric iron of closely related mesophiles, and their biotopes [8]. existing already since the Archean age, hyperther• Although not growing below 60 °C, hyperther• mophiles may have adapted to the hot environ• mophiles are able to survive at low temperatures ment already billions of years ago. at least for years. Some of the anaerobic hyper• thermophiles are able to tolerate oxygen much better at low (non-growth) temperatures than at 2. BIOTOPES high (growth) temperatures. This property may be essential for dispersal of these organisms through The hyperthermophilic bacteria known at pre• oxygen-rich low-temperature areas. sent have been isolated from submarine hydro- thermal areas and from continental solfataras. The surface of the solfataras is usually rich in sulfate 3. TAXONOMY OF HYPERTHERMOPHILIC and exhibits an acidic pH (0.5 to 6; [1,2]). In the BACTERIA depth, solfataras are less acidic or even neutral Up to now, about 35 different of hyper• (pH 5 to 7; [8]). Sometimes, solfataric fields may thermophilic bacteria are known. They belong to also contain some weakly alkaline hot springs (pH different taxa within the eu- and archaebacteria (Table 2). Within the eubacteria, hyperthermo• philes are within the Thermotogales order, which Correspondence to: K.O. Steuer, Lehrstuhl für Mikrobiologie, is the deepest phylogenetic branch-off based on Institute for Biochemistry, Genetics and Microbiology, Univer• sity of Regensburg, Universitätsstr. 31, P.O. Box 397, D-8400 16S rRNA sequences within this kingdom [7]. The Regensburg, F.R.G. phylogenetic tree within the archaebacterial king-

0168-6445/90/S03.50 © 1990 Federation of European Microbiological Societies dorn exhibits two main branches: the branch con• S° and S2", forming sulfuric acid (Table 3, 4). taining methanogens and halophiles and the Many Sulfolobus isolates are facultative hetero- branch of the sulfur metabolizing archaebacteria trophs and a few, still unnamed, are even strict [7]. The latter consists almost exclusively of hyper• heterotrophs (Stetter, unpublished). The type thermophiles [3,7] while there are only a few groups strain of Sulfolobus acidocaldarius (DSM 639) of hyperthermophiles within the other branch shows excellent growth on organic matter [11]. (Archaeoglobales, Thermococcales, Methano- Autotrophically cultured on S2~ and S°, however, thermaceae, Mc. jannaschii). Within the sulfur this strain shows only extremely weak growth and metabolizers, acidophilic (e.g. Sulfolobus, Metal- sulfuric acid formation [12-14]. Some members of losphaera, Acidianus) and neutrophilic (e.g. Pyro- the Sulfolobales like Metallosphaera sedula are dictium, Desulfurococcus, Staphylothermus, Ther- able to oxidize sulfidic ores like pyrite, chal- mococcus) genera were discovered during the last copyrite and sphalerite, forming sulfuric acid and years. Therefore, the old designation "Thermo- solubilizing the heavy metal ions (Table 3,4; [15]). acidophiles" which was primarily used to char• Under microaerophilic conditions, Sulfolobus is acterize the genera Sulfolobus and Thermoplasma able to reduce ferric iron and molybdate, which [1] is not suitable anymore for the designation of therefore act as electron acceptors under these this branch. conditions [16,17]. Members of the Sulfolo• bus grow only at very low ionic strength and were 3.1. Extremely acidophilic hyperthermophiles therefore not found within the marine environ• Extremely acidophilic hyperthermophiles were ment ([11]; Segerer and Stetter, unpublished). found up to now exclusively within continental Members of the genus Acidianus similar to solfataric fields. The organisms are coccoid-shaped Sulfolobus are able to grow by oxidation of S°, strict and facultative aerobes. They are extreme forming sulfuric acid [18,19]. Some strains grow acidophiles due to their requirement of acidic pH also on sulfidic ores, but much less efficient than (opt. approx. pH 3). Phylogenetically, they belong Metallosphaera (Table 4; [14]). In addition, mem• to the archaebacterial genera Sulfolobus, Metal- bers of Acidianus are able to grow by anaerobic losphaera, Acidianus, and Desulfurolobus. The less oxidation of H2, using S° as electron donor (Table extremely thermophilic archaebacterium Thermo• 4; [18]). A thermoacidophilic isolate, which had plasma is an extreme acidophile, too. It is a facul• been tentatively named "Sulfolobus ambiualens" tative anaerobe occurring both in smoldering coal [20] was later described as Desulfurolobus am- refuse piles and continental solfataric fields [1,10]. bivafens, representing a new genus [21]. By Members of the genus Sulfolobus are strict DNA : DNA hybridization, however, Desulfurolo• aerobes growing autotrophically by oxidation of bus ambivalens shows a close relationship to the

Table 1

Biotopes of hyperthermophilic bacteria

Characteristics Type of thermal area

Solfataric fields Submarine hydrothermal systems

Sites Steam-heated mud holes. Hot sediments and vents soils and surface waters; deep hot springs; geo- thermal power plants Temperatures Up to 100°C Up to about 374 °C pH 0.5 to 9 5 to 8.5

Presence of 02 Surface oxic; depth anoxic Anoxic

2_ + 2 Major gases and C02, CO, CH4, S04 , NH4 H2, H2S,S°,S03 - sulfur compounds 3.2. Slightly acidophilic and neutrophilic hyperther• Taxonomy of hyperthermophilic archaebäcteria mophiles

Order Genus Species Slightly acidophilic and neutrophilic hyperther• mophiles were found both in continental solfataric I. Eubacteria! kingdom Thermotogales Thermotoga T. maritima fields and in submarine hydrothermal systems and T. neapolitana they show specific adaptations to their environ• T. thermarum ments. All of them are strict anaerobes. Solfataric Thermosipho 8 T. africanus fields contain members of the genera Thermopro• 8 Fervidobacterium F. nodosum teus, Pyrobaculum, , Desulfurococcus, F. islandicum and (Table 3; [9,23-26]). Cells of II. Archaebacterial kingdom members of the genera Thermoproteus, Pyrobacu• Sulfolobales Sulfolobus S. acidocaldarius lum, and Thermofilum are characteristically stiff S. solfataricus rods with protruding spheres ("golf clubs") mainly Metallosphaera M. sedula Acidianus A. infernus at their ends, possibly due to a mode of budding. A. brierleyi 8 Cells of Thermofilum are only about 0.17 to 0.35 Desulfurolobus D. ambiualens /im in width and are therefore different from Thermoproteus T. tenax Thermoproteus and Pyrobaculum. Thermoproteus T. neutrophifus tenax, Thermoproteus neutrophilus and Pyrobacu• Pyrobaculum P. islandicum P. organotrophum lum islandicum are able to grow autotrophically by

Thermofilum T. pendens anaerobic reduction of S° with H2 as electron T. librum donor [9,27]. Strains of Thermofilum and Pyro• Desulfurococcus D. mobilis baculum organotrophum are obligate heterotrophs. D. mucosus They are growing by sulfur respiration, using dif• D. saccharovorans Staphylothermus • S. marinus ferent organic substrates (Table 4). Thermofilum Pyrodictialcs Pyrodictium P. occultum pendens shows an obligate requirement for a lipid P. brockii fraction of Thermoproteus tenax. Thermoproteus P. abyssum tenax and Pyrobaculum islandicum are also able to Tliermodiscus T. maritimus grow heterotrophically by sulfur respiration (Ta• Thermococcales Thermococcus T. celer T. stetteri ble 4). Also in solfataric fields coccoid hetero• Fyrococcus P. furiosus trophic organisms occur which gain energy by P. woésii respiring organic material using sulfur as electron Archaeoglobales Archaeoglobus A. fulgidus acceptor. They belong to the genus Desulfurococ• A. profundus cus [25]. From solfataras in the southwest of Ice• Thermoplasmales 8 Thermoplasma T. acidophilum land, rod-shaped methanogens growing at temper• T. volcanium b Methanothermus M. fervidus atures up to 97 °C (Table 3) were isolated [26,28]. M. sociabilis Two species are known: Methanococcales Methanococcus b M. thermolitho- and Methanothermus sociabilis, cells of the latter 8 trap hi cus growing in clusters up to 3 mm. Both species are M. jannaschii strict autotrophs, gaining energy by reduction of

' Growth only up to about 70°Cf mentioned due to their close C02 by H2 (Table 4). Within neutral continental relationship to hyperthermophiles hot springs in Djibouti, Africa, the extremely ther• b Contain also mesophilic species. mophilic eubacterial species Thermotoga ther• marum was found, which grows only at low ionic type species of Acidianus, Acidianus infernus [22]. strength (Table 3,4; [29]). Members of this group Members of the genus Acidianus are able to grow are strictly anaerobic heterotrophs, growing by in the presence of up to 4% salt. In agreement with fermentation of carbohydrates. Most of them are this result, they also have been isolated (rarely) found within the marine biotopes, however. from a marine hydrothermal system [19]. Many hyperthermophiles are adapted to the Growth conditions and morphological and biochemical features of hyperthermophiles

Species Growth conditions DNA Morphology (mol% Temperature (° C) pH Aerobic (ae) Habitat G-f C) anaerobic (an) Minimum Optimum Maximum (marine (m)/ solfataric (s))

Sulfo/obus acidocaldarius 60 80 85 1 -5 ae s 37 Lobed cocci Metallosphaera sedula 50 75 80 1 -4.5 ae s 45 Cocci Acidianus infernus 60 88 95 1.5-5 ae/an s 31 Lobed cocci

Thermoplasma uolcanium 33 60 67 1 -4 ae/an s 38 Irregular cocci

Thermoproteus tenax 70 88 97 2.5-6 an s 56 Rods with terminally protruding spheres Rods with terminally Pyrobaculum islandicum 74 100 103 5 -7 an s 46 protruding spheres Rods with terminally Pyrobaculum organotrophum78 100 102 5 -7 an s 46 protruding spheres Thermofilum pendens 70 88 95 4 -6.5 an s 57 Slender rods with terminal spheres Desulfurococcus mobilis 70 85 95 4.5-7 an s 51 Cocci Staphyhthermus marin us 65 92 98 4.5-8.5 an m 35 Cocci in aggregates

Pyrodictium occultum 82 105 110 5 -7 an m 62 Discs with fibres Pyrodictium abyssum 80 100 110 4.7-7.5 an m 60 Discs with fibres maritimus 75 88 98 5 -7 an m 49 Discs

Thermococcus celer 75 87 97 4 -7 an m 57 Cocci Pyrococcus furiosus 70 100 103 5 -9 an m 38 Cocci

Archaeoglobus fulgidus 60 83 95 5.5-7.5 an m 46 Irregular cocci

Methanothermus sociabiiis 65 88 97 5.5-7.5 an s 33 Rods in clusters

Methanococcus jannaschii 50 85 86 5.5-6.5 an m 31 Irregular cocci

Thermotoga maritima 55 80 90 5.5-9 an m 46 Rods with sheath Thermotoga t her ma rum 55 70 84 6 -9 an s 40 Rods with sheath

marine thermal environments (Table 3). They are by H2. Alternatively, these species are able to

represented by the genera Staphyhthermus, Pyro• grow mixotrophically on H2 and thiosulfate in the dictium, Thermodiscus, Thermococcus, Pyrococcus, presence of cell extracts (Table 4). In contrast, Archaeoglobus, and by members of the genera Pyrodictium abyssum is an obligate heterotroph, Methanococcus and Thermotoga (Table 3; gaining its energy from an up to now unknown [3,4,6,30-34]). The organisms with the highest type of fermentation. Elemental sulfur stimulates growth temperatures known are members of the growth, but is not essential (Table 4). A group of genus Pyrodictium, growing up to 110°C [4,35]. coccoid hyperthermophilic sulfate-reducing Cells of Pyrodictium are so well-adapted to high archaebacteria is represented by Archaeglobus temperatures that they do not even grow below fulgidus [33,36]. It is a facultative autotroph. Dur• 80 °C (Table 3). Cultures of Pyrodictium grow in ing autotrophic growth, Archaeoglobus fulgidus

floes. Cells are disc-shaped and are connected by a gains its energy by reduction of thiosulfate by H2 network of very thin hollow fibres. Pyrodictium (Table 4). Only very little growth is obtained when occultum and Pyrodictium brockii are able to grow thiosulfate is replaced by sulfate under auto• autotrophically, gaining energy by reduction of S° trophic conditions. During heterotrophic growth, Metabolism of hyperthermophiles

Species Autotrophic Substrates Electron acceptors End products growth *

H2S04; ? Sulfolobus acidocaldarius f S°; H2S; cell extracts; sugars o2

Metallosphaera sedula f S°; sulfidic ores; cell extracts o2 H2S04; ?

Acidianus infernus o S°; sulfidic ores; H2 02; S° H2S04; H2S

_ Thermoplasma volcanium Yeast extract; cell extracts 02; S° Acetate, C02;

H2S; ?

2 2- Thermoproteus tenax f H2; yeast extract; cell extracts S°; S203 "; SO H2S;?

2 2_ Pyrobaculum islandicum f H2; yeast extract; cell extracts S°; S^ -; S03 ; cystine H2S; ? 2- H S;? Pyrobaculum organotrophum - Yeast extract; cell extracts S°; SjO*""; SO ; cystine 2 H S; ? Thermofilum pendens - Yeast extract; tryptone; -f lipid S° 2 fraction

Desulfurococcus mobilis Yeast extract; tryptone; casein S° H2S; ? - H S; acetate; Staphylothermus marinus - Yeast extract; peptone; meat S° 2 extract isovalerate

b 2 Pyrodictium occultum f H2 (H2 +cell extract) S°; (S203 -) H2S Pyrodictium abyssum - Yeast extract; gelatine; starch; Fermentative? (S°) ? formate

Thermodiscus maritimus Yeast extract; (cell extracts 4- H ) S°; unknown respiration? H2S; ? - 2

Thermococcus celer Tryptone; yeast extract; casein S°; fermentation? H2S; ?

Pyrococcus furiosus Yeast extract; casein; starch; S°; fermentation? H2S; H2; C02; ? - maltose

c 2 2 2_ Archaeoglobus fulgidus f H2; formate; lactate; sugars; SO4 -; S203 -; S03 H2S; traces

proteins of CH4

Methanothermus sociabilis o co CH4 H2 2

Methanococcus jannaschii 0 C02 CH4 H2

Thermotoga maritima _ Yeast extract; sugars; starch; Fermentative L-Lactate; ace•

cellulose tate; H2; C02 Thermotoga (hermarum - Yeast extract; (yeast extract + Fermentative n.d. carbohydrates)

* o * obligately autotrophic; f = facultatively autotrophic; - = heterotrophic. b Autotrophic growth only with S° as electron acceptor.

c Autotrophic growth only with S203 ~ as electron acceptor, n.d. = not determined.

various substrates like lactate, formate, glucose, sion of factor 420 [36]. Due to its unique type of starch and proteins can be used. As electron RNA polymerase and its 16S rRNA sequence, acceptors, sulfate, thiosulfate, and sulfite can be Archaeoglobus forms a separate phylogenetic used heterotrophically. S° is inhibitory during au• branch situated between the thermophilic sulfur totrophic and heterotrophic growth. In addition, metabolizers and the methanogens [33,36,37]. A traces of methane (up to 0.1 /imol/ml) are formed further autotrophic marine hyperthermophile is via an unknown pathway [36]. Similar to the represented by Methanococcus jannaschii, which methanogens, cells of Archaeoglobus show a blue- had been isolated up to now only from submarine green fluorescence at 420 nm due to the posses• deep sea vents (34). Methanococcus jannaschii is a strictly autotrophic methanogen growing at tem• growing at temperatures up to 90 °C (Table 3; peratures up to 86 °C (Table 3, 4). [6,39]). They are using various carbohydrates as The marine thermal environment contains also energy source. As end products, L-lactate, acetate,

a variety of strictly heterotrophic hyperthermo• H2 and C02 are formed (Table 4). Hydrogen is philes. Members of the genus Staphyhthermus are inhibitory to growth and has to be removed during

obligate sulfur respirers. Cells of Staphyhthermus cultivation. In the presence of S°, H2S is formed

marinus are coccoid and grow in grape-like ag• and H2 inhibition is overcome [6]. gregates and form giant cells under special nutri• tional conditions [30]. Cells of isolates of the genus Thermodiscus are disc-shaped. In contrast to Pyro• dictium, they do not form fibres, grow only up to 4. CONCLUSIONS 98° C and show a much lower GC-content of their DNA (Table 3, 4). Growth occurs by sulfur respi• The isolation of various groups of hyperther• ration on yeast- and cell extracts and is stimulated mophilic bacteria from geothermally and hydro-

by H2. Good growth is also obtained without thermally heated environments demonstrates an sulfur possibly due to an unknown respiration of unexpected complexity of these up to now almost fermentation. The genera Thermococcus and Pyro• unexplored ecosystems. Within these, primary coccus are belonging to the Thermococcales, which production and consumption of organic matter is represent up to now the deepest branch-off within going on at temperatures around 100 °C. The en• the phylogenetic archaebacterial tree [7,31,32,38]. ergy-yielding reactions of primary production is The cells are coccoid shaped (Table 3). Thermo• based on reduction or oxidation of inorganic sulfur

coccus celer utilizes tryptone, yeast extract and compounds by H2 or 02, or in the case of meth-

protein as carbon sources. Growth is stimulated anogens on reduction of C02 by H2. Since H2,

by sucrose. In closed culture vessels, Thermococcus C02 and S° are formed within the environment, celer grows optimally in the presence of sulfur and anaerobic autotrophs using these compounds are

about 1.5 mol of H2S are formed per mole of C02 completely independent of sunlight. The con• [31]. Thermococcus can also grow without sulfur. sumers of organic matter are most likely using cell Under optimal conditions, the generation time of components of the decaying primary producers. Thermococcus celer is close to 50 min. Pyrococcus Most of them are growing by sulfur respiration or furiosus grows at temperatures up to 103°C and by unknown types of respiration and fermenta• shows a much lower GC content than Thermococ• tion. A great deal of the autotrophs are opportun• cus celer (Table 3). At 100° C, the doubling time is istic' heterotrophs, too. This property may be im• only 37 min [32]. Cells of Pyrococcus furiosus are portant for effective competition within the ex• highly motile due to monopolar polytrichous treme environment. flagellation, They grow on yeast extract, fieptone, The upper temperature border of life is still casein, starch and maltose and possess a powerful unknown. The existence of Pyrodictium and other protease and amylase. As metabolic products in hyperthermophiles demonstrates that still unre•

the absence of sulfur C02 and HU were found, the cognized thermostabilizing principles must exist. latter being inhibitory to growth. Hydrogen in• Since stability of biomolecules at temperatures hibition can be prevented by the addition of S°, above 100 °C decreases rapidly [40-42], the maxi•

whereupon H2S is formed in addition. The mode mal growth temperature at which microbial life of fermentation of Pyrococcus and Thermococcus can exist may be possibly found between 110 and is still unclear. Many submarine hydrothermal 150 °C. Within this temperature range, heat-sensi• fields contain also members of the eúbacterial tive biomolecules could possibly still be resynthe- genus Thermotoga which are thriving together with sized at biologically feasible rates. Beyond the hyperthermophilic archaebacteria in the same en• interesting questions in basic research, hyperther• vironment. Thermotoga maritima and Thermotoga mophiles may be well suited for the development neapolitana are fermentative hyperthermophiles of novel biotechnological processes due to their novel metabolic properties and the outstanding Torma, A., Eds.), pp. 71-81. Associazione Mineraria heat resistance of their cell components. Sarda, Iglesias. [13J Huber, G., Huber, H. and Stetter, K.O. 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