In: Microbial Growth on Compounds. Proceedings of the 5Th International Symposium. / (H.W. Van Verseveld, J.A. Duine Eds.) Pp. 44-51, Nijhoff Publ., Dordrecht (1987)

In: Microbial Growth on Compounds. Proceedings of the 5th International Symposium. / (H.W. van Verseveld, J.A. Duine eds.) pp. 44-51, Nijhoff Publ., Dordrecht (1987). AEROBIC AND ANAEROBIC EXTREMELY THERMOPHILIC AUTOTROPHS

R. HUBER G HUBS*. A. SEGERER J. SEGER and K.O. STETTER Lehrstuhl für MkrobJoiogie, Urwers/iát Regensburg, 8400 Regensburg, Federal Repubßc of Germany.

1. INTRODUCTION

During the last years, various extremely themioprÄ badend 80X rwe been i»latedfTOT 15,17}.Alof these organisms belong to the archaebacteria, the third kingdom of Be[19]. The mode of nutrition is Hthoautotrophlc or organotrophic, depenolng on the isolates. In this paper, limoautotrophic extreme thermophlles including some very recent isolates are described which are the primary producers of organic matter at high temperatures.

2. BIOTOPES

Up ta now, aft extremely therrnophfe autotrophic b^ fieWs and submarine hydPotherinalsysteni& about 400 to 2000 m wM the floor (10]. From tf^^

mainly C02. SO2 and H^S. escape and are heal the sol and surface water. The examination of so* profiles wiminsotfatarlcfields showed^ the top, there is an oxypen^ntaioing strongly aocfcs lay er of about 15 to 30 cm in thickness, which shows an ochre cotoir due to the presence of feró to neutraJ btoish-Wack 20m. which contains ferrous suTxJe. Submarine hydrcmermaJ syalems contain seawater of neutral to sfigMy acxScpH which remains Squid even above 100*C due to the prevenfion of boHmg by the hydros!^

sulfur which ts formed by the oxidation of H^S ana by the reaction of H2S with S02- Seawaler contains atwutjfnmotes of sufate per fitre.

3. ACIDOPHILIC AEROBIC ANO FACULTATIVELY ANAEROBIC AUTOTROPHS

The upper oxkfced layer within soff marie fields inducing solfataric mudholes contains coocoid lobe- shaped strictly aerobic organisms which belong to the genus Svffofoöus which was described by TO. Brock (4J> The type species Suffotobus ac&xaxiaws and most other isolates can gain their energy i% . 8 + c w < X

: i »9 O

» Ul • • •

i IS • <• + .+ • + .+ •

Sä Tí "O "O TJ T> c c cí «£ c

• • • •

3 « N S 8 S N if. tO IA 0» ^ *- O ^ 5? 8 c*> co ^ co o w «¡o co

8 I x S ui m > F > > X chemo lit hotroph»caiiy by oxidation oí S°, forming H2S04. Some isolates (Tab. 1) are able to grow

iithoautotrophicaJly in S°-containing mineral medium in the presence of C02 and oxygen, others require the addition of traces of yeast extract (e.g. 0.01%) , the function of which is not yet clear (source of minerals or of organic carbon). Alternatively both Suifclobus-speties and many Sulfolobus-shaped isolates (Tab. 1) grow without S°* organotrophtcally on yeast extract, sugars, peptone and amino acids [3. 4,15]. Many Sulfolobus isolates are able to oxidize ferrous iron [2, 5]. However, due to extensive precipitates of jarosite, even at pH 2 and in uninoculated controls, no significant growth could be observed [3, Huber and Stetter, unpublished]. Sulfolobus bherleyi, which has now been re named Adolanus brierieyi [12] and some still unnamed Suffolobus-shaped isolates [8] are moderate thermophiles, growing at temperatures up to 70°C. They are able to grow on surfkjic ores» forming sulfuric acid and soiubilizing heavy metals. New ore-leaching bacteria could be isolated aerobicafly in mineral medium in the presence of ore mixture G1, consisting of pyrite, chalcopynte. sphalerite and pitchblende [Tab. 1; 6]. tepyes HV5 and VE2 from samples of Hveragerthi and Hveraveflir in Iceland are strictly dependent on ores and can not grow on S°. Strains HV5 and VE6 are the most extremely ore- leaching bacteria known, growing at temperatures up to 95°C (Tab. 1). in contrast, Sulfolobus acidocaldanus and Sulfolobus solfatahcus are unable to grow by the oxidation of suffidic ores.

Recently, the new genus Acktianus was recognized (tentatively named 'Acidothermus', [14,17]. Members have been isolated from acidic soffatara fields in Italy, Iceland, the Azores and the United States, and from a submarine hydrothermal vent in Italy [11, 12]. Although the cefts of these thermoacidophfles have a coccoid, lobed shape similar to Sulfolobus (Fig. 1), they are different in DNA composition (G+C-content; DNA homology) and in metabolic properties.

Figure 1 .Thin section of AckManus internus contrasted with lead citrate and uranyl acetate.Bar 1 urn. Z7

1 P

5 S 5, 8 S 0)

CO CO ft ^ S CO N 3 3 S= 10 • in eo

I 1

"32 I .2 Tí 1 f I O •5 3 8 2 i Adátanos mfemus, the first species to be recognized (12], is able to grow aerobicaöy by S°-oxidation, forming sulfuric acid. Surprisingly, this organism is also able to grow under extremely anaerobic conditions by S°-reduction: H^S is formed from ^ and S° (Tab, 1. 2). Sulfolobus bnerteyi also processes this previously unrecognized metabolic versatility, and was therefore considered to also belong to the genus Aadianus (11, 12]. A further isolate, which contains a prophage (W. Zfllig, pers. comm.) and had been tentatively named "Sulfolobus ambtvalensT [21] exhibits 60% DNA homology with the type species, Adóianus infernas, indicating that it is another species of the genus AckSanus.

4. NEUTROPHILIC AND SLIGHTLY ACIDOPHILIC ANAEROBIC AUTOTROPHS

From the lower reduced zone of solfataric fields, extremely thermophilic methanogens and S°-

dependent autotrophs could be isolated. Previously, the methanogens could be only obtained from

Icelandic solfataric fieids. They are rod-shaped. Gram-positive lithoautotrophs, growing by formation of

methane from H2 and C02 at temperatures up to 97°C. Until now, the two species Methanothermus

fervidus and Methanothermus sociabilis (Fig. 2) are the only known representatives of

Methanoathermaceae [7.8].

Figure 2. Electron micrograph of Methanothermus sociabilis, platinum-shadowed. Bar 1 urn.

The S°-dependent autotrophs are rod-shaped organisms which divide by budding [20]. Unta now, members of the two new genera Thermoproteus and "Pyrobacukjm" (Fig. 3) could be isolated growing

iithoautotrophicairy on S°, H2 and C02. gaining energy by H¿S formation (Tab. 2). *Pyrobacukim* has a

G-kC-content of onry 46 mof% which is 10% lower than that of Thermoproteus [20]. In addition, isolates belonging to"Pyrobacvlum' show an optimal growth temperature of 10O°C which is 12°C higher than that of the Thermoproteus-isolales [17,20]. Alternatively. "Pyrobaculum islandicum" and

Thermoproteus tenax are able to grow without H2 organotrc^.icaliy on yeast extract in the presence of

S°, S2O32- and SO32- (Tab. 2).

Figure 3. Electron micrograph of 'Pyrobaculum islancScur-'Geo 3, platinum-shadowed. Bar 1 urn.

During organotrophic growth, onry tow amounts of H^S are formed compared with lithotrophic growth of these organisms, tn the case of "Pyrobaculum tslanöcum~. organotrophic growth is also obtained when S° is replaced by cysteine. It is most Beefy, these bacteria are growing organot rophcaüy by an

Electron micrograph of PyrocScäum occuJtum. aaimum-shadowed. Bar 5 urn. unknown fermentation rather than by sulfur respiration. From the submarine hydrothermal vents of VuJcano. Italy, the most extremely thermophilic organisms known until now have been isolated- They have an optimal growth temperature of 105°C, and a maximum at around 110°C [under slight overpressure, 13]. These disc- to dish-shaped cells form unusual networks of fibers connecting the celts (Fig.4).

The new genus m PyroaTctíum" currently contains the species Pyroofctium occuftum and Pyroäctium brockS, which are strict H^S-aiitotrophs (Tab. 2). During growth, the fiber networks cover the sulfur like cobwebs [13.16].

5. ISOLATION OF EXTREMELY THERMOPHILIC SULFATE-REDUCING ARCHAE• BACTERIA

Until now, no archaebacteria thriving by o^imüatory sulfato reduction were known [9J. During our recent to situ enrichments wimin the hydrothermal systems close to Vulcano, Italy, coccoid to disc- shaped irregular cafe could be isolated. These grew anaerobtcaty wihout S° at temperatures between 65 and 96°C with an optimum at around 83°C. They use formate, L(+) and D(-) lactate and, less efficiently, glycogen, starch, yeast extract, meat extract and bacterial homogenates as substrates. Sulfate, thtosuifate and sulfite are used as electron acceptors, while S° is toNbitory tor growth. As end products, H2S, CO2. and traces of methane (about 4 mmotos/l) could be detected. Growth is inhibäed by 0.1 mmoies/l of Na-motybdate. Under the UV microscope, the cells show a blue-green fluorescence due to the presence of F420 PI-,n addtion, rnethanopcerin, but not F430 [A. Pfaitz, pets, commjor coenzyme M could be detected. The RNA polymerase of isolate VC-16 shows homology to subunito B and B" of the rrtothanogens, but is unique to serological cross-reaction of the heaviest subunit with components A and C of methanogens and S-dependent archaebacteria. indicating an outstanding position for the new isolates within the phylogenetic tree of the archaebacteria.

References

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