t
Hyperthermophilic Bacterial Communities within Terrestrial and Marine Hydrothermal Areas
K. O. Steuer, G. Fiala, G. Huber, R. Huber, A. Neuner, and A. Segerer
Lehrstuhl für Mikrobiologie, Universität Regensburg, 8400 Regensburg, F.R.G.
Keywords: thermophilic, hyperthermophiles, hydrothermal, solfataras, archaebacteria
INTRODUCTION
D u r i n g t h e l a s t y e a r s , members o f communities o f h y p e r t h e r m o p h i l i c b a c t e r i a growing a t t e m p e r a t u r e s between 80 and 110°C have been i s o l a t e d ( 1 - 3 ) . As a r u l e , they a r e so w e l l adapted t o the h i g h t e m p e r a t u r e s t h a t they do n o t grow below 60°C. H y p e r t h e r m o p h i l i c b a c t e r i a m a i n l y b e l o n g t o the a r c h a e b a c t e r i a l kingdom ( 4 ) . Some o f them a r e a l s o p r e s e n t w i t h i n t h e e u b a c t e r i a ( 5 , 6 ) . The communities c o n s i s t of p r i m a r y p r o d u c e r s and consumers of o r g a n i c m a t t e r . Due t o t h e i r e x i s t e n c e w i t h i n p h y l o g e n e t i c a l l y d i v e r g e n t g r o u p s , the l a c k of c l o s e l y r e l a t e d m e s o p h i l e s and t h e i r b i o t o p e s h a v i n g e x i s t e d s i n c e t h e archaean age, h y p e r t h e r m o p h i l e s may have adapted to t h e h o t environment b i l l i o n s o f y e a r s ago.
I . BIOTOPES
Communities o f h y p e r t h e r m o p h i l i c b a c t e r i a a r e known t o e x i s t w i t h i n submarine h y d r o t h e r m a l a r e a s and c o n t i n e n t a l s o l f a t a r a s . The s u r f a c e s o f the s o l f a t a r i c f i e l d s a r e u s u a l l y r i c h i n s u l f a t e and e x h i b i t an a c i d i c pH v a l u e (0.5 t o 6 ) . A t d e p t h , s o l f a t a r a s a r e l e s s a c i d i c (pH 5 t o 7 ) . Sometimes, a c i d i c s o l f a t a r i c f i e l d s may a l s o h a r b o u r a few weakly a l k a l i n e h o t s p r i n g s (pH 7 t o 9 ) . Submarine hydro-^
t h e r m a l systems a r e s l i g h t l y a c i d i c t o a l k a l i n e (pH 5 t o 8.5) and n o r m a l l y c o n t a i n the h i g h amounts o f NaCl and SO4 p r e s e n t w i t h i n sea w a t e r . Due t o t h e low s o l u - b i l i t y of oxygen a t h i g h t e m p e r a t u r e s and the p r e s e n c e of r e d u c i n g g a s e s , most b i o t o p e s o f h y p e r t h e r m o p h i l e s a r e a n a e r o b i c . W i t h i n s o l f a t a r i c f i e l d s , oxygen i s p r e s e n t o n l y w i t h i n t h e upper ( a c i d i c ) l a y e r w h i c h appears o c h r e - c o l o r e d due t o t h e presence of f e r r i c i r o n ( 7 ) .
H y p e r t h e r m o p h i l e s may be a b l e t o s u r v i v e f o r y e a r s a t t e m p e r a t u r e s below 60°C, a l t h o u g h n o t growing under t h e s e c o n d i t i o n s . Some o f t h e a n a e r o b i c hyperthermo- p h i l e s t o l e r a t e oxygen much b e t t e r a t t h e low non-growth t e m p e r a t u r e s t h a n a t t h e growth t e m p e r a t u r e s . T h i s p r o p e r t y may be i m p o r t a n t f o r d i s s e m i n a t i o n o f t h e s e organisms t h r o u g h o x y g e n - r i c h low t e m p e r a t u r e a r e a s .
, I I . COMMUNITIES OF HYPERTHERMOPHILES WITHIN SOLFATARIC FIELDS A. Terrestrial Solfataras
The s t r o n g l y a c i d i c upper l a y e r w i t h i n t h e s o l f a t a r i c f i e l d s c o n t a i n s communi- t i e s of a e r o b i c and f a c u l t a t i v e l y a n a e r o b i c a c i d o p h i l e s w h i c h grow o n l y a t low i o n i c s t r e n g t h and a r e t h e r e f o r e non-marine o r g a n i s m s . They a r e extreme a c i d o p h i l e s (opt pH3). P h y l o g e n e t i c a l l y , t h e y b e l o n g t o t h e a r c h a e b a c t e r i a l genera Sulfolobus, Metallospharea, Acidianus and Desulfurolobus w h i c h a l l c o n s i s t o f c o c c o i d c e l l s
( 1 , 8-11). The m o d e r a t e l y t h e r m o p h i l i c , f a c u l t a t i v e l y a n a e r o b i c , p l e o m o r p h i c a r c h a e b a c t e r i u m Thermoplasma i s a l s o an extreme a c i d o p h i l e o c c u r r i n g w i t h i n t e r r e s - t r i a l s o l f a t a r i c f i e l d s ( 1 2 ) . Members o f t h e genus Sulfolobus a r e s t r i c t aerobes growing a u t o t r o p h i c a l l y by o x i d a t i o n o f S° and s T f o r m i n g s u l f u r i c a c i d .
K.O. STEUER ET AL.
Many Sulfolobus i s o l a t e s are f a c u l t a t i v e heterotrophs and are therefore "opportunis- t i cM consumers of organic matter within .the biotope. Some members of the
Sulfolobales l i k e Metallosphaera sedula are able to grow by oxidation of s u l f i d i c ores l i k e p y r i t e , chalcopyrite, and sphalerite forming s u l f u r i c acid and s o l u b i l i z - ing the heavy metal ions (9). S u l f i d i c ores l i k e p y r i t e are formed within s o l f a t a r i c f i e l d s ( S t e t t e r , unpublished). Similar to Sulfolobus, members of the genus Acidianus are able to grow by oxidation of S° (13). Some s t r a i n s of Acidianus grow also on s u l f i d i c ores, but less e f f i c i e n t l y than Metallosphaera (Huber and S t e t t e r , un- published). In contrast to a l l other members of the Sulfolobales9 Acidianus i s able to grow anaerobically on H and S° forming H2S. DesulfuroIoJbus i s very s i m i l a r to Acidianus i n i t s p h y s i o l o g i c a l properties and by DNA/DNA h y b r i d i z a t i o n (13).
Together with the obligate thermoacidophiles, s l i g h t l y a c i d o p h i l i c and n e u t r o p h i l i c hyperthermophiles are found within t e r r e s t r i a l s o l f a t a r i c f i e l d s . The l a t t e r are
s t r i c t anaerobes and may therefore mainly occur within the deeper more neutral an- aerobic zone within t e r r e s t r i a l s o l f a t a r i c f i e l d s . Members of the genera Thermopro- teus and Pyrobaculum, which consist of rod-shaped c e l l s (about 0.5 um i n width) are f a c u l t a t i v e or obligate autotrophs growing by formation of H2S from H2 and S°
(2, 14). A l t e r n a t i v e l y , the f a c u l t a t i v e autotrophs (e.g. Pyrobaculum islandium) grow by s u l f u r r e s p i r a t i o n of organic matter (15). The thin (0.17 pm i n width) filamen- tous Thermofilum and the coccoid Desulfurococcus are obligate s u l f u r r e s p i r e r s (2).
From s o l f a t a r a s i n the southwest of Iceland, rod-shaped lithoautotrophic methanogens growing at temperatures up to 97°C were i s o l a t e d which are obviously primary pro- ducers of organic matter within the biotope. Two species are known: Methanothermus fervidus and Methanothermus sociabilis (16). Within neutral continental hot springs i n D j i b o u t i , A f r i c a , was found the extremely thermophilic eubacterial species
Thermotoga thermarum which grows only at low i o n i c strength (17). Like the marine members of the genus Thermotoga, T. thermarum i s a s t r i c t l y anaerobic heterotroph growing by fermentation of carbohydrates.
S. Submarine Hydrothermal Systems
Many hyperthermophiles are adapted to the marine thermal environments. They are represented by primary producers of organic matter l i k e members of the genera
Pyrodictium, Archaeoglobus and Methanococcus and by consumers l i k e Staphylothermus9 Thermodiscus, Thermococcus, Pyrococcus and Thermotoga. The organisms with the highest growth temperatures are members of Pyrodictium, growing up to 110°C (18). C e l l s of Pyrodictium are so well adapted to high temperatures that they do not even grow below 80°C. Pyrodictium occultum and Pyrodictium brockii are able to grow a u t o t r o p h i c a l l y , gaining energy by reduction of S° by H^. Cultures of Pyrodictium grow i n f l o e s , c e l l s being disc-shaped and connected By a network of very thin hollow f i b r e s . Many submarine hydrothermal systems contain coccoid-shaped archaebacterial s u l f a t e reduces of the genus Archaeoglobus. Archaeoglobus fulgidus i s a f a c u l t a t i v e auto-
troph gaining energy'by reduction of s u l f a t e or t h i o s u l f a t e by H2. I t grows hetero- t r o p h i c a l l y by s u l f a t e r e s p i r a t i o n on various organic substances (19). Archaeoglobus members grow at temperatures up to 90°C. S i m i l a r to methanogens, c e l l s of Archae- oglobus show a blue-green fluorescence i n the UV l i g h t at 420 nm due to the posses- sion of f a c t o r 420 (19). A further autotrophic marine hyperthermophile i s
Methetnococcus jannaschii which grows at temperatures up to 86°C (20). Very recently, novel rod-shaped methanogens which grow at least at 110°C (Stetter, unpublished) were i s o l a t e d from an abyssal hydrothermal system. The marine thermal environment contains also a v a r i e t y of s t r i c t l y heterotrophic hyperthermophiles. Staphylothermus and Thermodiscus are coccoid and disc-shaped s u l f u r r e s p i r e r s growing on various kinds of organic matter (21). The genera Thermococcus and Pyrococcus are coccoid c e l l s which are widely d i s t r i b u t e d within marine hydrothermal systems (22, 23).
Thermococcus celer u t i l i z e s tryptone, yeast extract and protein as carbon sources.
Growth i s stimulated by sucrose. In closed culture vessels, optimal growth i s ob- tained i n the presence of s u l f u r and about 1.5 moles of H2S are formed per mole of C02 (22). Thermococcus and Pyrococcus can also grow without s u l f u r by an unknown type of fermantation. Pyrococcus furiosus grows at temperatures up to 103°C and shows a much lower GC-content than Thermococcus celer (23). At 100°C, the doubling
HYPERTHERMOPHILIC BACTERIAL COMMUNITIES
time i s o n l y 37 m i n u t e s . The mode of f e r m e n t a t i o n of Pyrococcus and Thermococcus i s s t i l l u n c l e a r . Many submarine h y d r o t h e r m a l f i e l d s c o n t a i n r e p r e s e n t a t i v e s of the e u b a c t e r i a l genus Thermotoga w h i c h t h r i v e t o g e t h e r w i t h h y p e r t h e r m o p h i l i c a r c h a e - b a c t e r i a i n the same e n v i r o n m e n t . Members of Thermotoga can be e a s i l y i d e n t i f i e d by t h e i r rod-shape and o u t e r s h e a t h - l i k e s t r u c t u r e o v e r b a l l o o n i n g a t the ends ( 5 ) . Thermotoga maritima and Thermotoga neapolitana are f e r m e n t a t i v e h y p e r t h e r m o p h i l e s growing a t t e m p e r a t u r e s up t o 90°C ( 5 , 2 4 ) . They use v a r i o u s c a r b o h y d r a t e s as energy s o u r c e s f o r m i n g as end p r o d u c t s , L - l a c t a t e , a c e t a t e , and ( 5 ) .
I I I . DISCUSSION
The i s o l a t i o n of d i f f e r e n t groups of a u t o t r o p h i c and h e t e r o t r o p h i c b a c t e r i a from g e o t h e r m a l l y and h y d r o t h e r m a l l y heated e n v i r o n m e n t s shows an unexpected v a r i e t y of organisms w i t h i n t h e s e almost u n e x p l o r e d e c o s y s t e m s . W i t h i n t h e s e , the p r i m a r y p r o - d u c t i o n of o r g a n i c m a t t e r and consumption p r o c e e d s a t t e m p e r a t u r e s up t o about
110°C. The e n e r g y - y i e l d i n g r e a c t i o n s a r e based on o x i d a t i o n or r e d u c t i o n of i n o r - g a n i c s u l f u r compounds by 0^ or H^. I n the case of methanogens, C 0o i s r e d u c e d by
A e r o b i c h y p e r t h e r m o p h i l i c a u t o t r o p h s seem t o o c c u r o n l y w i t h i n " " a c i d i c t e r r e s - t r i a l s o l f a t a r i c f i e l d s . The a n a e r o b i c h y p e r t h e r m o p h i l i c a u t o t r o p h s use H^, C 0? and S° w h i c h a r e formed w i t h i n the e n v i r o n m e n t . These o r g a n i s m s are t h e r e f o r e com- p l e t e l y independent of any sun. The consumers of o r g a n i c m a t t e r a r e most l i k e l y u s i n g c e l l components of d e c a y i n g p r i m a r y p r o d u c e r s . Most of them grow by s u l f u r r e s p i r a t i o n and f e r m e n t a t i o n . Many s t r a i n s of h y p e r t h e r m o p h i l i c a u t o t r o p h s a r e
f a c u l t a t i v e l y h e t e r o t r o p h i c . T h i s p r o p e r t y may be an " o p p o r t u n i s t i c " f e a t u r e and may be i m p o r t a n t f o r s u c c e s s f u l c o m p e t i t i o n w i t h i n t h i s extreme e n v i r o n m e n t .
SUMMARY
H y p e r t h e r m o p h i l i c b a c t e r i a l communities w i t h i n t e r r e s t r i a l and m a r i n e t h e r m a l areas are v e r y complex. They c o n s i s t of chemol t o i h o a u t o t r o p h i c and h e t e r o t r o p h i c b a c t e r i a , growing o p t i m a l l y between 80° and 110°C. P r i m a r y p r o d u c t i o n and consumption of o r g a n i c m a t t e r i s g o i n g on a t these h i g h t e m p e r a t u r e s .
REFERENCES
1. Brock, T.D. (1978) T h e r m o p h i l i c m i c r o o r g a n i s m s and l i f e a t h i g h t e m p e r a t u r e s . S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New Y o r k .
2. S t e t t e r , K. 0. & Z i l l i n g , W. (1985) Thermoplasma and t h e t e r m o p h i l i c s u l f u r - dependent a r c h a e b a c t e r i a . In The B a c t e r i a , V o l . V I I I . ( W o l f e , R. S. & Woese, C R . , e d . ) , pp. 85-170, Academic P r e s s , New Y o r k .
3. S t e t t e r , K.O. (1986) D i v e r s i t y of e x t r e m e l y t h e r m o p h i l i c a r c h a e b a c t e r i a . In T h e r m o p h i l e s , G e n e r a l , M o l e c u l a r and A p p l i e d M i c r o b i o l o g y ( B r o c k , T. D. e d . ) . pp. 39-74, J . W i l e y and Sons I n c . , New Y o r k , London, Sydney, T o r o n t o .
4. Woese, C. R., Magrum, L. J . & Fox, G. E. (1978) J . M o l e c . E v o l u t i o n 11, 245-252.
5. Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., S l e y t r , U. B. & S t e t t e r , K. 0. (1986) A r c h . M i c r o b i o l . 144, 324-333.
6. Woese, C. R. (1987) M i c r o b i o l . Rev. 51, 221-271.
7. S t e t t e r , K. 0., S e g e r e r , A., Z i l l i g , W., Huber, G., F i a l a , G., Huber, R. &
König, H. (1986) System. A p p l . M i c r o b i o l . 7, 393-397.
8. B r o c k , T. D., B r o c k , K. M., B e l l y , R. T. & W e i s s , R. L. (1972) A r c h . M i k r o b i o l . 84, 54-68.
9. Huber, G., S p i n n l e r , C , Gambacorta, A. & S t e t t e r , K. 0. (1989) System. A p p l . M i c r o b i o l . , i n p r e s s .
110. S e g e r e r , A., Neuner, A., K r i s t j a n s s o n , J . K. & S t e t t e r , K. 0. (1986) I n t . J . S y s t . B a c t . 36, 559-564.
K.O. STETTER ET A L
11. Z i l l i g , W., Y e a t s , S., H o l z , I . , Böck, A., Gropp, F. & Simon, G. (1987) S y s t . A p p l . M i c r o b i o l . 8, 197-203.
\2. S e g e r e r , A., Lanfcvorthy, T. A. & S t e t t e r , K* 0, (1988) System, Appl* M i c r o b i o l . 10, 161-171.
13. Huber, R., Huber, G., S e g e r e r , A., S e g e r , J . & S t e t t e r , K. 0. (1987) A e r o b i c and a n a e r o b i c e x t r e m e l y t h e r m o p h i l i c a u t o t r o p h s . In M i c r o b i a l growth on C1 compounds
(van V e r s e v e l d , H. W. & D u i n e , J . A., e d . ) , pp. 44-51, P r o c e e d i n g s o f t h e 5 t h I n t e r n a t i o n a l Symposium, M a r t i n u s N i j h o f f P u b l . , D o r d r e c h t .
14. Z i l l i g , W., S t e t t e r , K. 0., Schäfer, W., J a n e k o v i c , D., W u n d e r l , S., H o l z , I.
& Palm, P. (1981) Z b l . B a k t . Hyg., I . A b t . O r i g . C 2, 205-227.
15 Huber, R., K r i s t j a n s s o n , J . K. & S t e t t e r , K. 0. (1987) A r c h . M i c r o b i o l . 10, 161-171.
16. L a u e r e r , G., K r i s t j a n s s o n , J . K., L a n g w o r t h y , T. A., König, H. & S t e t t e r , K.O.
(1986) System. A p p l . M i c r o b i o l . 8, 100-105.
17. W i n d b e r g e r , E., Huber, R., T r i n c o n e , A., F r i c k e , H. & S t e t t e r , K. 0. (1989) A r c h , M i c r o b i o l . 151, 506-512.
18. S t e t t e r , K. 0., König, H. & S t a c k e b r a n d t , E. (1983) System. A p p l . M i c r o b i o l . 4, 535-551.
19. S t e t t e r , K. 0. (1988) System. A p p l . M i c r o b i o l . 10, 172-173.
20. J o n e s , W. L., L e i g h , J . A., Mayer, P., Woese, C. R. & W o l f e , R. S (1983) A r c h M i c r o b i o l . 136, 254-261.
21. F i a l a , G., S t e t t e r , K. 0., J a n n a s c h , H. W., L a n g w o r t h y , T. A. & Madon, J . (1986) System A p p l . M i c r o b i o l . 8, 106-113.
22. Z i l l i g , W., H o l z , I . , J a n e k o v i c , D., Schäfer, W. & R e i t e r , W. D. (1983) System. A p p l . M i c r o b i o l . 4, 88-94.
23. F i a l a , G. & S t e t t e r , K. 0. (1986) A r c h . M i c r o b i o l . 145, 56-61.
24. J a n n a s c h , H. W., Huber, R., B e l k i n , S. & S t e t t e r , K. 0. (1988) A r c h . M i c r o b i o l . 150, 103-104.
