Alkalithermophiles - Biochemical Society Transactions

Thermophiles 2003

Alkalithermophiles J. Wiegel*1 and V.V. Kevbrin*† *Department of Microbiology, University of Georgia, Athens, GA 30602 2605, U.S.A., and †Institute of Microbiology RAS, Prospect 60 letija Octiabria, 7/2, 117312 Moscow, Russia

Abstract Alkalithermophiles are an exciting subset of extremophilic organisms and represent extremophiles that are adapted to two extreme conditions, i.e. to a combination of alkaline and thermobiotic growth conditions. Among the anaerobic alkalithermophiles are representatives of both Bacteria and Archaea within a wide variety of physiological types and systematic groups, although a great majority belongs to the Firmicutes. Alkaliphiles have been isolated from a variety of niches including mesobiotic and neutrophilic soils and sediments. Interestingly anaerobic isolates from mesobiotic and neutrophilic niches exhibit shorter doubling times than isolates from thermobiotic niches; some anaerobic alkalithermophiles exhibit extremely fast growth rates, i.e. doubling times as short as 10 min. Their adaptation to both high pH and high temperature draws our attention not only because they are potential sources of industrially valuable enzymes but also because of their adaptive mechanisms to external environmental parameters. They could thus function as model organisms for extraterrestrial life in some environments and for theories on the origin of life. Alkalithermophiles, as far we know, do not represent the most thermophilic nor the most alkaliphilic of micro-organisms but represent the most alkaliphilic ones among the thermophiles and vice versa. We believe that the presently known species are only the tip of the iceberg and therefore that they do not represent the true boundaries under which life can thrive in respect to high temperature in alkaline environments.

Alkalithermophiles, a subset of extremophiles Micro-organisms not only growing under but requiring unusual, thus extreme, environmental conditions for growth are grouped together under the term extremophiles. They come in all kinds of forms. Many of them cannot even survive under what we call, from an anthropogenic point of view, ‘normal conditions’, i.e. conditions conducive to the human physiology and for most of the micro-organisms associated with humans, which are mesophilic neutrophilic micro-organisms. Micro-organisms preferring extreme conditions include: heat-loving, i.e. hyper (T opt above 80◦ C), extreme (T opt above 65◦ C) and thermophiles (T opt above 55◦ C); cold-loving, i.e. psychrophiles (T opt < 15◦ C); acid- or alkaline-loving, i.e. acidophiles (pHopt < 5.0) and alkaliphiles (pHopt > 8.0), respectively; salt-loving, i.e. halophiles; extremely low-substrate-concentration-preferring or -requiring, i.e. oligophiles or ‘oligotrophs’; and high-pressureloving, i.e. barophiles or piezophiles. Interestingly, the opposite to piezophiles, so far, have not been isolated, i.e. no bacteria have been described which require reduced pressure for optimal growth, despite micro-organisms existing at extremely high altitudes. Also micro-organisms growing at high concentrations of heavy metal ions, under high doses of γ and UV radiation, high solvent concentrations or very low-water-activity conditions, i.e. dry-resistant microorganisms from the desert, are regarded as extremophiles. We Key words: aerobe, alkalithermophile, anaerobe, biodiversity, extremophile. 1 To whom correspondence should be addressed (e-mail [email protected]).

maintain furthermore that bacteria that can grow extremely fast with doubling times below 15 min (‘hyperauxanophiles’ i.e. significantly faster than Escherichia coli) should be regarded also as extremophiles. Examples include some of the alkalithermophiles with doubling times around 10 min (see below) or the neutrophilic marine mesophile Vibrio natriegans with doubling times below 10 min. In other words what is ‘normal’ for E. coli and similar micro-organisms is extreme for the extremophiles and vice versa. Thus mesophilic and neutrophilic conditions and atmospheric pressure are just physicochemical parameters in the ‘middle of the road’. Nearly all presently known physiological types are represented among the extremophiles, e.g. phototrophs, organoheterotrophs, chemo-organotrophs and chemolithoautotrophs. With respect to microbial diversity, the word on what the prevalent microbial living conditions are is still undecided. This is because (i) we assume that we only know and can culture around 1–2% of the probably existing species and (ii) the diversity of micro-organisms in the huge environments of the deep cold ocean or deep subsurface with oligotrophic growth conditions are very little explored. However, according to Whitman et al. [1], these reservoirs harbour the highest numbers of microbial cells. The mesophiles associated with humans, animals and to some extent with plants have captured most of the attention in the past, despite the fact that some extremophiles have been used for decades in leather making, dye making and food-fermenting processes. Yet only during the last 10–15 years have various extremophiles, especially (hyper)thermophiles, entered more into mainstream research in microbiology and biochemistry. C 2004

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This is in part due to the potential for use in industrial applications at a time of renewed interest in environmentally friendly biotechnology applications as well as to the theory that life began under thermobiotic conditions. Although in the past regarded as a curiosity, moderate thermophiles, such as the aerobic thermophiles Geobacillus stearothermophilus and other related species, have been known for more than 100 years. Species such as the anaerobic, cellulolytic thermophile Clostrididum thermocellum were in the 1920s already included in patents for industrial fermentations to produce ethanol and other chemicals. Many novel species have been described recently, and the rate of discovery is increasing. This is fostered by the relative ease of using 16 S rDNA sequence analysis to determine whether olate represents a novel species/genus or not. Furthermore, environmental 16 S rDNA sequence analyses are showing that there are hundreds of unknown micro-organisms in even 1 g of ordinary garden soil. Many of the described extremophiles, known either for 100 years or only recently isolated, are characterized by only one distinctive ‘extreme’, as indicated by the above list of extremophiles. One of the exceptions are the obligately aerobic hyperthermophiles, which are all acidothermophiles requiring elevated temperatures above 80◦ C and pH values below 4.0. Apparently, combinations of extreme parameters are making it more difficult for micro-organisms to maintain optimal growth within a wider range of conditions. On the other hand, many environments exhibit various combinations of extreme environmental conditions, such as elevated temperature, non-neutral pH, high salt and/or high UV radiation. These environments contain micro-organisms able to cope with combinations of stress factors. 16 S rDNA sequence analyses have indicated that the biodiversity in extreme environments is lower than in mesobiotic, neutral soil or sediments. Whereas this is apparently true for several studied environments, for some others, e.g. the African Rift Valley lakes, this assumption might be incorrect. One example of multiextremophiles are the alkalithermophiles, to be discussed here: micro-organisms which grow at elevated temperatures and alkaline pH. Two species among these even require additional elevated salt concentrations. We believe that in the future, when more extreme environments are thoroughly investigated and unusual conditions are applied for isolating micro-organisms, many novel extremophiles will be found requiring multiple extreme conditions for growth. The biodiversity will be further extended beyond the present physicochemical boundaries for microbial growth.

Boundaries for life: the combination of elevated growth temperatures and alkaline pH as represented by isolated alkalithermophiles The comparison of published data for alkalithermophiles (as well as for acidothermophiles) has to be done cautiously. In most cases the media pH values have been determined C 2004

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using pH meters and electrodes calibrated at 25◦ C (or less, defined as room temperature) with or without a temperature probe for corrections. However, temperature probes cannot compensate for the effect of temperatures on the pK values of media components. In rich media this can make a difference of 1 pH unit when comparing, for example, values measured at room temperature with values determined using the pH electrode equilibrated and the pH meter calibrated at a growth temperature of 60◦ C. An example is the data for Clostridium paradoxum: the pHopt determined at 25◦ C is 10.1 with a pHmax of 11.3, whereas, if correctly determined at the T opt of 56◦ C, the values are 9.3 and 10.3 [2,3]. As suggested by Wiegel [4,5,5a] the marginal pH data for these extremophiles should always contain, in the form of a superscript, the temperature at which the pH was determined and the pH meter calibrated ◦ (e.g. pH55 C opt ). Although the alkalithermophilic species described to date neither represent the most thermophilic nor the most alkaliphilic micro-organisms known, they are the most alkaliphilic among the thermophiles and the most thermophilic among the alkaliphiles. In other words, from the very limited data presently available, the combination of two (or three, if one looks at the halophilic alkalithermophiles) ‘extremes’ of physicochemical growth parameters restricts the range in which micro-organisms can proliferate more than does a single extreme. Graphs depicting the optimal growth temperature versus optimal growth pH clearly show that with increasing temperature, the pH range becomes more limited [6]. The most alkalithermophilic anaerobe, ◦ C. paradoxum, with a pH25 C opt for growth at 10.3 and a maximal pH of 11.3, has its T opt around 56◦ C, whereas the most thermophilic ones, Thermococcus alkaliphilus and Thermococcus acidaminovorans [7,8], growing optimally around 85◦ C only exhibit a pHopt of 9.0. Among the aerobic alkalithermophiles, Bacillus alcalophilus B-M20 represents the most alkaliphilic, with a pHopt around 10.5 at a T opt around 65◦ C. With increasing T opt the pHopt becomes less alkaline again: at a T opt of 72◦ C the pHopt is around 9.2 (Bacillus sp. TA2.A1) and at a T opt of 80◦ C, the pHopt is only around pH 8.0 (‘Bacillus caldotenax’ YT G). However, the authors believe that these presently observed boundaries are probably not the true boundaries for growth of alkalithermophiles but rather reflect the isolation and culturing limits of the past and present investigators. Interestingly, very little is known about alkalipsychrophiles, another unexplored group of extremophiles, and whether or not a similar apparent correlation exists, i.e. the lower the temperature, the more restricted the pH range becomes [9].

Habitats of isolated alkalithermophiles At a first glance, one would assume that the major environments from which to isolate alkalithermophiles would be alkaliphilic and thermobiotic environments such as alkaline hot springs, the new alkaline hydrothermal vents of the ‘Lost City’ or alkaline lakes like Lake Bogoria (Africa)

Thermophiles 2003

Table 1 Anaerobic alkalithermophiles n, not known; T opt , temperature optimum for growth rate. Species

Determined pHopt at ◦ C T opt (◦ C)

Clostridium paradoxum

10.3

55

54–58*

Clostridium thermoalcaliphilum Anaerobranca gottschalkii ‘Thermopallium natronophilum’

9.8 9.5 9.5

50 n n

49 53 70

Anaerobranca sp. KS5Y Thermosyntropha lipolytica Desulfotomaculum alkaliphilum

8.7 8.5 8.6

n 25 n

57 63 53

Anaerobranca horikoshii Halonatronum saccharophilum Thermobrachium celere

8.5 8.3 8.2

60 n 60

57 46 65–67*

Caloramator indicus Thermococcus acidaminovorans Thermococcus alkaliphilus

8.1 9 9

n n n

63 85 85

Thermococcus fumicolans Methanothermobacter thermautotrophicus AC60

8.5 8

n n

85 60

Methanothermobacter thermoflexus

8

n

55

*Depending on the strain.

containing hot springs from which three anaerobic alkalithermophiles have been isolated [10] (Table 1). However, many alkalithermophiles have also been isolated from mesobiotic, slightly acidic to neutrophilic habitats [5]. For instance the anaerobic alkalithermophile with the highest pH optimum isolated so far comes exclusively from sewage sludges with maximally observed bulk temperatures below 35◦ C and bulk pH values never above 7.4 (data for sewage plants in Athens and Atlanta, GA, U.S.A.). All samples from analysed sewage plants from four continents contained C. paradoxum/Clostridium thermoalkaliphilus-like bacteria. It is interesting to note that also the aerobic alkalithermophiles, Bacillus thermocloacae [11], Sphaerobacter thermophilus [12], and the methanogenic Archaea Methanobacterium thermoflexum and Methanobacterium defluvii [13] have been isolated from sewage sludge, but no attempts were made to determine their MPNs (most probable numbers) or distribution. Also Thermobrachium celere, the alkalithermophile with the shortest doubling time of 10 min, has been isolated not only from many mesobiotic river and lake sediments, but also various hot springs (see discussion below) [14]. In general terms, one can categorize alkalithermophiles according to the combinations of geographical distribution and the geochemistry of the niches from which they have been isolated. Thus, there are alkalithermophiles found: (i) only in one place (restricted biogeography) but with a relaxed biogeochemistry requirement, e.g. Anaerobranca horikoshi [15] is found only in one place in Yellowstone National Park but from both alkaline and acidic springs at various temperatures; (ii) in one type of environment (restricted biogeochemistry) but in various continents of the globe (relaxed biogeography), e.g. C. paradoxum and C.

thermoalkaliphilum only in sewage plants or hot springs receiving grey water [2,16]; (iii) in many different kinds of places and locations, i.e. ubiquitous species (relaxed biogeochemistry and relaxed biogeography), e.g. Bacillus spp. and T. celere, which have been isolated from alkaline hot springs, mesobiotic lake and river sediments, wet meadow grounds and manure. In short, the environment where alkalithermophiles have been isolated encompasses mesobiotic and thermobiotic environment of natural (sediments, soil, manure piles), and anthropogenic origin. Since many of the aerobic species and some of the anaerobes are spore formers, one can conclude that this group of the alkalithermophiles is ubiquitous with respect to geographical distribution. The question of whether Beijering’s hypothesis that every micro-organism is (potentially) everywhere and the environment selects, as reported by Baas Becking [17], cannot really be answered. There are not enough isolates obtained and the environments have not been analysed in enough detail. Furthermore, the question is whether indigenous strains successfully out-compete similar but introduced strains. This situation is similar to the observations that introduced Rhizobium strains which are genetically engineered for higher nitrogen-fixing activity are usually out-competed within a year by the indigenous strains. Thus small differences in the physicochemical properties in an alkalithermobiotic niche would not allow the introduced strains to compete with the indigenous micro-organisms and to grow into a sizable, detectable population. However, the introduced strain might be present in very small numbers and thus not detected by culturing or 16 S rDNA sequence analysis. Recently it has been demonstrated that for soil samples, more than 800 clones would have to be sequenced to come close to saturation in a saturation plot, i.e. to find the sequences for all microorganisms present in a given environment. Is a species present in a sediment if, e.g. 10 cells of it exist among 109 cells of other micro-organisms in a gram of soil or sediment or as a few dormant spores? Thus the answer to Beijering’s question [17] depends on the available detection limits and on the definition of what constitutes ‘presence’.

Relationship between habitat and growth rates A truly general correlation cannot be established at this time due to the restricted number of isolated alkalithermophiles within the different physiological groups. However, among the anaerobic heterotrophic alkalithermophiles, an interesting trend was observed. Strains isolated from mesobiotic environments have short doubling times below 30 min and as short as 10 min, whereas all the strains isolated from thermobiotic environments have doubling times above 30 min. This correlation is observed for different species and for strains within the same species (based on 16 S rDNA sequence analyses for isolates from T. celere). A plausible explanation is that the organisms in these mesobiotic environments exist there due to temporary C 2004

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Figure 1 Clostridium paradoxum (A) pH profile for three strains. (B) Corresponding temperature profile of strains from (A). (C) Internal pH versus external pH.

alkaline and thermobiotic microniches. To be able to grow in these temporary microniches (e.g. created by degradation of biomass releasing thermal energy and ammonia), the organisms have to be able to grow fast, whereas there is no such selection in thermobiotic environments in which microorganisms grow continuously, as in a chemostat.

Physiology and energy metabolisms Among the aerobic and microaerophilic alkalithermophilic Bacillus and related species, the major unifying physiology is the capability to degrade proteinaceous material, i.e. possessing potential enzymes for industrial applications. As indicated above, this property is due to the chosen isolation procedures. Many of these species can also utilize acids and sugars as carbon and energy sources. Except for the Anoxybacillus, they are typical aerobes, whereas the latter is a microaerophilic and facultative aerobe as we have recently demonstrated for Anoxybacillus pushinoensis [18] and strain Anoxybacillus KG4 from Kamchatka (V. Kevbrin and J. Wiegel, unpublished work). In contrast to this, the diversity of the anaerobic alkaliphiles is much broader. It also includes proteolytic species isolated on casein (C. paradoxum, C. thermoalkaliphilum, A. horikoshii, T. celere) [5,5a]. The main fermentation products are acetate and H2 /CO2 . Most strains also grow well on carbohydrates, although frequently with a restricted substrate spectrum. Fermentation products include acetate as the main product, plus lactate, ethanol, CO2 and H2 . The saccharolytic species Thermoanaerobacter and Caloramator spp. produce mainly acetate, ethanol and lactate besides H2 /CO2 . Thermoanaerobacter ethanolicus, a facultative alkalithermophile with a very broad pH optimum between pH 5.5 and nearly 9, produces up to 1.9 mol of ethanol/mol of glucose utilized. Furthermore it includes the lipolytic, nonsaccharolytic syntroph Thermosyntropha lipolytica and the sulphate reducer Desulfotomaculum alkaliphilum [19] and recently isolated iron reducers closely related to non-Fe C 2004

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reducing utilizer CO ([20] and T. Sokolova and J. Wiegel, unpublished work). The anaerobic alkalithermophiles also include the chemolithoautotrophic methanogenic Archaea Methanothermobacter thermoflexus, M. thermoautotrophicus strain thermoalkaliphilus [21,22] and Methanohalophilus zhilinae [23]. It also includes three heterotrophic archaeal Thermococcus species. For the aerobic mesophilic alkaliphilic Bacillus spp., a pH homoeostasis has been observed. They even can adjust the medium pH to their optimum from either side [24]. In contrast, the anaerobic alkalithermophile C. paradoxum [3] as well as the aerobic alkalithermophile Bacillus strain TA2.A1 [25,26] exhibit a change in the intracellular pH with changing medium pH; they maintain a pH gradient. The largest pH occurs around the pH optimum for growth, at which the ATP concentration in the cell is also high. However, in contrast to neutrophiles, the pH is reversed with a more acidic pH inside, thus reducing the total proton-motive force (p). At the corresponding medium pH where no pH is observed, the bacterium does not grow (Figure 1). However, the pH is smaller than that observed in mesophilic alkaliphiles [27– 29]. In tests so far, all alkaliphiles still synthesize ATP via a proton coupled, membrane bound ATPase. Driessen et al. [30] showed that thermophiles are leaky for protons, further reducing the efficiency in alkalithermophiles. No indications for the presence of a PEP (phosphoenolpyruvate) transfer system for sugar uptake has been observed; however, the functioning of sodium-dependent transport systems could be shown. All alkalithermophiles grow better in the presence of NaCl, although the levels vary greatly among the different taxa [3,6,24,28,29,31,32]

Alkalithermophiles: potential models of primitive micro-organisms It is believed that some of the optimal growth conditions for alkalithermophiles could represent conditions under which life evolved [4], e.g. in the late Hadean Sea or in extraterrestrial

Thermophiles 2003

Table 2 Facultative and aerobic alkalithermophiles For taxonomically identified bacteria, names are given according to the most recent validation lists published in http://www.bacterio.cict.fr/ac.html based on publications in the International Journal of Systematic and Evolutionary Microbiology. T opt , temperature optimum for growth rate. Species

Comments

pH (range)

T opt (◦ C) Enzymes produced

Bacillus alcalophilus B-M20

Tolerates up to 7.5% NaCl

10.6 (8–12)

60–65

Extracellular lipase (pH 10.6 and 60◦ C)

Thermoactinomyces sp. HS682

Tolerates up to 10% NaCl

10.3 (7.5–11.5)

50

10.0* (ND)

60*

Extracellular serine protease. Purified: 11.5 and 70 Purified: 9.0 and 75◦ C. Extracellular protease

Geobacillus stearothermophilus F1 Anoxybacillus pushchinoensis K1T Bacillus halodurans

Reduces nitrate to nitrite 9.5–9.7 (8.0–10.5) 62 1 out of 16 strains; tolerates 9–10 (ND) 55 up to 12% NaCl

Has been studied in terms of systematic position

Bacillus sp. TA2-A1 Sphaerobacter thermophilus S6022T Bacillus thermocloaceae S6025T

100 mM NaCl is required

Has been studied in terms of bioenergetics

9.2 (7.7–10.5) 8.5 (ND) 8–9 (7–?)

70 55 55

Thermomicrobium roseum ATCC 27502T Contains carotenoids Bacillus pallidus H12T At 10% NaCl

8.2–8.5 (6.0–9.4) 8.0–8.5 (ND)

70–75 60–65

Bacillus ‘caldotenax’ YT G

7.5–8.5 (ND)

80

Anoxybacillus ‘kamchatkensis’ KG4T

6.8–8.5 (5.7–9.9)

57–62

Thermoactinomyces sacchari A-1

8.5* (ND)

50*

Extracellular amylase, alkaline phosphatase, protease: 70◦ C Cellulase free extracellular endo-1,4-β-xylanase (EC 3.2.1.8)

*The conditions at which cultivation was performed were not unambiguously described.

environments such as on Mars. Conflicting hypotheses have emerged regarding ocean chemistry during this early period of the Earth’s history. Holland [33] and Walker [34] proposed that the early ocean chemistry was similar to that observed today. Kempe and Kazmierczak ([35] and literature cited therein), on the other hand, proposed that the chemistry of the early Precambrian ocean was similar to that of soda lakes (NaCO3 -rich), such as the lakes in the modern rift valley systems (e.g. East Africa) with a pH in excess of 9 [36,37]. Morse and Marion [37] discuss the earliest Hadean ocean (4.3–3.8 Gyr) being hot (70–100◦ C) and slightly acidic (pH 5.8 ± 0.2), but not alkaline. The present-day alkaline pH and ocean water chemistry evolved by about 3.8 Gyr. Alkaline springs could have contributed dissolved solids to the Hadean oceans, facilitating the evolution of life as discussed by Morse and Marion [37], Schwartzman in [4] (pp. 33–46) and by Russell, Daia and Hall in [4] (pp. 77–126). It will be interesting to see what kind of diverse micro-organisms will be obtained from the recently discovered alkaline hot system of the ‘Lost City’, which represents a novel system, and whether these microorganisms represent deep branches in the 16 S rDNA-based phylogenetic tree. We observed isolated Fe(III) reducers, grown under chemolithoautotrophic conditions, reducing Fe(III) (leading to the formation of magnetite) at pH values above pH 10.0 and 60◦ C at geologically and ecologically relevant rates of around 1 µmol · ml−1 · day−1 . Subsequently we hypothesize that this type of bacterium could have been responsible for origin of the Precambrian banded iron formations [20].

Presently more and more evidence suggests that liquid water has flowed [38] or is still today flowing under the surface of Mars. Seeps or even ocean-like bodies of water, as Martian analogues to the Earth’s salt pans and saline lakes, may have existed. Thus salt-tolerant alkali thermophiles such as Halonatronum saccharophilum [39] and ‘Bacillus thermoalkaliphilus’ [40] could be examples of analogous extremophilic micro-organisms from such extraterrestrial environments. The physiology of some of the presently described alkalithermophiles (e.g. T. celere or C. paradoxum with doubling times of and below 15 min) makes them interesting models for studying extraterrestrial life and hypothetical models of the origin of life.

Industrial applications Alkalithermophiles are promising sources for a multitude of enzymes for biotechnological applications [41] (Table 2). These include mainly extracellular enzymes: (i) saccharolytic (hydrolysing starch and xylan-related polymers), (ii) proteolytic enzymes (from various Bacillus isolates, growing optimally above 55◦ C and at pH values above 8.5) and (iii) lipases (T. lipolytica) [6,39,42–47]. To describe the different extracellular hydrolytic enzymes from various alkalithermophilic Bacillus strains is beyond the scope of this summary and the reader is referred to other reviews [46]. To some extent, intracellular enzymes, such as isomerases [48], oxidoreductases and hydrogenases, are also of interest for combined enzymic chemical synthesis reactions for reactions otherwise difficult to carry out solely by C 2004

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chemical processes. There is a need to find enzymes working at elevated temperatures, alkaline pH values, and in the presence of organic solvents for synthesis of, e.g. stereospecific compounds. Since the intracellular pH of alkaliphiles is more acidic than the medium (see above), intracellular enzymes are expected to be somewhat less alkaliphilic/ alkali-stable. However, as discussed above (see Figure 1C) ◦ the intracellular pH still can be above pH55 C 8.5 under the optimal growth conditions and therefore cells should contain enzymes which are more alkaline-stable than their counterparts from neutrophilic micro-organisms. Almost no research has been done so far on these enzymes from alkalithermophiles. However, after the genome sequence for Anaerobranca gottschalkii has been obtained, this should change in the near future ([31], and presentation at Extremophiles 2003). Applications for alkalithermophilic enzymes include modification of starchy and fat-containing materials in the food industry, leather making, the pulp and paper industries and semi-chemical synthesis processes [49].

Conclusion In conclusion, the further study of this interesting, but rarely studied, subgroup of thermophiles should be warranted due to their unique properties of being the most alkaliphilic thermophiles and containing some of the fastest growing micro-organisms. Some of the taxa are of interest for different scientific aspects including biodiversity and originof-life studies. Several taxa exhibit the potential to be useful in biotechnological processes. It is expected that in the near future further alkalithermophilic micro-organisms will be isolated with growth ranges beyond those presently observed.

References 1 Whitman, W.B., Coleman, D.C. and Wiebe, W.J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6578–6583 2 Li, Y., Mandelco, L. and Wiegel, J. (1993) Int. J. Syst. Bacteriol. 43, 450–460 3 Cook, G.M., Russell, J.B., Reichert, A. and Wiegel, J. (1996) Appl. Env. Microbiol. 62, 4576–4579 4 Wiegel, J. and Adams, M.W.W. (eds.) (1998) Thermophiles, the Keys to Molecular Evolution and Origin of Life, Taylor and Francis, London 5 Wiegel, J. (1998) Extremophiles 2, 257–267 5a Wiegel, J. (2002) in Encyclopedia of Environmental Microbiology (Bitton, G., ed.), pp. 3127–3140, John Wiley and Sons, New York 6 Reference deleted 7 Keller, M., Braun, F.J., Dirmeier, R., Hafenbradl, D., Burggraf, S., Rachel, R. and Stetter, K. (1995) Arch. Microbiol. 164, 390–395 8 Dirmeier, R., Keller, M., Hafenbradl, D., Braun, F.J., Rachel, R., Burggraf, S. and Stetter, K.O. (1998) Extremophiles 2, 109–114 9 Zavarzin, G.A., Zhilina, T.N. and Kevbrin, V.V. (1999) Microbiology 68, 503–521 10 Jones, B.E., Grant, W.D., Duckworth, A.W. and Owenson, G.G. (1998) Extremophiles 2, 191–200 11 Demharter, W. and Hensel, R. (1989) Syst. Appl. Microbiol. 11, 272–276 12 Demharter, W., Hensel, R., Smida, J. and Stackebrandt, E. (1989) Appl. Microbiol. 11, 261–266 13 Kotelnikova, S.V., Obraztsova, A.Y., Gongadze, G.M. and Laurinavichius, K.S. (1993) Syst. Appl. Microbiol. 14, 427–434

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14 Engle, M., Li, Y., Rainey, F., DeBlois, S., Mai, V., Reichert, A., Mayer, F., Messmer, P. and Wiegel, J. (1996) Int. J. Syst. Bacteriol. 46, 1025–1033 15 Engle, M., Li, Y., Woese, C. and Wiegel, J. (1995) Int. J. Syst. Bacteriol. 45, 454–461 16 Li, Y., Engle, M., Mandelco, L. and Wiegel, J. (1994) Int. J. Syst. Bacteriol. 44, 111–118 17 Baas Becking, L.M.G. (1934) Geobiologie of inleiding tot de miliekunde (van Stockumand, W.P., ed.), pp. 13 and 15, N.V. Zoon, The Hague 18 Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G. and Laurinavichius, K. (2000) Int. J. Syst. Evol. Microbiol. 50, 2109–2117 19 Pikuta, E., Lysenko, A., Suzina, N., Osipov, G., Kuznetsov, B., Tourova, T., Akimenko, V. and Laurinavichius, K. (2000) Int. J. Syst. Evol. Microbiol. 50, 25–33 20 Wiegel, J., Ayres, K. and Hanel, J. (2003) in Biology and Physiology of Anaerobic Bacteria (Ljungdahl, L.G., Adams, M.W.W., Johnson, M., Ferry, G. and Barton, L., eds.), pp. 235–251, Springer Verlag, New York 21 Blotevogel, K. H., Fischer, U., Mocha, M. and Jannsen, S. (1985) Arch. Microbiol. 142, 211–217 22 Wasserfallen, A., Nolling, ¨ J., Pfister, P., Reeve, J. and Conway de Macario, E. (2000) Int. J. Syst. Evol. Microbiol. 50, 43–53 23 Boone, D.R., Worakit, S., Mathrani, I.M. and Mah, R.A. (1986) Appl. Env. Microbiol. 7, 230–234 24 Horikoshi, K. (1991) Microorganisms in Alkaline Environments, Kodansha VCH, Tokyo 25 Olsson, K., Keis, S., Morgan, H.W., Dimroth, P. and Cook, G.M. (2003) J. Bacteriol. 185, 461–465 26 Peddie, C.J., Cook, G.M. and Morgan, H.W. (1999) J. Bacteriol. 181, 3172–3177 27 Krulwich, T.A. (2000) The Prokaryotes: an Evolving Electronic Database for the Microbiological Community, 3rd edn, version 3.1 (Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H. and Stackebrandt, E., eds.), Springer Verlag, Berlin 28 Krulwich, T.A., Ito, M. and Guffanti, A.A. (2001) Biochim. Biophys. Acta 1505, 158–168 29 Yumoto, Y. (2002) J. Biosci. Bioeng. 93, 342–353 30 Driessen, A.J.M., van de Vossenberg, J.L.C.M. and Konings, W.N. (1996) FEMS Microbiol. Rev. 18, 139–148 31 Prowe, S.G., van de Vossenberg, J.L.C.M., Driessen, A.J.M., Antranikian, G. and Konings, W.N. (1996) J. Bacteriol. 178, 4099–4104 32 Prowe, S.G. and Antranikian, G. (2001) Int. J. Syst. Evol. Microbiol. 51, 457–465 33 Holland, H.D. (1984) The Chemical Evolution of the Atmosphere and Oceans, Princeton University Press, Princeton, N.J 34 Walker, J.G.G. (1977) Evolution of the Atmosphere, Macmillan, New York 35 Kempe, S. and Kazmierczak, J. (1997) Planetary Space Sci. 45, 1493 36 Eugster, H.P. and Hardie, L.A. (1978) in Lakes Chemistry, Geology, Physics (Lerman, A., ed.), pp. 237–293, Springer Verlag, New York 37 Morse, J.W. and Marion, G.M. (1999) Am. J. Sci. 299, 738–761 38 Zuber, M.T., Slomon, S.C., Phillips, R.J. et al. (2000) Science 287, 1788–1793 39 Zhilina, T.N., Garnova, E.S., Tourova, T.P., Kostrikina, N.A. and Zavarzin, G.A. (2001) Microbiology (RU) 70, 64–72 40 Sarkar, A. (1991) Geomicrobiol. J. 9, 225–232 41 Horikoshi, K. (1999) Microbiol. Mol. Biol. Rev. 63, 735–750 42 Rainey, F., Fritze, D. and Stackebrandt, E. (1994) FEMS Microbiol. Lett. 115, 205–212 43 Nazina, T.N., Tourova, T.P., Poltaraus, A.B., Novikova, E.V., Grigoryan, A.A., Ivanova, A.E., Lysenko, A.M., Petrunyaka, V.V., Osipov, G.A., Belyaev, S.S. and Ivanov, M.V. (2001) Int. J. Syst. Evol. Microbiol. 51, 433–446 44 Nielsen, P., Fritze, D. and Priest, F.G. (1995) Microbiology (UK) 141, 1745–1761 45 Gupta, R., Beg, Q.K. and Lorenz, P. (2002) Appl. Microbiol. Biotechnol. 59, 15–32 46 Bertoldo, C. and Antranikian, G. (2002) Curr. Opin. Chem. Biol. 6, 151–160 47 Svetlitshnyi, V., Rainey, F. and Wiegel, J. (1996) Int. J. Syst. Bacteriol. 46, 1131–1137 48 Chauthaiwale, J. and Rao, M. (1994) Appl. Environ. Microbiol. 60, 4495–4499 49 Takami, H. and Horikoshi, K. (2000) Extremophiles 4, 99–108 Received 19 September 2003