Chemolithotrophic haloalkaliphiles from soda lakes

Abstract

This paper summarizes recent data on the occurrence and properties of lithotrophic prokaryotes found in extremely alkaline, saline (soda) lakes. Among the chemolithotrophs found in these lakes the obligately autotrophic sulfur-oxidizing bacteria were the dominant, most diverse group, best adapted to haloalkaline conditions. The culturable forms are represented by three new genera, Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira in the Gammaproteobacteria. Among them, the genus Thioalkalivibrio was most metabolically diverse, including denitrifying, thiocyanate-oxidizing and facultatively alkaliphilic species. Culturable methane-oxidizing populations in the soda lakes belong to the type I methanotroph group in the Gammaproteobacteria, mostly in the genus Methylomicrobium. The nitrifying bacteria in hyposaline soda lakes were represented by a new species Nitrobacter alkalicus (Alphaproteobacteria), and by an alkaliphilic subspecies of Nitrosomonas halophila (Betaproteobacteria). Both belonged to the low salt-tolerant alkaliphiles. The facultatively autotrophic haloalkaliphilic isolates able to grow with hydrogen as electron donor were identified as representatives of the α-3 subclass of the Proteobacteria (aerobic) and of the Natronolimnicola–Alkalispirillum group in the gammaproteobacteria (nitrate-reducing). While all chemolithotrophic isolates from soda lakes belong to the alkaliphiles with a pH optimum for growth around 10, only the sulfur-oxidizing group included species able to grow under hypersaline conditions. This indicates that carbon and nitrogen cycles in the hypersaline alkaline lakes might not be closed.

Soda lakes, Haloalkaliphilic, Sulfur-oxidizing bacteria, Nitrifying bacteria, Methanotrophic bacteria, Hydrogenotrophs

1 Introduction

Chemolithotrophic bacteria, which utilize reduced inorganic compounds as electron donors, are important players in the element cycles of natural and industrial environments. They are widely distributed in various habitats, associating mostly with the interface zones where opposed fluxes of reduced (substrates) and oxidized (acceptors) inorganic compounds meet. The chemolithotrophs include aerobic, facultatively anaerobic and obligate anaerobic bacteria. This review is focused on the aerobic and denitrifying chemolithotrophs including sulfur-oxidizing, nitrifying, hydrogenotrophic and methanotrophic bacteria active at extremely high pH, found recently in saline alkaline (soda) lakes.

2 Soda lake habitat

Soda lakes represent a specific type of salt lakes, which contain an alkaline sodium carbonate/bicarbonate fraction among the dominant salts. They are mostly confined to dry areas with high evaporation rates that facilitate salt accumulation in local depressions. The main geochemical conditions of their formation include leaching of rock material, rich in sodium but low in Ca and Mg, by CO₂-saturated waters in an area with a dry and warm climate, facilitating evaporative concentration of the brines in natural depressions [1,2]. Under such conditions, sodium becomes dominant among the cations and , Cl⁻ and – are the dominant anions in the solution. The presence of sodium carbonate in variable combinations with sodium chloride and sodium sulfate creates a unique, buffered haloalkaline habitat appropriate for a stable development of obligately (halo)alkaliphilic microorganisms growing optimally at pH around 10. Apart from the high salt/high pH effects, which demand adaptations well known for haloalkaliphiles (osmolytes, pH homeostasis, elements of sodium cycle), there are other extreme factors associated with high alkalinity, which might be important specifically for the chemolithotrophs (Fig. 1). In particular, the availability of metal cofactors could be limited at highly alkaline conditions. Fortunately, the carbonate ion forms alkaline complexes with the metal ions, which are much more soluble than the respective hydroxides [3], as in case of the moderately soluble basic magnesium carbonate (Mg₂(OH)₂CO₃]. At pH 10 and higher, is mostly converted to toxic and volatile NH₃. On the other hand, nitrite becomes less toxic at high pH, to the benefit of the nitrite-metabolizing bacteria. A high carbonate/bicarbonate ratio at pH > 10 may result in limitation of autotrophic bacteria by their carbon source as has been suggested, for example, for alkaliphilic cyanobacteria [4,5]. On the other hand, the high alkaline buffering capacity might be advantageous for the growth of chemolithotrophic bacteria that neutralize acids produced during oxidation of reduced inorganic compounds. With respect to the sulfur cycle, the nucleophilic attack of HS⁻ and on elemental sulfur at high pH results in the spontaneous formation of polysulfides (⁻S–S_x–S⁻) and thiosulfate, respectively. While the latter is also common for neutral conditions, the polysulfides are specific for highly alkaline conditions.

1

Schematic representation of the possible impact of carbonate alkalinity on microbial element cycling in soda lakes.

The soda lakes are populated almost exclusively by prokaryotes, which can form dense communities (possibly due to the absence of grazing pressure) even in saturated alkaline brines [6–11]. Alkaliphilic cyanobacteria (Spirulina, Arthrospira, Anabaenopsis, Cyanospira) are responsible for the generally high level of primary production and nitrogen fixation in soda lakes [12–14]. The polymers produced by the primary producers are degraded by the aerobic and anaerobic hydrolytics, such as haloalkaliphilic Bacillus spp. and Clostridia, respectively [8,10]. The main groups of primary and secondary anaerobes using monomers and oligomers, such as fermentative (Spirochaeta alkalica, Spirochaeta asiatica, Spirochaeta africana, Tindalia magadii, Alkalibacter, Alkaliflexus), acetogenic (Natroniella acetigena, Natronoincola histidinovorans), methanogenic (Methanohalophilus zhilinae, Methanosalsus zhilinaeae) and sulfate-reducing bacteria (Desulfonatronovibrio, Desufonatronum), well adapted to haloalkaline conditions, have also been identified [10,15]. The microbial sulfur cycle appears to be very active in the soda lakes. It is driven by the extremely haloalkaliphilic anaerobic purple sulfur bacteria (Ectothiorhodospira and Halorhodospira) and hydrogenotrophic sulfate-reducing bacteria [6,10]. Haloalkaliphilic sulfur/polysulfide-reducing hydrogenotrophs however, remained to be identified.

Until recently, the bacterial “filter” responsible for the reoxidation of reduced inorganic compounds, such as methane, hydrogen, sulfide and ammonia, produced by the haloalkaliphilic anaerobes in the soda lake sediments, remained unknown despite some evidence of their activity [16,17], and of functional genes for these organisms [18] being found in different soda lakes. Our investigation of the soda lakes in various geographic areas (Table 1) revealed culturable representatives of all missing groups of aerobic and denitrifying chemolithotrophs. Among them, only the methanotrophs have been studied in detail by another group [19].

1

Soda lake samples used for study of haloalkaliphilic chemolithotrophs

Area	Number of investigated lakes	Total salts (g l−1)	pH	Total carbonate alkalinity (M)
Kenya (sampled by B. Jones and W. Grant)	6	20–220	9.5–11.0	0.12–1.16
Wadi Natrun (Egypt)	8	200–380	9.5–10.3	0.11–0.75
Mono Lake, California (sampled by V. Gorlenko)	1	90	9.7	0.4
Tuva, Russia (sampled by T.N. Zhilina)	1	20	10.0	nd
Kunkur steppe, Russia	10	5–40	9.5–10.2	0.02–0.11
North-eastern Mongolia	16	5–360	9.2–10.5	0.02–1.20
Kulunda steppe, Altai (Russia)	20	20–380	9.3–10.6	0.02–5.20

3 Sulfur-oxidizing bacteria in soda lakes

One of the important questions at the beginning of this study was what composition of mineral base medium would be suitable for the successful enrichment and isolation of a wide range of the soda lake lithotrophs. The optimized mineral medium is based on the sodium bicarbonate/carbonate buffer with pH 10–10.1, variable NaCl concentration and a total Na⁺ content 0.5–4.3 M. This medium, even at minimal salt content, was sufficient to keep the pH above 9.2 during massive production of sulfuric acid by sulfur-oxidizing bacteria (SOB).

With thiosulfate as electron donor, the wide distribution of haloalkaliphilic SOB in soda lakes of different geographic locations has been demonstrated. Their abundances range from 10³ to 10⁸ viable cells/cm³ sediments. Based on the activity of sulfide oxidation by pure cultures (8–57 μmol (10⁹ cells)⁻¹ d⁻¹], this density might provide the rate of sulfide removal in the sediments within the range of 0.125–4700 μmol S kg⁻¹ d⁻¹. The measured rates of sulfate reduction varied from 3.1 to 780 μmol S kg⁻¹ d⁻¹ with the maximal values in hyposaline lakes ([11]; N. Pimenov, personal communication). This means that in some cases the SOB population density might be insufficient to keep the sulfur cycling in the soda lakes balanced.

Under aerobic or denitrifying conditions, more than 100 strains of obligately chemolithoautotrophic alkaliphilic sulfur-oxidizing bacteria actively growing at pH 10 have been obtained in pure culture. They grouped into three distinct clusters described as new genera in the Gammaproteobacteria–Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira[20–23]. The genus Thioalkalimicrobium includes three valid species and is closely affiliated with the neutrophilic marine sulfur-oxidizing bacteria of the genus Thiomicrospira. The genus Thioalkalivibrio is much more diverse, presently containing nine species. It is a member of the family Ectothiorhodospiraceae, which accommodates phototrophic purple sulfur bacteria, most of which are extremely (halo)alkaliphilic. The single-species genus Thioalkalispira clusters only loosely with the Ectothiorhodospiraceae, but its exact position cannot be easily established at the moment. The genus Thialkalimicrobium dominated in the low-salt enrichment cultures from the hyposaline soda lakes of Central Asia on low-saline medium. Enrichments from the hypersaline lakes on high-salt medium invariably resulted in the domination of the genus Thialkalivibrio. Thioalkalispira microaerophila was isolated under microoxic conditions from a denitrifying enrichment culture.

Both growth rate and growth yield of the soda lake SOB were maximal at pH values around 10, which differentiated these species from all previously known aerobic chemolithoautotrophs. The pH range for growth in chemostat cultures was 7.5–10.6, while the pH range of the respiratory activity was broader at both extremes. The failure of the bacteria to grow at pH > 10.6 might be explained by an anabolic constraint, most probably the unavailability of carbon in the form of bicarbonate . This is also evident from the presence of high number of carboxysomes in the cells of haloalkaliphilic SOB [20]. Molecular analysis of the genera Thioalkalivibrio and Thioalkalispira demonstrated the presence of the “green-like” type of RuBisCO form I in all species [24].

On the basis of their salt tolerance/requirement the sulfur-oxidizing bacteria in soda lakes included three groups. All Thioalkalimicrobium and some of the Thioalkalivibrio isolates belong to a moderately salt tolerant type, growing up to 1.2–1.5 M total Na⁺. Many of the Thioalkalivibrio isolates were extremely salt-tolerant, able to grow in saturated soda brines (4.5 M Na⁺). However, most of them grew optimally at moderate salt concentrations (0.5–1 M Na⁺). Only a few Thioalkalivibrio isolates belong to the true extreme halophiles growing only above 1 M Na⁺. Overall, the cultivated forms of SOB isolated from the soda lakes cover the full range of pH/salt concentrations typical for their environment. Furthermore, a definite tendency towards a preference of sodium carbonates over sodium chloride was evident from the growth and respiratory experiments with different strains of the extremely salt tolerant Thioalkalivibrio, which also indicated unique physiological adjustment of these bacteria to the soda lake conditions (natronophily instead of halophily) [25].

The genus Thioalkalivibrio includes fully aerobic (dominating), microaerophilic (isolated in sulfide-oxygen gradient cultures) and denitrifying species. Three organisms described as different species of the genus Thioalkalivibrio with different denitrifying potential have been found in soda lakes. When growing in coculture, Thialkavibrio nitratireducens[26] and Thialkavibrio denitrificans[27] catalyzed the complete denitrification of nitrate to dinitrogen in presence of thiosulfate as the electron donor at pH 10 [28]. The former can only reduce nitrate to nitrite and the latter reduced nitrite and N₂O. While Tv. denitrificans depended on Tv. nitratireducens for its supply of nitrite, the latter was able to grow independently, being extremely tolerant to high nitrite concentrations. The Tv. denitrificans grew best with polysulfide as the electron donor and N₂O as electron acceptor – a combination not shown previously for any other chemolithotrophic bacterium. The third thiodenitrifying strain, described as Thioalkalivibrio thiocyanodenitrificans[29], was isolated from the soda lakes using thiocyanate as electron donor and nitrate as the electron acceptor. In contrast to the others, it was able to completely reduce nitrate to dinitrogen. Anaerobic oxidation of thiocyanate under denitrifying conditions represents a specialized type of extremely slow chemolithotrophic metabolism, which was hypothesized, but never confirmed, to be used by the neutrophilic Thiobacillus denitrificans[30].

Use of thiocyanate (NC–S⁻) as an electron donor instead of thiosulfate resulted in the enrichment from various soda lakes and isolation in pure culture of two different phenotypes of haloalkaliphilic SOB described as Thioalkalivibrio thiocyanoxidans and Thioalkalivibrio paradoxus[31]. These bacteria degraded thiocyanate through cyanate (N=C=O⁻) with the final production of ammonia, CO₂ and sulfate [32]. But, in contrast to anaerobic hydrolysis suggested previously as a mechanism of primary thiocyanate degradation for the neutrophilic thiobacilli [33], in alkaliphiles the responsible enzyme appears to act as a dehydrogenase, utilizing oxidized cytochrome c as an immediate electron acceptor. The new enzyme is a soluble 57-kDa monomer with a unique primary peptide structure (unpublished results).

4 Methanotrophic alkaliphiles

Positive methanotrophic enrichments at pH 9–10 from soda lake sediments, even from the hypersaline ones, were successful only at a salt concentration below 1.5 M total Na⁺. This finding was in agreement with a very low methane-oxidizing activity in hypersaline lakes measured with ¹⁴CH₄. Only in the hyposaline and moderately saline lakes, as in the case of the north-eastern Mongolia, were the observed rates of methane oxidation (1.1–6.5 μmol CH₄ kg⁻¹ d⁻¹) sufficient to balance the rates of methane formation (0.06–3.4 μmol CH₄ kg⁻¹ d⁻¹) [11,34]. The methane cycle in hypersaline soda lakes with salt content >10% might be unbalanced.

Molecular probing, including ¹³C-labelling of the DNA, of the methanotrophic population in the soda lakes of Central Asia demonstrated the ubiquitous domination and the activity of the type I methanotrophs belonging to the Methylomicrobium-Methylobacter group of the gammaproteobacteria in moderately saline soda lakes [11,19,35].

We obtained a pure culture of an obligate methanotroph from a Kenyan soda lake using standard sodium carbonate-based mineral medium containing 0.6 M total Na⁺ at pH 10. Strain AMO 1 was identified as a member of the genus Methylomicrobium in the Gammaproteobacteria. According to a recent phylogenetic comparison [19,36], all salt-dependent neutrophilic and alkaliphilic methanotrophic strains are clustered together within the genus Methylomicrobium. With respect to its ultrastructure and biochemical properties, strain AMO 1 is a typical representative of the type I methanotrophs with lamellar intracellular membrane structures and the RuMP pathway of carbon assimilation [37]. The bacterium utilized methane and, much less actively, methanol as carbon and energy sources. It had a very narrow pH range for growth: between 9 and 10.2 and an optimum at pH 9.9–10. The washed cells however, oxidized methane and methanol within much broader pH range, i.e. from 6.0 to 11.0, with an optimum at pH 10. Strain AMO 1 had remarkably low formaldehyde- and formate-oxidizing activities, which was probably the reason for the accumulation of formaldehyde in the medium. Inhibition of the formate metabolism by sodium carbonate was suggested to be a specific effect of the soda lake environment on methanotrophic isolates [19]. Strain AMO 1 was able to grow in sodium carbonate media at a very low background concentration of Cl⁻ ions that caused slight stimulation up to 0.1 M, which is a common characteristic of the soda lake bacteria. The total salt concentration in the form of sodium carbonate/bicarbonate and NaCl suitable for growth ranged from 0.2 to 1.2 M total Na⁺. The ecological role of the AMO-like alkaliphiles might not be limited to methane oxidation only, since it was found to possess an additional potential to oxidize carbon disulfide (CS₂) and NH₃. CS₂ was converted to polysulfide, indicating possible formation of elemental sulfur and sulfide as immediate products with further spontaneous production of polysulfide at high pH. The addition of low numbers of the alkaliphilic SOB (Thioalkalimicrobium) to the cells of AMO 1 increased the rate of the CS₂ oxidation and changed the product to elemental sulfur, mimicking a possible variant of interaction of these two groups of haloalkaliphiles. Strain AMO 1 co-oxidized NH₃ to nitrite in the presence of moderate concentrations of methane or methanol only at very alkaline pH between 10 and 11, with the maximum rate amounting to 17% of the rate of methane oxidation. Both CS₂ and NH₃ might be unspecific substrates of the pMMO. The potential for ammonium oxidation has also been demonstrated for alkaliphilic Methylomicrobium buryatense isolated from hyposaline soda lake in Buriatia (Transbaikal area) [34,36].

5 Nitrifying alkaliphiles

Nitrification is one of the essential activities in natural and man-made environments. This process supplies oxidized forms of nitrogen for assimilation and anaerobic respiration and contributes to the primary production. Until recently, very little was known about its operation in extremely alkaline environments, such as soda lakes and soda soils. Indirect evidence, such as the presence of active populations of denitrifying and nitrate-utilizing bacteria, including heterotrophic haloalkaliphilic Halomonas spp. [38] and chemolithoautotrophic SOB (see above), indicated that nitrogen oxides might be produced in the alkaline habitats. Furthermore, the in situ activity of ammonia oxidation and the presence of the 16S rRNA gene sequences related to Nitrosomonas europea has been demonstrated in the chemocline of the saline alkaline Mono Lake and Big Soda Lake [16,17,39]. Additional evidence on the presence of nitrifiers in the Mono Lake has been obtained recently by cloning the sequences amplified with RuBisCO-specific primers [18], although the cbbL gene is not as conserved as the 16S rRNA gene and therefore the identification of the host is not so certain.

Preliminary tests on the presence of nitrifying populations, using low-salt soda medium, demonstrated production of nitrate from urea but not from ammonia in the Kenyan soda lakes. Nitrite oxidation to nitrate occurred in the Siberian soda lakes and soda soils [40]. However, attempts to obtain stable enrichment cultures of lithotrophic nitrifying bacteria from those experiments failed. It was speculated that the methane-oxidizing haloalkaliphiles, such as Methylomicrobium AMO 1 and M. buryatense, might be responsible for the first stage of nitrification in soda lakes [34,37]. However, eventually it appeared that the concentrations of ammonium used for enrichments had been too high for stable cultivation of the specialized ammonia oxidizers. At an NH₃ concentration <4 mM and salt concentration of 0.6 M total Na⁺, five stable enrichment cultures of ammonia-oxidizing bacteria growing at pH 10 have been obtained from sediment samples of the north-eastern Mongolian soda lakes. Nitrite production ceased at higher NH₃ concentrations and a salt content >1 M total Na⁺[11,41]. The same conditions favored NH₃ oxidation in an enrichment from Wadi Natrun lake Fazda (unpublished data]. Isolation of pure cultures was performed using solid double-layered agar plates to allow ammonia to diffuse gradually from the bottom layer to the top layer of alkaline mineral medium. The five strains obtained (ANs 1–5) were genetically very close and, surprisingly, also very closely related to the known marine species Nitrosomonas halophila Nm 1 [41].

Batch culture tests demonstrated that the soda lake isolates belong to obligate alkaliphiles with a pH optimum around pH 9. In the ammonia-limited continuous culture with pH control one of the isolates grew at pH values up to 11.4 – an absolute maximum not only among chemolithotrophs but also close to the maximum proven for alkaliphilic heterotrophs (11.5) [42]. It appears that the NH₃-oxidizing alkaliphiles somehow managed to overcome the major problem (e.g. carbon limitation) of autotrophic metabolism at extremely high carbonate/bicarbonate ratio. The high salt concentration, however, was a much bigger problem for the growth of the ANs isolates, than was high pH. Growth was possible within a range of total Na⁺ content from 0.1 to 0.9 M. The cultures did not require Cl⁻, they could grow in pure sodium carbonate media. Another problem for this specific type of nitrifier was the ammonium concentration. At pH 10, they could tolerate no higher than 8 mM of NH₄Cl and grew without a lag phase only when the NH₃ concentration was <4 mM. The ammonia toxicity increased with increasing pH. For example, the K_i50 for NH₃-dependent respiration at pH 10 was 16 times higher than at pH 11.0. Washed cells respired actively within a pH range between 7.0 and 11.5 with a maximum at pH 9.0–10.0, which is typical for the soda lake alkaliphiles.

The isolation of nitrite-oxidizing bacteria from the alkaline habitats was less difficult, most probably because of the absence of substrate toxicity. In fact, nitrite toxicity at circumneutral pH is determined by the presence of undissociated nitric acid, which is virtually absent at pH as high as 10. This made it possible to obtain several stable enrichment cultures from the soda lake sediments and soda soils using low-saline (0.6 M Na⁺) Na-carbonate mineral medium supplemented with 10–20 mM nitrite. The five pure cultures obtained through single colony isolation possessed a typical Nitrobacter phenotype. The genus identification was confirmed by phylogenetic analysis. However, despite the very high sequence similarity to the type species Nitrobacter winogradskii, there was very little DNA homology between the soda lake isolates and the type strain, which, together with their distinct alkalitolerance prompted us to describe a new species Nitrobacter alkalicus[43]. In contrast to the neutrophilic Nitrobacter species, the new isolates formed a thick globular S-layer, did not show any signs of heterotrophy and grew at high pH. On the other hand, unlike the NH₃-oxidizing isolates, the nitrite-oxidizers still grew at neutral pH 7–8, albeit much slower than within the alkaline region between 9 and 10.2. Therefore, they can be classified as facultative alkaliphiles. Nb. alkalicus was even less salt-tolerant than the NH₃-oxidizing alkaliphiles, being able to grow within the range of Na⁺ content from 0.05 to 0.5 M. Similar to the ANs strains, though, the nitrite-oxidizing isolates preferred a carbonate anionic environment to Cl⁻.

It may be concluded that salt content, rather than high pH values, control development of nitrifying bacteria in saline alkaline lakes and that the nitrogen cycle in hypersaline soda lakes might be impaired.

6 Aerobic and denitrifying hydrogenotrophs in soda lakes

The anaerobic sediments in soda lakes are inhabited by an active and diverse population of haloalkaliphilic anaerobic bacteria, which can produce dihydrogen as an end product of their catabolism [10,15]. Despite the fact that the “secondary” anaerobes such as methanogens and sulfate-reducing bacteria are also present, part of the hydrogen might escape to the more oxidized zone and thus become available for aerobic and denitrifying hydrogenotrophs.

One such bacterium, strain AHO 1, was obtained from an aerobic enrichment inoculated with mixed sediment sample from Kenyan soda lakes, using sodium carbonate-based mineral medium with 0.6 M total Na⁺ at pH 10 [44]. The isolate was identified as a member of the Alphaproteobacteria. It grew chemolithoautotrophically with hydrogen, heterotrophically with various organic acids and sugars and lithoheterotrophically with hydrogen or sulfide as the energy source and organic compounds as the carbon source. Growth with dihydrogen was optimal at pH 9.5–9.8 and possible up to pH 10.25, although washed cells still actively consumed oxygen in the presence of dihydrogen up to pH 11.0. At least 0.2 M of Na⁺ was necessary for autotrophic growth with hydrogen, but the salt tolerance was not higher than 1 M total Na⁺.

The development of enrichment culture with dihydrogen under denitrifying conditions inoculated with the sediment mixture from Kenya, Mongolia and Siberia, was extremely slow with copious accumulation of nitrite. Only one enrichment yielded a pure culture, strain AHN 1, which grew autotrophically with hydrogen and nitrate at pH 10 and a salt concentration up to 1.5 M total Na⁺ (unpublished results). On the other hand, it could grow even in saturated soda brines (4 M total Na⁺) aerobically with acetate. Strain AHN 1 is a partial denitrifier with a gap in the middle of its denitrification pathway, e.g. it can reduce nitrate to nitrite and N₂O to dinitrogen, but cannot grow anaerobically with nitrite. It is a member of the cluster Alkalispirillum-Alcalilimnicola in the gammaproteobacteria. Based on our observations and the literature data, this cluster appears to contain basically heterotrophic, high salt-tolerant alkaliphilic bacteria with a diverse lithotrophic potential and it might be important and specific for hypersaline soda lakes. In our experiments representatives of this cluster frequently dominated in denitrifying enrichments with hydrogen or polysulfide as the electron donors and nitrate as an electron acceptor. Another haloalkaliphilic strain belonging to this cluster has recently been obtained from Mono Lake using a somewhat “exotic” combination of substrates, previously unknown to sustain lithoautotrophic growth. Strain MLHE-1 grew autotrophically by coupling the oxidation of arsenite to arsenate with nitrate reduction to nitrite [45]. All mentioned isolates and the type strains of the Alkalispirillum and the Alcalilimnicola contain the type I RuBisCO ([45]; T. Tourova, personal communication).

7 Conclusions and prospectives

Soda lakes contain all major groups of chemolithotrophic bacteria. Their place in the elemental cycling in soda lakes is shown in Fig. 2. These bacteria can be cultivated at least at pH 10 and sometimes higher using mineral medium highly buffered with sodium carbonate. On the basis of combined pH and salt tolerance we can conclude that the SOB group is adapted best to the conditions of soda lakes (Fig. 3). Their clear preference for the sodium carbonate environment with its extremely high pH and alkalinity is actually the main property discriminating these bacteria from their neutrophilic counterparts. Interestingly, in two of the groups we found an intermediate type with enhanced alkalitolerance and a certain phylogenetic relationship to the alkaliphiles. Based on a comparison of the pH profiles, three different ecotypes can be discriminated among the salt-tolerant chemolithotrophs: NaCl-, NaHCO₃-, and Na₂CO₃-preferring types (Fig. 4). The NaCl-preferring type is capable of functioning in the marine environment, whilst the NaHCO₃-type is probably adapted to elevated NaHCO₃ alkalinity in photosynthetic microbial mats of the tidal flats, where there are diurnal fluctuations of pH/. Neither of these two types, however, can tolerate a sodium carbonate-rich environment of pH 10 or higher, which is the real domain of the soda lake haloalkaliphiles.

2

Roles of aerobic and denitrifying chemolithotrophic bacteria in the elemental cycling in soda lakes. VFA – volatile fatty acids and alcohols, MA – methylamines.

3

Maximum pH and salt tolerance for growth of various lithotrophic bacteria from soda lakes. 1 –Nitrobacter alkalicus; 2 –Nitrosomonas halophilus ANs 5; 3 – hydrogenotrophic strain AHO 1; 4 –Methylomicrobium sp. AMO 1; 5 –Thioalkalivibrio.

4

pH profiles for respiratory activity of three salt-tolerant ecotypes of sulfur-oxidizing (a) and nitrifying (b) bacteria. NaCl-type (open triangles) is represented by Halothiobacillus neapolitanus and Nitrosomonas eutropha, which were isolated from neutral habitats; the NaHCO₃-type (open circles) is represented by Thiomicrospira pelophila and Nitrosomonas halophila Nm1, isolated from the North Sea; the Na₂CO₃-type (closed circles) is represented by Thioalkalimicrobium aerophilum and Nitrosomonas halophila ANs 5, isolated from soda lakes.

The second characteristic vitally important for the functioning of bacteria in soda lakes is the salt tolerance. Judging from the direct incubations of the sediments with different electron donors and enrichment at different salts and from the salt tolerance of the isolated lithotrophic alkaliphiles, only the SOB group is fully capable to function in hypersaline soda lakes, while activity of the other groups might be either partially (methanotrophs) or completely (nitrifiers) restricted to the hypo/moderately saline lakes (below 10% salt). One of the most probable reasons for this is large energy cost for a life under double extreme conditions [46]. In the future, it would therefore be interesting to undertake a complete genome sequence of the most advanced haloalkaliphilic autotroph, namely a representative of the genus Thioalkalivibrio, to identify what set of genes is sufficient for haloalkaliphilic phenotype in chemolithoautotrophs.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (04-04-48647) and by the Program of the Russian Academy of Sciences “Molecular and Cell Biology” and partly by the Dutch Technology Foundation (STW). We would like to thank our colleagues B. Jones, G. Zavarzin and V. Gorlenko for the possibility to work with their samples from the soda lakes in Kenya and USA.