The microbial sulfur cycle at extremely haloalkaline conditions of soda lakes

Abstract

Soda lakes represent a unique ecosystem with extremely high pH (up to 11) and salinity (up to saturation) due to the presence of high concentrations of sodium carbonate in brines. Despite these double extreme conditions, most of the lakes are highly productive and contain a fully functional microbial system. The microbial sulfur cycle is among the most active in soda lakes. One of the explanations for that is high-energy efficiency of dissimilatory conversions of inorganic sulfur compounds, both oxidative and reductive, sufficient to cope with costly life at double extreme conditions. The oxidative part of the sulfur cycle is driven by chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria (SOB), which are unique for soda lakes. The haloalkaliphilic SOB are present in the surface sediment layer of various soda lakes at high numbers of up to 10(6) viable cells/cm(3). The culturable forms are so far represented by four novel genera within the Gammaproteobacteria, including the genera Thioalkalivibrio, Thioalkalimicrobium, Thioalkalispira, and Thioalkalibacter. The latter two were only found occasionally and each includes a single species, while the former two are widely distributed in various soda lakes over the world. The genus Thioalkalivibrio is the most physiologically diverse and covers the whole spectrum of salt/pH conditions present in soda lakes. Most importantly, the dominant subgroup of this genus is able to grow in saturated soda brines containing 4 M total Na(+) - a so far unique property for any known aerobic chemolithoautotroph. Furthermore, some species can use thiocyanate as a sole energy source and three out of nine species can grow anaerobically with nitrogen oxides as electron acceptor. The reductive part of the sulfur cycle is active in the anoxic layers of the sediments of soda lakes. The in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries using sulfate, thiosulfate, or elemental sulfur as electron acceptors demonstrated relatively high sulfate reduction rates only hampered by salt-saturated conditions. However, the highest rates of sulfidogenesis were observed not with sulfate, but with elemental sulfur followed by thiosulfate. Formate, but not hydrogen, was the most efficient electron donor with all three sulfur electron acceptors, while acetate was only utilized as an electron donor under sulfur-reducing conditions. The native sulfidogenic populations of soda lakes showed a typical obligately alkaliphilic pH response, which corresponded well to the in situ pH conditions. Microbiological analysis indicated a domination of three groups of haloalkaliphilic autotrophic sulfate-reducing bacteria belonging to the order Desulfovibrionales (genera Desulfonatronovibrio, Desulfonatronum, and Desulfonatronospira) with a clear tendency to grow by thiosulfate disproportionation in the absence of external electron donor even at salt-saturating conditions. Few novel representatives of the order Desulfobacterales capable of heterotrophic growth with volatile fatty acids and alcohols at high pH and moderate salinity have also been found, while acetate oxidation was a function of a specialized group of haloalkaliphilic sulfur-reducing bacteria, which belong to the phylum Chrysiogenetes.

Keywords: soda lakes; sulfate-reducing bacteria; sulfidogenesis; sulfur reduction; sulfur-oxidizing bacteria; thiosulfate reduction.

Figure 1

Phylogenetic position of haloalkaliphilic SOB from soda lakes (in bold) within the Gammaproteobacteria based on 16S rRNA sequence analysis. The strain abbreviations are as follows: AL, strains from Transbaikal region (Russia); AKL, strains from Kulunda Steppe (Altai, Russia); ALMg, strains from north-eastern Mongolia; ALJ, strains from Kenya; ALE, strains from Wadi Natrun in Egypt; ASL, strain from Soap Lake (USA); ASLr, strain from Searles Lake (USA); ALOw, strains from Owens lake (California); ALN1, extremely haloalkaliphilic nitrate-reducing strain from Wadi Natrun; ALR, strains from a lab-scale sulfide-oxidizing bioreactor; HL-EbGr7, a strain from a full-scale sulfide-oxidizing bioreactor. In red are the strains with sequenced genome and in pink are strains which genome sequencing is in progress. Numbers at the nodes indicate the percentage of bootstrap values for the clade of this group in 1000 replications (the values for maximum-likelihood method are given in parentheses). Only values above 70% are shown. Bar, 5 substitutions per 100 nucleotides (nt).

Figure 2

Illustration of the three different anion-pH types of closely related SOB species from saline habitats. Thiomicrospira crunogena is a deep-see marine halophile, Thiomicrospira pelophila – a bicarbonate-loving alkalitolerant type from an littoral marine habitat, which may have temporal pH increase due to the phototrophic activity, and Thioalkalimicrobium aerophilum is a typical carbonate-loving type from a soda lake. 100% rates were 0.8, 2.2, and 3.8 μmol O₂ (mg protein min)⁻¹ for Tm. crunogena, Tm. Pelophila, and Tm aerophilum, respectively.

Figure 3

Illustration of the concept of natronophily among the extremely salt-tolerant soda lake SOB, showing the influence of counter-anion of sodium on growth of two different types of extremely salt-tolerant Thialkalivibrio isolates. The strains were grown at 4 M total Na⁺ and pH 10. Sodium carbonate and sodium sulfate represent weak electrolytes in contrast to the strong electrolyte NaCl. Thioalkalivibrio K90mix represent a typical natronophile from soda lakes in the Kulunda Steppe, while Thioalkalivibrio ALE20 represents a haloalkaliphile from hypersaline alkaline lakes of Wadi Natrun. Na-c, medium with 90% carbonates/10% Cl⁻ Na-s, medium with 90% sulfate/10% carbonates; NaCl50, medium with 50% Cl⁻/50% carbonate; NaCl75, medium with 75% Cl⁻/25% carbonate.

Figure 4

Examples of pH (A) and salt (B,C) on the rate of sulfidogenesis (VHS⁻) in benthic microbial communities of soda lakes. (A,B), moderately saline lake Tanatar-5; (C), hypersaline lake Tanatar-1 (both in Kulunda Steppe, Altai, Russia). Electron donor – formate.

Figure 5

16S rRNA-based phylogeny of haloalkaliphilic SRB from soda lakes within the Deltaproteobacteria (sequences are in bold). Numbers at the nodes indicate the percentage of bootstrap values for the clade of this group in 1000 replications (the values for maximum-likelihood method are given in parentheses). Only values above 70% are shown. Bar, 5 substitution per 100 nt. E. coli was used as an outgroup.

Figure 6

Salt [(A), at pH 10] and pH [(B), at optimum salinity] profiles for growth in three subgroups of lithotrophic SRB from soda lakes. Dsn, Desulfonatronum isolates, Dsnv, Desulfonatronovibrio isolates, Dsnsp, Desulfonatronospira thiodismutans. 100% growth rates were 0.05, 0.04, and 0.02 h⁻¹ for Dsn, Dsnv, and Dsnp, respectively.

Figure 7

Possible adaptation scenario of prokaryotes to functioning in saturated soda brines: case of sulfur cycling.

Figure 8

A generalized microbial sulfidogenic system detected in soda lakes at pH 9.5–11.

Figure 9

Proposed scheme of microbial sulfur cycling in soda lakes. Dashed lines indicate abiotic reactions and solid lines show microbial conversions. The horizontal color gradient shows increase in salinity from left to right.