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Keywords:

  • hypersaline soda lakes;
  • natronophilic;
  • acetogens;
  • sulfidogens;
  • Tindallia;
  • Natroniella

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Microbial sulfidogenesis is the main dissimilatory anaerobic process in anoxic sediments of extremely haloalkaline soda lakes. In soda lakes with a salinity >2 M of the total Na+ sulfate reduction is depressed, while thiosulfate- and sulfur-dependent sulfidogenesis may still be very active. Anaerobic enrichments at pH 10 and a salinity of 2–4 M total Na+ from sediments of hypersaline soda lakes with thiosulfate and elemental sulfur as electron acceptors and simple nonfermentable electron donors resulted in the isolation of two groups of haloalkaliphilic bacteria capable of dissimilatory sulfidogenesis. Both were closely related to obligately heterotrophic fermentative homoacetogens from soda lakes. The salt-tolerant alkaliphilic thiosulfate-reducing isolates were identified as representatives of Tindallia magadiensis, while the extremely natronophilic obligate sulfur/polysulfide-respiring strains belonged to the genus Natroniella and are proposed here as a novel species Natroniella sulfidigena. Despite the close phylogenetic relation to Natroniella acetigena, it drastically differed from the type strain phenotypically (chemolithoautotrophic and acetate-dependent sulfur respiration, absence of acetate as the final metabolic product). Apparently, in the absence of specialized respiratory sulfidogens, primarily fermentative bacteria that are well adapted to extreme salinity may take over an uncharacteristic ecological function. This finding, once again, exemplifies the importance of isolation and phenotypic investigation of pure cultures.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Hypersaline soda lakes represent habitats on Earth maintaining stable highly alkaline pH due to the presence of high concentrations of soluble sodium carbonates. Furthermore, some of the soda lakes are hypersaline, which makes them double extreme (hypersaline and hyperalkaline) habitats. Because of these harsh conditions, only a limited number of prokaryotic groups, known as haloalkaliphiles, are thriving in saturated soda brines. The microbial systems in such lakes include all major trophic blocks, such as primary producers (mostly cyanobacteria and anoxygenic phototrophs), aerobic heterotrophs (natronoarchaea, Halomonas), fermentative (primary) and respiratory (secondary) anaerobes [sulfate-reducing bacteria (SRB), acetogens, methanogens; Duckworth et al., 1996; Mesbah et al., 2006; Zavarzin, 2007]. The microbial sulfur cycle in soda lakes is particularly active (Sorokin et al., 2006, 2011). However, while the extremely haloalkaliphilic sulfur-oxidizing bacteria are widely distributed in hypersaline lakes, currently, only a single group of haloalkaliphilic SRB belonging to the genus Desulfonatronospira has been found in soda lakes able to grow at salinity >2 M Na+ that preferred thiosulfate over sulfate as an electron acceptor (Sorokin et al., 2008). Furthermore, our recent measurements of the rates of sulfidogenesis in sediments of hypersaline soda lakes in south-eastern Siberia clearly indicated that sulfate reduction was depressed at salt concentrations >2 M of total Na+ (Sorokin et al., 2010). In contrast, thiosulfate and, especially, sulfur reduction were active up to salt-saturating conditions. This led us to look at the identity of microorganisms acting as thiosulfate and sulfur reducers at extremely high salinity and pH in hypersaline soda lakes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Samples

Three sediment samples were obtained from hypersaline soda lakes in south-western Siberia (Kulunda Steppe, Altai region, Russia; brine pH 10.1–11.05, total salt concentration 18–40% w/v and total soluble alkalinity 2.1–4.0 M) and seven sediment samples from hypersaline alkaline lakes in Wadi Natrun (Lybian desert in Egypt; pH 9.1–10.0, total salts 20–36% w/w and total soluble alkalinity 0.2–2.0 M). For the purpose of enrichment, the individual samples were combined together in equal proportions to prepare a single mixed sample for each geographical location. After mechanical homogenization, the samples were subjected to low-speed centrifugation (2000 g 1 min) to remove coarse particles.

Medium composition and growth conditions

A mineral medium based on sodium carbonate/bicarbonate buffer with pH 10 containing 2–4 M total Na+ was used for enrichments and pure culture growth experiments (final concentration in g L−1): Na2CO3, 95–180; NaHCO3, 15–35; NaCl, 16; K2HPO4, 1. After sterilization, the medium was supplemented with 4 mM NH4Cl, 1 mM MgSO4, 20 mg L−1 of yeast and 1 mL L−1 each of trace metal and vitamin solutions (Pfennig & Lippert, 1966) and Se/W mix (Plugge, 2005). Sodium acetate (20 mM), sodium formate (50 mM), ethanol (20 mM) and hydrogen (H2) (100% gas phase) were used as electron donors (individually) for enrichments and for pure cultures. Elemental sulfur (Fluka) was sterilized in closed bottles at 110 °C for 40 min and added in excess of approximately 3 g L−1. Other electron acceptors used were Na2S2O3 (20 mM), Na2SO3, KNO3, KNO2, sodium selenate and selenite, sodium arsenate (5 mM each), sodium fumarate (20 mM) and freshly prepared ferrihydrite (20 mM). In all cases, except for selenite and ferrihydrite, the medium was reduced by adding Na2S at a final concentration of 1 mM. Cultivation was performed either in 15-mL Hungate tube with 5–10 mL medium or in 50-mL serum bottle with 10–40 mL medium under an argon or an H2 gas phase. The pH dependence was examined at a Na+ content of 0.6 M, using the following filter-sterilized buffers: for pH 6–8, 0.1 M HEPES and NaCl/NaHCO3, and for pH 8.5–11, a mixture of sodium bicarbonate/sodium carbonate. All buffers contained 50 mM K2HPO4. To study the influence of salt concentration on growth and activity, sodium carbonate buffers with pH 10, containing 0.2 and 4.0 M of total Na+, were mixed in different proportions. Natroniella acetigena DSM9952 was grown in a medium containing 2.5 M total Na+, pH 10, with lactate (Zhilina et al., 1995).

Analyses

Free sulfide and the sulfane content of polysulfides were measured colorimetrically (Trüper & Schlegel, 1964) after precipitation in 10% w/v Zn acetate. Thiosulfate and sulfite were analyzed by iodimetric titration (with formaldehyde to bind sulfite) in the supernatant after separation from ZnS. Internal zero-valent sulfur of polysulfides was precipitated by acidification of the sample to pH<3 by concentrated HCl, washed with distilled water, dried, extracted from the pellet with acetone overnight and analyzed by cyanolysis (Sörbo, 1957). The protein content was determined according to Lowry et al. (1951) after the removal of sulfide/polysulfide and washing the cell pellet several times with 1–2 M NaCl. Acetate and formate were detected in the filtrated supernatant after neutralization by HPLC-anionic chromatography [HPX-87-H column (Bio-Rad) at 60 °C with UV detection and a 5 mM H2SO4 solution at 0.6 mL min−1 as an eluent]. The fatty acid composition of cellular polar lipids was determined by GC–MS according to Zhilina et al. (1997). Phase-contrast microphotographs were obtained using a Zeiss Axioplan Imaging 2 microscope (Göttingen, Germany). For electron microscopy, the cells were separated from the alkaline brine by centrifugation, resuspended in an NaCl solution of the same molarity, fixed in glutaraldehyde (3% final, v/v) and negatively stained with 1% w/v neutralized phosphotungstic acid.

Genetic and phylogenetic analysis

Genomic DNA was isolated according to Marmur (1961). Determination of the G+C content of the DNA and DNA–DNA hybridization were performed using the thermal denaturation/reassociation technique (Marmur & Doty, 1962; De Ley et al., 1970). 16S rRNA genes were amplified using general bacterial primers 11F-1492R (Lane, 1991). Sequencing was performed using the Big Dye Terminator v.3.1 sequencing reaction kit of an ABI 3730 DNA automatic sequencer (Applied Biosystems Inc.). The sequences were first compared with those stored in GenBank using the blast algorithm and were consequently aligned using clustalw. A phylogenetic tree was reconstructed using the treecon w package and the neighbor-joining algorithm.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Enrichment and isolation of thiosulfate-reducing haloalkaliphiles at high salinity

Enrichments at 2 M Na+ and pH 10 with ethanol as an electron donor and thiosulfate as an acceptor resulted in a sulfidogenic culture from the Kulunda lake sediments consisting of curved motile rods and large motile vibrios. The replacement of ethanol by formate reversed the domination in favor of the vibrio morphotype and eventually resulted in the isolation of the extremely natronophilic SRB strain AHT8 described previously as Desulfonatronospira thiodismutans (Sorokin et al., 2008). The curved rods were purified from single colonies on the original medium with ethanol and thiosulfate and the resulting strain was designated AHT5. A similar strain, ASP-p, was obtained from another Kulunda lake enrichment at 2 M Na+ and pH 10 with formate/acetate as an electron donor/carbon source and thiosulfate as an electron acceptor. In that case, the enrichment was dominated by the SRB Desulfonatronovibrio sp., while the curved rods became dominant after the replacement of formate/acetate with pyruvate.

According to 16S rRNA gene sequence analysis, both rod-shaped isolates were very close to each other and to Tindallia magadiensis (99% sequence similarity), which was found previously in the hypersaline soda lake Magadi (Kenya) and described as an obligately heterotrophic haloalkaliphilic acetogen preferentially fermenting amino acids (Kevbrin et al., 1998). DNA–DNA hybridization showed that the novel isolates were nearly identical (around 95% DNA similarity) and belonged to T. magadiensis species (85% DNA hybridization value with the type strain). However, despite this close relation, anaerobic respiration has not been demonstrated previously in the genus Tindallia, except for the ability to reduce ferric iron, which was not coupled to growth in T. magadiensis and Tindallia texcoconensis (Kevbrin et al., 1998; Alazard et al., 2007). Examination of the type species T. magadiensis confirmed the absence of growth by thiosulfate respiration in this organism.

There were two ecologically important differences of Tindallia sp. strains AHT5 and ASP-p from the previously described acetogenic Tindallia species. First, they both grew lithoautotrophically with H2 and formate. Second, they were capable of true anaerobic respiration with H2, formate, pyruvate, lactate and glycerol as electron donors using thiosulfate, sulfur or fumarate as an electron acceptor (Table 1). Interestingly, formate was detected as a product of anaerobic H2 metabolism instead of acetate, which is expected for an acetogen. It might be speculated that formate in this case is a product of reversed formate lyase reaction, but the significance of its formation is not clear and needs further investigation. Recently, a possibility of anaerobic growth by the opposite reaction (conversion of formate to H2) has been demonstrated for a thermophilic archaeon (Kim et al., 2010) and for syntrophic cultures of acetogens and methanogens (Dolfing et al., 2008). In the presence of an external electron acceptor (thiosulfate), formate accumulation decreased and the growth efficiency with H2 increased, indicating additional energy conservation in comparison with ‘formagenic’ growth (Table 1). The same was true for growth on pyruvate, which could either be fermented or could serve as an electron donor with thiosulfate as an electron acceptor. Thiosulfate reduction in both strains was incomplete, with stoichiometric formation of sulfide and sulfite due to the absence of sulfite reductase.

Table 1.   Influence of electron acceptors on anaerobic growth of Tindallia sp. AHT5 at pH 10 and 2 M Na+ (incubation time 20–40 days)
Electron donorElectron acceptorBiomass (OD600 nm× 100)Products (mM)
HSSO32−FormateAcetate
  • *

    No growth without thiosulfate.

H236  12.40.8
S2O32−508.26.89.20
S8589.0010.50
Fumarate55  10.55.5
Formate*S2O32−508.06.2  
Lactate* 586.05.0  
Pyruvate42  14.02.1
S2O32−657.25.919.21.8
Glycerol*S2O32−454.02.5  

Enrichment and isolation of sulfur-reducing haloalkaliphiles at high salinity

Enrichments under soda-saturating conditions were positive with sulfur as an electron acceptor and resulted in the isolation of three pure cultures. Two identical strains, AHT3 and AHT4, were obtained under chemolithoautotrophic conditions using H2 (Kulunda sample) or formate (Wadi Natrun sample) as an electron donor, respectively. Another strain, AHT18, was enriched and isolated from the Kulunda Steppe sample with acetate as a carbon and energy source. All three isolates were similar in morphology. Young cultures consisted of long flexible rod-shaped cells with peritrichous flagellation. In the late exponential growth phase, cells started to form round bodies and lysed. Upon exposure to oxygen, the cells grown with polysulfide as an electron acceptor formed multiple sulfur globes (Fig. 1). This might be a result of the reverse action of polysulfide reductase, which, in the presence of an oxidized acceptor, such as menaquinones, can oxidize polysulfide to sulfur in sulfur-respiring bacteria (Dietrich & Klimmek, 2002).

image

Figure 1.  Cell morphology of strain AHT3 grown anaerobically at pH 10 and 4 M total Na+ with formate/acetate and sulfur. (a–c) Phase contrast; (d) electron microphotograph of the total preparation. (a) Cells from the logarithmic growth phase; (b) sphaeroplast formation in the late logarithmic phase; (c) reoxidation of cell-bound polysulfide in cells exposed to oxygen.

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Phylogenetic analyses based on 16S rRNA gene sequences placed the isolates into the genus Natroniella with a similarity 96–97% to its single species N. acetigena (Fig. 2). This was somewhat unexpected, because N. acetigena has been described as an obligate heterotrophic homoacetogen (Zhilina et al., 1995), while the novel sulfur-reducing isolates can grow autotrophically, obtaining electrons from H2 and formate and, in one case, even from acetate – the final metabolic product of N. acetigena. The level of sequence similarity (99%) and the results of DNA–DNA hybridization between the sulfur-reducing isolates (more than 85% similarity) demonstrated that all isolates belong to a single species. Analyses of cellular fatty acids showed the presence of three dominating species constituting more than 60% of the total: C14:0, C16:1ω7 and C16:1ω9. Two of these were also dominant in the type species, N. acetigena, but it also contained high concentrations of two other C16 species totally lacking in the sulfur-reducing isolate (Supporting Information, Table S1), confirming that the novel isolates are significantly different from the type strain of the genus.

image

Figure 2.  Neighbor-joining tree based on the 16S rRNA gene sequences showing the phylogenetic position of sulfur-reducing extremely natronophilic isolates from soda lakes within the order Halanaerobiales. The numbers on the branches indicate bootstrap values >70% after 1000 times of resampling. Scale bar corresponds to 5% sequence divergence. The organisms with respiratory metabolism are shown in bold.

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Metabolism of the sulfur-reducing isolates was limited to anaerobic respiration with sulfur/polysulfide (Fig. 3) and fumarate as electron acceptors (Table 2). No fermentative growth was observed, which represents a drastic difference from their closest phylogenetic relative N. acetigena. Furthermore, the ability of strain AHT18 to respire acetate during anaerobic growth with sulfur in saturated soda brines (Fig. 3) has an important ecological implication and deserves special attention. This is the only organism known so far that is capable of such a function under soda-saturated conditions among sulfidogens from soda lakes. Although the pathway of acetate utilization needs to be studied in detail, one of the possibilities is that it might be used by reversing the acetogenic Wood cycle. A test for the ability of the type species N. acetigena to grow by sulfur respiration either organotrophically with EtOH or lactate or lithotrophically with H2 and formate yielded negative results.

image

Figure 3.  Dynamics of anaerobic growth and polysulfide formation at pH 10 and 3.0 M total Na+ in strain AHT3 grown with formate (50 mM)/acetate (2 mM) and sulfur (a) and strain AHT18 grown with acetate (20 mM) and sulfur (b). The average formula of produced polysulfide was S3.22−. Circles, biomass; open triangles, total sulfane; closed triangles, zero-valent sulfur dissolved in polysulfide.

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Table 2.   Comparative properties of novel extremely natronophilic sulfur-reducing isolates and Natroniella acetigena (Zhilina et al., 1995, 1998)
PropertyN. ‘sulfidogena’ (three strains)N. acetigena
  • *

    This study.

Cell morphologyLong flexible rods motile by multiple peritrichous flagella and rapidly forming sphaeroplasts
Endospore formation+
AcetogenesisLactate, pyruvate, glutamate, ethanol, propanol
Autotrophic growth with H2 and formateWith sulfur/polysulfide as an electron acceptor*
Utilization of acetate as an electron donorStrain AHT18*
Heterotrophic respiration with sulfur/polysulfide as an acceptorLactate, pyruvate, glycerol, glucose, fructose, maltose, sucrose*
pH range (optimum) for growth8.4–10.4 (9.8–10.0)8.1–10.7 (9.7–10.0)
Salinity range (optimum), M Na+1.5–4.0 (3.0)1.5–4.2 (2.0–2.5)
Dominant fatty acids in membrane polar lipids14:0, 16:1ω7, 16:1ω916:1ω9, 14:0, ald16:1ω9, 16:0
G+C content in DNA31.3–32.031.9
Isolated fromSoda lakes in Wadi Natrun and Kulunda SteppeSoda lake Magadi

Thus, significant physiological differences within a single phylotype highlight the necessity of combining molecular ecology with the isolation and physiological investigation of pure cultures in order to understand the function of microbial communities. In other words, multiple closely related phylotypes detected using a culture-independent approach may correspond to physiological diversification and, therefore, both aspects need to be studied in parallel. A recent example of such a trait has been revealed by an extensive polyphasic analysis of two extremely halophilic members of Salinibacter ruber (Peña et al., 2010). Fermentative members of the order Halanaerobiales dominate the anaerobic bacterial community under hypersaline conditions due to their relatively ‘cheap’ K+-based osmoadaptation strategy (Oren, 1999, 2011). According to the hypothesis of A. Oren, in prokaryotes, there is a direct correlation between the energy yield of catabolism and the ability to grow at high salinity. Because the inorganic osmolyte strategy based on potassium import needs much less energy input than de novo synthesis of organic osmolytes, it confers an advantage to such organisms to exploit low energy yield catabolic reactions at extreme salinity. On the basis of the work presented here and also based on other recently published results, it seems that some members of the order Haloanaerobiales use an energy metabolism that has until now been considered rather uncharacteristic for this group. In the absence of more specialized extremely halophilic dissimilatory sulfur-dissimilatory respirers, these organisms are able to perform anaerobic respiration in addition to or even instead of fermentation. Such examples are represented by the extremely halophilic Selenihalanaerobacter shriftii (Switzer-Blum et al., 2001), the recently described extremely haloalkaliphilic arsenate- and sulfur-reducing Halarsenatibacter silvermanii (Switzer-Blum et al., 2009) and the Natroniella strains AHT3, AHT4 and AHT18, described here. The latter, however, advanced further in their specialization by adopting a lithoautotrophic lifestyle. Both possibilities (autotrophy and respiratory catabolism) are basically present in some of the nonextremophilic acetogens. For example, some species are able to reversibly switch from acetogenesis to energetically more favorable respiratory catabolism, such as nitrate reduction, double-bond reduction and thiosulfate-dependent respiration (Beaty & Ljungdahl, 1991; Matties et al., 1993; Seifritz et al., 1993; Müller, 2003). This can already be considered as a step towards further specialization, when respiratory metabolism becomes irreversible, just as we observed in extreme natronophiles.

On the basis of phylogenetic distance and significant physiological differences, we propose to accommodate the extremely natronophilic sulfur-respiring strains from soda lakes in a novel species within the genus Natroniella with the name Natroniella sulfidigena sp. nov.

Description of N. sulfidigena sp. nov.

[sul.fi.di'ge.na N.L. n. sulfidum, sulfide; N.L. suff. -gena (from Gr. v. gennaô, to produce), producing; N.L. part. adj. sulfidigena, sulfide-producing].

Cells are Gram-negative long flexible rods, 0.3–0.5 × 3–30 μm, motile with multiple peritrichous flagella. Cells have the tendency to form sphaeroplasts and rapidly lyse toward the stationary phase. Spore formation is not observed. Strictly anaerobic, growing by respiration with sulfur/polysulfide and fumarate. Can grow autotrophically with either H2 or formate as an electron donor. Also utilize the following compounds as electron donors: pyruvate, lactate, glycerol, glucose, fructose, maltose and sucrose. The utilization of acetate as the electron donor is shown for one of the strains. Fermentative growth was not observed with the following substrates: EtOH, PrOH, lactate, glucose, fructose and yeast extract. Extremely haloalkaliphilic with a pH range for growth between 8.1 and 10.6 (optimum 10) and a salinity range of 1.5–2.0 to 4.0 M Na+ (optimum 3.0 M). Mesophilic with an optimal growth temperature at 35 °C and a maximum at 41 °C. The dominant fatty acids include C14:0, C16:1ω7 and C16:1ω9. The G+C content in the genomic DNA is 31.3–32.0 mol% (Tm). Isolated from hypersaline soda lakes. The type strain is AHT3T (DSM22104T=UNIQEM U268T). The GenBank accession numbers of the 16S rRNA gene of strains AHT3T and AHT18 are GU452711 and GU452712, respectively.

Emended description of the genus Natroniella

In addition to the original description by Zhilina et al. (1995), some of the genus representatives have obligate sulfur-dependent respiratory metabolism and are able to grow autotrophically or with acetate as an electron donor when sulfur serves as an electron acceptor.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Table S1. Comparative composition of cellular fatty acids in strain AHT3T and Natroniella acetigena.

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