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Applied and Environmental Microbiology, January 2007, p. 451-455, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.02087-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Sulfur-Oxidizing Bacteria in Soap Lake (Washington State), a Meromictic, Haloalkaline Lake with an Unprecedented High Sulfide Content

Dimitry Y. Sorokin,^1,2* Mirjam Foti,¹ Holly C. Pinkart,³ and Gerard Muyzer¹

Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands,¹ Winogradsky Institute of Microbiology, Russian Academy of Sciences, 117811 Moscow, Russia,² Department of Biological Sciences, Central Washington University, Ellensburg, Washington 98926³

Received 4 September 2006/ Accepted 7 November 2006

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genus Thioalkalimicrobium, which are common inhabitants of soda lakes. A dense population of Thioalkalimicrobium (up to 10⁷ cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. Twelve Thioalkalimicrobium strains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related to T. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from known Thioalkalimicrobium species and unique to Soap Lake. It represented a new species, for which we suggest the name Thioalkalimicrobium microaerophilum sp. nov.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Soap Lake, located in central Washington State, is the terminal lake at the end of a chain of lakes characterized by increasing salinity and alkalinity. It contains high concentrations of sodium carbonate and sulfate, resulting in a high alkalinity and pH (around 10), typical for soda lakes. However, two features distinguish this lake from many other soda lakes: (i) its sharp stratification into two layers with different features and (ii) an unprecedentedly high sulfide concentration in the anaerobic layer. The bottom layer of the lake, termed the monimolimnion, is hypersaline (140 g liter^–1), cold (6 to 8°C), and highly sulfidic, with anaerobic waters containing up to 200 mM sulfide, the highest concentration ever recorded in natural water. In contrast, the top layer, termed the mixolimnion, is brackish (around 15 g liter^–1) and aerobic. It is separated from the monimolimnion by a chemocline with oxygen concentrations changing from saturation to zero (1, 7, 13). Despite the large difference in density between the two layers, sulfide diffuses into the aerobic layer and, therefore, the role of sulfide-oxidizing bacteria (SOB) must be particularly important in this unusual lake.

Previous work has demonstrated the presence of obligately chemolithoautotrophic SOB in soda lakes, capable of growth at extremely high pH values and variable salinity (11). The genus Thioalkalimicrobium was found mostly in hyposaline lakes of south Siberia and northeastern Mongolia and dominated the enrichments at low-salt conditions (i.e., below 1 M of total sodium). In contrast, the genus Thioalkalivibrio was dominant in hypersaline soda lakes and was the only representative of chemolithoautotrophs known so far capable of growing in saturated soda brines (4 M of total sodium). Use of culture-independent molecular methods allowed the detection of Thioalkalivibrio in soda lake sediments, while the presence of the Thioalkalimicrobium population was obviously below the detection limit (M. Foti and D. Sorokin, unpublished results).

Here we describe the diversity of SOB in Soap Lake, a haloalkaline, meromictic lake with an unusually high sulfide concentration. We used both culture-dependent and -independent techniques to study the SOB present in this lake and found evidence for members belonging to the genera Thioalkalimicrobium and Thioalkalivibrio. A group of microaerophilic isolates affiliated with Thioalkalimicrobium belongs to a new species, for which we suggest the name Thioalkalimicrobium microaerophilum.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Samples.
Soap Lake is permanently stratified into two layers, an upper layer (mixolimnion) and lower layer (monimolimnion), separated by a chemocline at 20.5 m (Fig. 1). Five samples were obtained from Soap Lake (Washington State) in fall 2004, including samples from the mixolimnion, the chemocline, and the monimolimnion; from the deep sediment; and from the mixolimnion sediment at the fringe of the lake (Table 1). Part of the water samples was used for molecular analysis, for sulfide analysis, and for cultivation.

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FIG. 1. Depth profiles of dissolved oxygen (open circles), total dissolved solids (TDS; filled triangles), and sulfide (filled circles).

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TABLE 1. Samples from Soap Lake

Cultivation of sulfur-oxidizing bacteria.
Enrichments and cultivation of haloalkaliphilic SOB were performed using a mineral medium buffered with sodium carbonate-bicarbonate mixture (0.6 and 4 M total Na⁺) at pH 10 to 10.1 as described previously (12). Thiosulfate (20 to 40 mM) served as the energy source and NH₄Cl (4 mM) as the nitrogen source. The medium was supplemented with 1 ml liter^–1 of trace metal solution (6) and 1 mM MgCl₂. Microaerophilic cultivation was performed in 100-ml serum bottles with 10 ml medium in which the air was replaced with argon, containing 1 to 5% (vol/vol) O₂. Solid alkaline media with final salt concentrations of 0.6 and 2 M of total Na⁺ were prepared by 1:1 mixing of 4% (wt/vol) agar and double-strength mineral medium at 50°C. The plates were incubated in closed jars in microaerophilic (2% oxygen) conditions. Enrichments for denitrifying SOB were performed in 100-ml serum bottles filled with 50 ml of the alkaline base media and sealed with butyl rubber stoppers. The medium was supplemented either with 20 mM thiosulfate-20 mM nitrate, 20 mM thiosulfate-50 mM N₂O, or 5 mM HS^–-20 mM nitrate. Anaerobic conditions were achieved by five cycles of evacuation and flushing with argon. The cultivation was performed at 22 (enrichments) and 28°C (pure cultures).

Chemical and other analyses.
Chemical analysis of sulfur compounds (i.e., sulfide, thiosulfate, and sulfur) and cell protein and respiration tests were performed as described previously (12). The dissolved oxygen in-lake measurements were performed with an oxygen probe associated with a Hydrolab (Hach Environmental, Loveland, CO). Phase-contrast microphotographs were obtained with a Zeiss Axioplan Imaging 2 microscope (Göttingen, Germany). For electron microscopy, the cells were fixed with glutaraldehyde (final concentration, 3% [vol/vol]) and positively contrasted with 1% (wt/vol) uranyl acetate. The isolation of the DNA from pure cultures was performed according to Marmur (4). Determination of the G+C content of the DNA and DNA-DNA hybridization were performed by the thermal denaturation/reassociation technique (2).

DNA extraction and PCR amplification of 16S rRNA genes.
Ten-milliliter water samples, 2-cm³ sediment samples, and 1-ml cultures were used for DNA extraction after centrifugation and washing of the pellet with 0.5 M NaCl. Genomic DNA was extracted from the cells using the UltraClean soil DNA extraction kit (Mo Bio Laboratories, West Carlsbad, CA) by following the manufacturer's instructions.

The partial 16S rRNA gene was amplified using bacterial primers 341F-GC and 907R and a touchdown protocol (8). The nearly complete 16S rRNA gene was obtained from pure cultures using bacterial primers GM3F and GM4R (5).

DGGE of PCR products.
Denaturing gradient gel electrophoresis (DGGE) was performed as described by Schäfer and Muyzer (8) with minor modifications. The PCR products were separated on polyacrylamide gel with a linear gradient of 35 to 70% urea and formamide and run at a constant voltage of 100 V for 16 h. Subsequently, the gels were photographed with the Gel Doc 2000 system (Bio-Rad, Hercules, CA) after 30 min of ethidium bromide staining and 30 min of rising in Milli-Q water. Bands of interest were excised, reamplified, and checked for purity in a second DGGE. PCR products were then purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and sequenced.

Phylogenetic analysis.
The sequences obtained in this study were first compared to sequences stored in GenBank using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST). Subsequently, the sequences were imported into the ARB software program (3), aligned, and added to a phylogenetic tree using the QUICK_ADD_TO_EXISTING_TREE tool. The alignment was further corrected by eye, and a tree was calculated using the neighbor-joining algorithm with Felsenstein correction.

Nucleotide sequence accession numbers.
All sequences obtained in this work were deposited in GenBank under the accession numbers DQ900619 to DQ900627.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Enrichment and enumeration of haloalkaliphilic SOB.
Results of a first screening demonstrated the presence of an active population of aerobic, low-salt-tolerant, alkaliphilic, lithoautotrophic SOB in the different samples from Soap Lake, able to grow at 0.6 M of total Na⁺ and pH 10 (Table 2). The most abundant populations were isolated from the chemocline samples. Smaller populations were detected in the anaerobic, high-sulfide samples from the monimolimnion. This was likely a result of sedimentation of the SOB developing in the oxygen-containing layers of the lake, since no growth of anaerobic (denitrifying) SOB was observed in the enrichments. In this sampling period, there was no evidence of a mass development of anaerobic phototrophic sulfur bacteria, commonly present below the chemoclines of meromictic lakes. This was not unexpected since the chemocline does not experience any light penetration (13). Based on these observations, it seems likely that the aerobic chemotrophic SOB are responsible for most of the biological oxidation of sulfide and other reduced sulfur compounds in the lake.

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TABLE 2. Isolation of halotolerant alkaliphilic SOB from Soap Lake

In contrast to the low-salt medium, enrichments in saturated soda brine at 4 M Na⁺ demonstrated much smaller populations, indicating a minor role of these bacteria at the time of sampling.

Isolation and identification of pure cultures.
Enrichments at low salt (0.6 M Na⁺) yielded 12 pure cultures of obligately chemolithoautotrophic SOB with three different morphotypes (Table 2). Seven strains with morphotype A were obtained from all samples except the deep sediments. They were short motile rods. Longer, motile, and slightly curved rods of morphotype B were represented by a single strain from the chemocline. Our main target for the isolation, however, was morphotype C, since the cells of this type were seen in water samples of the chemocline and, after concentration, in samples of the monimolimnion. From four pure cultures of this type, three were long, bent, motile rods at pH 10, turning to fat vibrios at pH 8 to 9. The isolate ASL8-1 was similar in morphology, but the cells were significantly smaller.

DGGE analysis of PCR-amplified 16S rRNA gene fragments from pure cultures and samples of different layers of Soap Lake (Fig. 2) demonstrated that (i) the genetic diversity in Soap Lake was higher in the sediments than in the water column, at least during the sampling period, and (ii) dominant DNA fragments from the DGGE analysis detected in the chemocline, mixolimnion, and monimolimnion sediments were related to low-salt SOB isolates, while the PCR product obtained from the monimolimnion water layer was related to high-salt SOB isolates.

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FIG. 2. DGGE analysis of 16S rRNA gene fragments amplified from water samples (SL) and bacterial isolates (ASL) of Soap Lake. SL1, chemocline; SL2, mixolimnion, sediment pore water; SL3, monimolimnion, water; SL4, monimolimnion, sediment pore water. Sequenced bands that were used for phylogenetic analysis (see Fig. 3) are indicated by white circles and numbers.

Phylogenetic analysis of the sequences of the dominant DGGE bands from the lake samples and from the representative SOB isolates with different morphologies isolated at low-salt conditions identified them as members of the genus Thioalkalimicrobium (Fig. 3). More specifically, the dominant band sequences from the chemocline and the monimolimnion sediments and the morphotype A and B isolates (Table 2) were closely related to Thioalkalimicrobium cyclicum, found previously in the chemocline water of the Mono Lake in California (10) and absent in soda lakes of central Asia (Siberia and Mongolia) and Africa (Kenya and Egypt) (11). It appears that this particular species of haloalkaliphilic SOB is endemic to the North American continent. The morphotype C isolates, together with the dominant sequence from the mixolimnion sediments, formed a separate cluster with a sequence difference of 3% or more with other members of the genus Thioalkalimicrobium. The results of DNA-DNA hybridization analysis of the low-salt-tolerant SOB isolates (Table 3) confirmed their phylogenetic identification, demonstrating the intraspecies relation of morphotypes A and B with T. cyclicum and the separate species position of the morphotype C isolates.

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FIG. 3. Neighbor-joining tree based on 16S rRNA gene sequences, showing the phylogenetic affiliation of bacteria from Soap Lake. Names of the sequences determined in this study are in boldface. Sequences with DGGE-SL are obtained from DGGE bands as indicated in Fig. 1; sequences with ASL are from pure cultures. The sequence accession numbers are in parentheses. The nodes on the branches indicate bootstrap values above 90% from 1,000 replications. The bar indicates 10% sequence variation.

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TABLE 3. Total DNA analysis of the haloalkaliphilic SOB isolates from Soap Lake

High-salt enrichments at 4 M Na⁺ resulted in the isolation of two morphologically similar strains from the chemocline and monimolimnion water samples. Both strains contained a yellow pigment with an absorption spectrum typical for the high-salt-tolerant representatives of the genus Thioalkalivibrio (11). The PCR products obtained from these strains had a migration distance identical to that of the dominant band obtained from the monimolimnion water sample (Fig. 2, band 3). A band at the same position in the gel, although not so intense, was also visible in the profile obtained from the monimolimnion sediment sample (SL4).

Both phylogenetic (Fig. 2) and total DNA (Table 3) analyses of the two high-salt-tolerant SOB isolates confirmed their affiliation with the genus Thioalkalivibrio and in particular to the core group of this genus, most of which are extremely salt tolerant (11). It is interesting, however, that there was very low DNA homology between the Soap Lake Thioalkalivibrio isolates and Thioalkalivibrio jannaschii isolated from Mono Lake (10).

Some of the other bands from the DGGE profiles of the environmental samples have also been successfully identified. Since general diversity was not a subject of this work, we can only briefly mention that most of the sequences belonged to the Clostridiales, but with relatively low homology (usually less than 90%) to sequences deposited in the GenBank.

Influence of environmental parameters on growth and activity of the Thioalkalimicrobium isolates.
Strains ASL1 and ASL8-2, representatives of the two different morphotypes and the genetic groups of the low-salt-tolerant SOB from Soap Lake, responded differently to increasing pH. While ASL1 had a pH profile for growth and respiratory activity typical for obligately alkaliphilic Thioalkalimicrobium species, with an optimum at 9.5 to 10 (data not shown), strain ASL8-2 grew and respired best at pH 8.5 to 9.0. However, no growth occurred at neutral pH values in either strain, similar to the known obligately alkaliphilic Thioalkalimicrobium species (Table 4). Therefore, strain ASL8-2 can be qualified as a moderate, but obligate, alkaliphile. The respiratory profile of strain ASL8-2 was typical for the representatives of the genus Thioalkalimicrobium (9, 12), with extremely high oxidation rates for thiosulfate and sulfide (1.6 and 2.4 µmol O₂ mg protein^–1 min^–1 at pH 10, respectively) and no activity with tetrathionate, elemental sulfur, and sulfite. In respect to its salt tolerance, both strains had low salt tolerance, with a range for growth (at pH 9) from 0.2 to 1.2 M total Na⁺ (optimum at 0.4 to 0.5 M). In contrast to ASL1, ASL8-2 (and all other isolates of this type) was incapable of initiating growth at fully aerobic conditions. The best way to grow these bacteria was to start a culture in closed bottles with a gas phase containing 2% O₂. After one-half of the thiosulfate was consumed, and the biomass level exceeded 30 to 40 mg protein liter^–1, oxygen content in the gas phase could be increased up to normal atmospheric concentrations. However, active aeration on the rotary shaker inhibited growth and eventually caused cell lysis.

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TABLE 4. Influence of pH on growth and respiration of strain ASL8-2

Despite extremely high sulfide concentrations in anaerobic parts of the lake, the relevance of extraordinary sulfide tolerance seems to be not of utmost importance for the obligately aerobic SOB, such as the Thioalkalimicrobium population, dominating in Soap Lake, in contrast to the obvious necessity of such an adaptation among anaerobic inhabitants of the lake. The aerobic SOB populations develop and are active only in the interface, i.e., within a sulfide-oxygen gradient, where sulfide concentration is low. Furthermore, our experiments with soda solutions at pH 10 demonstrated that sulfide at concentrations above 1 mM starts to react with oxygen spontaneously. Therefore, the role of SOB in sulfide oxidation in the lake is important at micromolar concentrations. Two other factors are certainly important at so high a sulfide content: (i) the ability to retain activity at short-term exposure to high sulfide, in case of temporary exposure to anaerobic high-sulfide waters, and (ii) extremely high sulfide-oxidizing capacity, which allows this particular group of haloalkaliphilic SOB to cope with high upward flux of sulfide. In a short-term respiratory experiment at pH 10, strain ASL8-2 showed only slightly higher tolerance to sulfide than the type strain, Thioalkalimicrobium aerophilum AL3, isolated from a low-sulfide soda lake (50 and 70% inhibition of sulfide respiration at 2 mM sulfide, respectively). In contrast, the tolerance of sulfide of anaerobic haloalkaliphiles can be extraordinarily high. For example, strains of a fermentative Amphibacillus sp. isolated from soda soils can grow at pH 10 in the presence of up 100 mM sulfide (our unpublished data).

Soap Lake harbors relatively dense populations of obligately chemolithoautotrophic haloalkaliphilic SOB which, similar to other soda lakes, are represented by the genera Thioalkalimicrobium and Thioalkalivibrio. The former is clearly dominant in the low-salinity, aerobic parts of the mixolimnion and seems to play an important role in the oxidation of sulfide diffusing across the chemocline from the extremely concentrated anaerobic brines of the monimolimnion. This is not surprising, owing to the remarkably high sulfide-oxidizing capacity typical for the haloalkaliphilic SOB of the genus Thioalkalimicrobium (11). The environmental importance of this particular group is confirmed by its detection as a dominant in bacterial populations of three out of four analyzed environmental samples from Soap Lake using PCR-DGGE.

One of the genotypes of Thioalkalimicrobium (represented by four isolates) detected in Soap Lake was not detected previously in any other soda lakes. In general, this group has much in common with the other Thioalkalimicrobium species. Particularly, it could not grow without reduced-sulfur compounds on purely organic medium (acetate and yeast extract) and could not utilize organic carbon instead of bicarbonate. Only reduced sulfur compounds, such as sulfide and thiosulfate, but not H₂ or formate, could be used as electron donors. On the other hand the group clearly differed from the known species in morphology, phylogeny, and some physiological properties and therefore is proposed to form a new species, Thioalkalimicrobium microaerophilum.

Description of Thioalkalimicrobium microaerophilum sp. nov.
Thioalkalimicrobium microaerophilum (mic.ro.ae.ro.phi'lum. Gr. adj. micros, small, little; Gr. masc. n. aër, gas; Gr. adj. philum, loving; M.L. n. microaerophilum, loving low-oxygen conditions). The cell morphology varies from thick vibrios, 1 to 2 µm by 2 to 5 µm, at optimal pH values (pH 8 to 9) to long rods, 0.6 to 0.8 µm by 3 to 8 µm, which are motile by means of a single polar flagellum (Fig. 4c). It is obligately chemolithoautotrophic and microaerophilic. It grows best in soda-buffered culture medium at pH 8.5 to 9. Thiosulfate or sulfide is used as an electron donor. It grows within a pH range from 8 to 10 and at a salt concentration equivalent to 0.2 to 1.2 M of total Na⁺. The temperature optimum for growth at pH 9 and 0.6 M Na⁺ is between 25 and 28°C. It oxidizes sulfide and thiosulfate to sulfate at high rates within a pH range of 6 to 10.5, with an optimum at 9 to 9.5. The G+C content in the DNA is 49.3 to 49.8 mol% (melting temperature). Other properties are similar to those described for the genus. It was isolated from the saline, alkaline Soap Lake (Grant County, Washington State). It is represented by four strains with a DNA-DNA homology level of more than 70%. The type strain is ASL8-2^T (DSM 17327, UNIQEM U242). The accession number of the 16S rRNA gene sequence is DQ900623.

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FIG. 4. Cell morphology of Thioalkalimicrobium microaerophilum sp. nov. strain ASL8-2T. (a and b) Phase-contrast photographs of the cells grown at pH 8.5 (a) and at pH 10.0 (b); (c) electron-microscopic photograph of the cell, grown at pH 8.5. Scale bar, 1 µm.

 
This work was supported by NWO-RFBR grant 47.011.2004.010, by the Program of the Russian Academy of Sciences "Molecular and Cell Biology," by the Dutch Foundation for Applied Research (STW, grant WBC 5939), and by a United States National Science Foundation Microbial Observatories program (MCB-0132158).

 
* Corresponding author. Mailing address: Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Moscow, Russia. Phone: (7095)1350109. Fax: (7095)1356530. E-mail: soroc{at}inmi.host.ru.

Published ahead of print on 17 November 2006.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, January 2007, p. 451-455, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.02087-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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