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Halophilic microbial communities and their environments
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ScienceDirect Halophilic microbial communities and their environments Aharon Oren Abstract Use of culture-independent studies have greatly increased our understanding of the microbiology of hypersaline lakes (the Dead Sea, Great Salt Lake) and saltern ponds in recent years. Exciting new information has become available on the microbial processes in Antarctic lakes and in deep-sea brines. These studies led to the recognition of many new lineages of microorganisms not yet available for study in culture, and their cultivation in the laboratory is now a major challenge. Studies of the metabolic potentials of different halophilic microorganisms, Archaea as well as Bacteria, shed light on the possibilities and the limitations of life at high salt concentrations, and also show their potential for applications in bioremediation. Address Department of Plant & Environmental Sciences, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat-Ram, Jerusalem 91904, Israel Corresponding author: Oren, Aharon ([email protected])
and metagenomics led to a better understanding of the biology of hypersaline lakes such as Great Salt Lake, Utah, the Dead Sea, and salterns evaporation and crystallizer ponds. Studies of unusual and previously unexplored hypersaline environments in Australia, Antarctica and in the deep sea have also added interesting information. Culture-dependent methods were not forgotten: recognition of interesting and numerically important phylotypes in the DNA recovered from these environments has triggered attempts to isolate the relevant organisms, and at least in one case this strategy has been successful. The ability of some halophilic microorganisms to degrade hydrocarbons and other toxic chemicals is shown in bioremediation studies of polluted hypersaline environments.
Studies in salt lakes The best-studied hypersaline lakes are Great Salt Lake and the Dead Sea. But in recent years interesting studies have been published on salt lakes in Australia, Romania, and in other locations in hot and temperate climates.
Current Opinion in Biotechnology 2015, 33:119–124 This review comes from a themed issue on Environmental biotechnology Edited by Spiros N Agathos and Nico Boon
http://dx.doi.org/10.1016/j.copbio.2015.02.005 0958-1669/# 2015 Elsevier Ltd. All rights reserved.
Introduction Natural and man-made hypersaline environments (here defined as having salt concentrations more than twice that of seawater) such as salt lakes and evaporation ponds for the production of salt from seawater are found on all continents. They are inhabited by a great diversity of microorganisms adapted to life at high salt concentrations. Growth of representatives of all three domains of life — Archaea, Bacteria and Eukarya — is possible even in NaCl-saturated brines [1,2] (Figure 1). In the past few years our insight in the microbial processes in high-salt environments and the possibilities and limitations of the biota to adapt to extreme salinities has greatly improved. Application of state-of-the-art cultureindependent techniques of high-throughput sequencing www.sciencedirect.com
Great Salt Lake was the object of a number of cultureindependent studies of both the less saline south arm and the north arm approaching NaCl saturation. The primary producers are cyanobacteria in the lower salinity areas and Dunaliella spp. at the highest salt concentrations. Microcosm experiments showed salinity as a strong determinant of phytoplankton diversity. Species richness decreased with increasing salinity and increased with nutrient enrichment [3]. Inventories of bacterial and archaeal diversity at sites of different salinities and at different depths along the vertical salinity gradient in the south arm showed the majority of 16S rRNA gene sequences within the clone libraries to be distantly related to previously described halophilic archaeal and bacterial taxa, and they represent novel phylotypes [4,5]. A 16S rRNA oligonucleotide microarray (PhyloChip) and a functional gene array (GeoChip) were used to study the correlation between the phylogenetic diversity within the community and increased salt concentration [6,7]. The lake is also an excellent model to study horizontal gene transfer, as shown by monitoring chromium resistance genes within the microbial community [7]. Understanding the Dead Sea ecosystem is important in view of the planned implementation of a water conveyor from the Red Sea that will raise the water level and dilute the upper water layers [8]. Because of the extreme salinity (35%) and divalent cation concentrations (2 M Mg2+, 0.5 M Ca2+) the lake does not currently support dense Current Opinion in Biotechnology 2015, 33:119–124
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Figure 1
Current Opinion in Biotechnology
Examples of hypersaline environments and their biota discussed in this review: the Dead Sea (upper left panel), a saltern evaporation pond populated by different eukaryotic algae and cyanobacteria (Atlit, Israel) (upper middle panel), a saltern crystallizer pond (Eilat, Israel) colored red by dense communities of Archaea and Dunaliella cells (upper left panel), a benthic cyanobacterial mat from a saltern pond (Secˇovlje, Slovenia; lower left panel), and a bundle of filaments of the cyanobacterium Coleofasciculus chthonoplastes from this mat, enclosed in a common sheath (lower right panel). Photographs by the author.
microbial communities. The last archaeal bloom was observed in 1992–1995, triggered by dilution by rain floods. Culture-independent studies showed that that bloom was dominated by a single, yet-uncultured phylotype, while the small residual population 15 years later was highly diverse [9,10]. Large amounts of bacteriorhodopsin were present in a bloom of Archaea that developed in the lake in 1980–1981, but no rhodopsin genes were detected in the 1992 bloom. Thus, bacteriorhodopsin probably does not play a role during that bloom; however, novel bacteriorhodopsin and sensory rhodopsin genes were found in samples collected in 2007 and 2010 [11]. Underwater freshwater to brackish springs are inhabited by interesting microbial communities including chemolithotrophs, phototrophs, sulfate reducers, nitrifiers, iron oxidizers, iron reducers, and others. The springs supply nitrogen, phosphorus and organic matter to the Dead Sea microbial communities. Due to frequent fluctuations in the freshwater flow and local salinity, microorganisms that inhabit these springs must be capable of withstanding large and rapid salinity fluctuations [12,13]. Microbial communities of a number of hypersaline lakes not or poorly explored in the past were recently studied. Current Opinion in Biotechnology 2015, 33:119–124
Metagenomic assembly of DNA libraries from Lake Tyrrell, Victoria, Australia enabled reconstruction of two unusual archaeal genomes. These belong to an uncultured but abundant type of very small halophiles (0.6 mm diameter). These ‘Nanohaloarchaea’ form a distinct euryarchaeotal lineage, distantly related to the Halobacteriaceae [14]. Sequences related to this group were first reported from Lake Magadi, Kenya, in 1999, and they were since detected in hypersaline environments elsewhere. Seasonal succession was examined over a twoyear period, using a metagenomic approach [15]. Eukaryotic diversity was investigated based on 18S rRNA gene sequences; a novel cluster of the Alveolate Colpodella spp. were the dominant planktonic eukaryotes, while the eukaryotic phototroph Dunaliella was abundant in the salt crusts [16]. The hypersaline stratified and heliothermal Lake Ursu, Romania, was explored using culture-based and cultivation-independent techniques [17], and the archaeal community of Ocnei Lake, a salt mine-associated Romanian salt lake, was assessed from 16S rRNA libraries. Archaeal diversity increased with water depth and salinity [18]. Hot Lake, Washington, is an unusual MgSO4-dominated meromictic lake, unexplored since the 1950s [19]. www.sciencedirect.com
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Renewed study of the site demonstrated complex seasonal phototrophic microbial mats that are subject to >10-fold variation in salinity. 16S RNA gene clone libraries yielded different types of Cyanobacteria, Actinobacteria, and groups of Proteobacteria involved in the sulfur cycle. A great diversity of Bacteria growing in either 1.7 M NaCl or 2 M MgSO4 was isolated from the lake. Archaea are scarce, despite nearsaturated salinities [20,21]. Metagenomic sequencing was also reported for the Salton Sea, California [22].
Studies in saltern ponds Understanding the microbiology of saltern ponds is important as the biota directly influence the salt production process [23]. From the early days of molecular microbial ecology, saltern crystallizer ponds have been popular study objects for culture-independent studies [24,25]. In a 19% salinity pond of a Spanish saltern, novel microbial groups were abundantly found, including a group of Actinobacteria and Euryarchaeota related to the recently described Nanohaloarchaea [14], which probably lead a photoheterotrophic and polysaccharide-degrading lifestyle [26]. More than half of the 16S rRNA genes retrieved from a 13% salinity pond could not be assigned to previously described genera, and they belonged to many groups, mainly of Euryarchaeota, Gammaproteobacteria, Alphaproteobacteria, Actinobacteria and Bacteroidetes. The abundance of genes for bacteriorhodopsins and other retinal proteins suggests that light absorbed by retinal proteins may contribute to the energy supply of the prokaryote community also at these relatively low salinities [27]. A novel gammaproteobacterium, present in high numbers and first detected by its 16S rRNA sequence in a culture-independent study [27], was brought into culture and characterized as Spiribacter salinus. It is an aerobic heterotroph growing optimally at 2.6 M NaCl [28]. Comparative metagenomic studies were performed in Mediterranean and Atlantic saltern ponds in Spain [29] and along the salinity gradient of the Guerrero Negro, Baja California salterns [30]. Saltern evaporation ponds typically contain benthic mats with colorful layers of cyanobacteria and anoxygenic phototrophs, stratified due to sharp gradients of light, oxygen, and sulfide. The most intensively investigated mat system is that at Guerrero Negro (salinity 9%) [31]. High-throughput 16S rRNA sequencing of the different layers led to the recognition of several new phylum-level groups of Bacteria and many novel lower-level taxa [32]. Reconstruction of the proteins encoded by the metagenomes showed near-identical, acid-shifted isoelectric point profiles in all layers. This was interpreted as molecular convergence of amino-acid usage dictated by hypersalinity [33]; however, similar isoelectric point profiles are common in marine Proteobacteria [34]. Trace metal concentrations vary on a millimeter-scale, ascribed to different geochemical processes and bacterial population distributions [35]. During daytime the cyanobacteria www.sciencedirect.com
(Coleofasciculus, Lyngbya) perform oxygenic photosynthesis, but at nighttime they produce molecular hydrogen and organic fermentation products from photosynthetic storage products. Most hydrogenase transcripts were attributed to cyanobacterial [NiFe]-hydrogenases. Chloroflexi and sulfate reducing bacteria are the main consumers of the cyanobacterial fermentation products [36]. A study of lipids in a higher-salinity, gypsum-containing mat at Guerrero Negro provided a wealth of biomarkers witnessing the vertical stratification of different prokaryotes [37]. A metagenomic study of the benthic community of a crystallizer pond on Mallorca, Spain, was carried out with special emphasis on the detection of yet-uncultured methanogenic Archaea adapted to high salt [38].
Antarctic lakes and deep-sea brines Metagenomic analysis of the microbial communities in Organic Lake (Vestfold Hills, Antarctica) was made in view of its high dimethylsulfide concentration. Bacteria were dominated by Marinobacter, Roseovarius and Psychroflexus. Abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, and rhodopsins indicate the use of phototrophy and lithoheterotrophy. Dimethylsulfoniopropionate lyase genes were abundant [39]. A metagenomic study of the cold and hypersaline Deep Lake, Antarctica, showed an ecosystem dominated by four halophilic Archaea whose complete genomes could be reconstructed: Halorubrum lacusprofundi (10% of the community) and the types designated tADL (isolated and described as Halohasta litchfieldiae) (44%), DL31 (18%), and DL1 (0.3%). The organisms differ in their abilities to degrade organic substrates [40]. Below the permanent ice cover of the Antarctic Lake Vida a diverse Bacteria-dominated aphotic ecosystem was found at 13 8C and 20% salt. Abiotic interactions between the brine and the rocks may be responsible for the generation of hydrogen as an energy source for the biota [41]. The anoxic brine pools on the bottom of the Red Sea and the Eastern Mediterranean have been objects of intensive studies lately [42]. The Red Sea Atlantis II and Discovery brines are characterized by steep salinity as well as temperature gradients. Alphaproteobacteria dominated the water above the brines, Planctomycetaceae and Deferribacteres abounded at the interfaces, and Gammaproteobacteria and several Euryarchaeota groups increased in the brine [43]. Metagenomic study of the Atlantis II brine (>2000 m depth, pH 5.3, 68 8C, salinity 26%, high heavy metal concentrations) led to the identification of a novel mercuric reductase, thermostable, functional in high salt, and efficiently detoxifying Hg2+ in vivo [44]. The main components of the biota of the recently discovered Lake Medee in the Eastern Mediterranean belong to the euryarchaeal MSBL1 and the bacterial KB1 candidate divisions. These organisms at least partially rely on reductive cleavage of the osmoprotectant glycine Current Opinion in Biotechnology 2015, 33:119–124
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betaine [45], but many other phylotypes are present [46]. Anaerobic ammonium oxidation (anammox) is functional in the chemoclines of the Bannock and L’Atalante brines in the eastern Mediterranean [47]. Mediterranean deepsea brines are also inhabited by a surprising diversity of ciliates, stramenopiles, and other eukaryotes [48].
Metabolic diversity at the highest salt salinities Aerobic heterotrophic halophiles are very versatile with respect to the organic substrates catabolized. Even the Archaea of the family Halobacteriaceae have a surprisingly large range of metabolizable substrates [49]. A thus far poorly explored substrate is chitin, often abundant in hypersaline environments inhabited by brine shrimp and brine flies. Chitin degrading aerobic (Saccharospirillum and Arhodomonas spp.) and anaerobic bacteria (Orenia chitinitropha) were enriched from hypersaline neutral-pH lakes of the Kulunda Steppe (Altai, Russia) in 2 M NaCl media [50]. In alkaline hypersaline lakes chitin degradation was demonstrated up to 3.5–4 M Na+, and a number of alkaliphilic chitin-degrading aerobes were isolated from soda lake sediments and surrounding soils [51]. To live at high salt is energetically expensive, and the salt concentration limit supporting different dissimilatory processes may be determined by bioenergetic constraints. Further studies of the dynamics of different processes in hypersaline environments are needed to test and refine current insights [52,53]. Sulfate reduction by ‘complete oxidizers’ using acetate as electron donor is not known above 12% salt, while ‘incomplete oxidizers’ can oxidize organic substrates to acetate + CO2 at much higher salinities [52,53]. However, a recent study of coastal salt pans in South Africa, based on amplification of dissimilatory sulfite reductase genes, suggested activity of Desulfobacteraceae (complete oxidizers) and Desulfohalobiaceae (incomplete oxidizers) at salinities as high as 300–400% [54]. In anoxic sediments of hypersaline lakes in the Kulunda Steppe (Altai, Russia), thiosulfate and elemental sulfur were better electron acceptors than sulfate. No growth of sulfate reducing bacteria was observed at 4 M NaCl. Enrichments with lactate, propionate, acetate, or butyrate using sulfate or thiosulfate as electron acceptors yielded isolates related to Desulfosalsimonas propionicica, Desulfohalobium utahense, and Desulfocella halophila. Sulfur-reducing enrichments at 4 M NaCl with acetate yielded Archaea closest related to the genus Halobacterium [55]. Another interesting novel type of metabolism detected in haloarchaea is oxidation of arsenite to arsenate, observed in samples from Salar de Punta Negra, Chile [56].
Bioremediation of polluted hypersaline environments A great diversity of hydrocarbonoclastic Bacteria and Archaea were found in oil-contaminated hypersaline (3–4 M NaCl ambient salinity) coastal areas in Kuwait, Current Opinion in Biotechnology 2015, 33:119–124
using culture-dependent methods [57,58]. Amino acids, vitamins and light enhanced hydrocarbon removal in laboratory microcosms [58,59]. Two Marinobacter spp. isolated from Kuwait grew optimally at 1–1.5 M NaCl, but maintained hydrocarbonoclastic activity up to 5 M NaCl. Both use aliphatic hydrocarbons (C9–C40), benzene, biphenyl, phenanthrene, anthracene and naphthalene as sole carbon and energy sources, and both fix nitrogen. Their use in bioremediation was successfully tested in hypersaline microcosms [60].
Concluding remarks Culture-independent studies, using the rapidly developing methods for ever faster and higher-throughput sequencing, and the ever-improving tools to evaluate the results and to make sense of the wealth of data obtained, have clearly dominated the field of hypersaline environmental microbiology in the past years. This situation is similar to what has happened in other areas of microbial ecology. It is encouraging to note that culture-dependent studies were not completely neglected [50,51,60]. Studying the relevant organisms in culture is indispensable to obtain an insight into the functioning of the ecosystems and the interactions between its components. The isolation in the first years of this century of Haloquadratum and Salinibacter has greatly increased our understanding of saltern crystallizer ecosystems. The case of Spiribacter salinus gives an excellent recent example of a novel type of halophile isolated after its importance in the ecosystem was ascertained by culture-independent techniques [27,28]. The earlier isolation of Salinibacter ruber represents a similar case [61]. We must hope that representatives of the nanohaloarchaea, a group discovered by sequencing of environmental DNA [14,26], will soon become available in culture.
Acknowledgement This study was supported by Grants no. 1103/10 and 343/13 from the Israel Science Foundation.
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40. Williams TJ, Allen MA, DeMaere MZ, Kyrpides NC, Tringe SG, Woyke T, Cavicchioli R: Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea. ISME J 2014, 8:1645-1658. A metagenomic study of the cold and hypersaline Deep Lake, Antarctica, showed the presence of a simple ecosystem dominated by four halophilic Archaea, whose complete genomes could be reconstructed: the earlier isolated Halorubrum lacusprofundi (10% of the community) and the types designated tADL (isolated and described as Halohasta litchfieldiae) (44%), DL31 (18%), and DL1 (0.3%). The organisms differ in their abilities to degrade organic substrates and may occupy different niches in the lake.
55. Sorokin DY, Zacharova EE, Pimenov NV, Tourova TP, Panteleeva AN, Muyzer G: Sulfidogenesis in hypersaline chloride–sulfate lakes of Kulunda Steppe (Altai, Russia). FEMS Microbiol Ecol 2012, 79:445-453.
41. Murray AE, Kenig F, Fritsen CH, McKay CP, Cawley KM, Edwards R, Kuhn E, McKnight DM, Ostrom NE, Peng V et al.: Microbial life at S13 -C in the brine of an ice-sealed Antarctic lake. Proc Natl Acad Sci U S A 2012, 109:20626-20631. Below the permanent ice cover of Lake Vida (Antarctica) a diverse microbial ecosystem dominated by Bacteria is found at 13 8C and 20% salt. This aphotic ecosystem, rich in reduced metals, ammonia, molecular hydrogen, and dissolved organic carbon, has been isolated from external sources of energy for thousands of years. Abiotic interactions between the brine and the rocks may be responsible for the generation of hydrogen as an energy source for the biota. 42. Antunes A, Ngugi DK, Stingl U: Microbiology of the Red Sea (and other) deep-sea anoxic brine lakes. Environ Microbiol Rep 2011, 3:416-433. 43. Bougouffa S, Yang JK, Lee OO, Wang Y, Batang Z, Al-Suwailem A, Qian PY: Distinctive microbial community structure in highly stratified deep-sea brine water columns. Appl Environ Microbiol 2013, 79:3425-3437. 44. Sayed A, Ghazy MA, Ferreira AJS, Setubal JC, Chambergo FS, Ouf A, Adel M, Dawe AS, Archer JAC, Bajic VB et al.: A novel
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54. Roychoudhury AN, Cowan D, Porter D, Valverde A: Dissimilatory sulphate reduction in hypersaline coastal pans: an integrated microbiological and geochemical study. Geobiology 2013, 11:224-233.
56. Dı´az-Palma P, Alfaro G, Hengst M, Pozo P, Stegen S, Queirolo F, Rojo G, Silva P, Arias D, Gallardo K et al.: Kinetics of arsenite removal by halobacteria from a highland Andean Chilean Salar. Aquat Biosyst 2013, 9:8. 57. Al-Mailem DM, Eliyas M, Khanafer M, Radwan SS: Culturedependent and culture-independent analysis of hydrocarbonoclastic microorganisms indigenous to hypersaline environments in Kuwait. Microb Ecol 2014, 67:857-865. 58. Al-Mailem DM, Eliyas M, Radwan SS: Enhanced haloarchaeal oil removal in hypersaline environments via organic nitrogen fertilization and illumination. Extremophiles 2012, 16:751-758. 59. Al-Mailem DM, Eliyas M, Radwan SS: Enhanced bioremediation of oil-polluted, hypersaline, coastal areas in Kuwait via vitamin-fertilization. Environ Sci Pollut Res 2014, 21:3386-3394. 60. Al-Mailem DM, Eliyas M, Radwan SS: Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulf coasts. Extremophiles 2013, 17:463-470. 61. Anto´n J, Oren A, Benlloch S, Rodrı´guez-Valera F, Amann R, Rossello´-Mora R: Salinibacter ruber gen. nov., sp. nov., a novel extreme halophilic member of the Bacteria from saltern crystallizer ponds. Int J Syst Evol Microbiol 2002, 52:485-491.
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ScienceDirect Halophilic microbial communities and their environments Aharon Oren Abstract Use of culture-independent studies have greatly increased our understanding of the microbiology of hypersaline lakes (the Dead Sea, Great Salt Lake) and saltern ponds in recent years. Exciting new information has become available on the microbial processes in Antarctic lakes and in deep-sea brines. These studies led to the recognition of many new lineages of microorganisms not yet available for study in culture, and their cultivation in the laboratory is now a major challenge. Studies of the metabolic potentials of different halophilic microorganisms, Archaea as well as Bacteria, shed light on the possibilities and the limitations of life at high salt concentrations, and also show their potential for applications in bioremediation. Address Department of Plant & Environmental Sciences, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat-Ram, Jerusalem 91904, Israel Corresponding author: Oren, Aharon ([email protected])
and metagenomics led to a better understanding of the biology of hypersaline lakes such as Great Salt Lake, Utah, the Dead Sea, and salterns evaporation and crystallizer ponds. Studies of unusual and previously unexplored hypersaline environments in Australia, Antarctica and in the deep sea have also added interesting information. Culture-dependent methods were not forgotten: recognition of interesting and numerically important phylotypes in the DNA recovered from these environments has triggered attempts to isolate the relevant organisms, and at least in one case this strategy has been successful. The ability of some halophilic microorganisms to degrade hydrocarbons and other toxic chemicals is shown in bioremediation studies of polluted hypersaline environments.
Studies in salt lakes The best-studied hypersaline lakes are Great Salt Lake and the Dead Sea. But in recent years interesting studies have been published on salt lakes in Australia, Romania, and in other locations in hot and temperate climates.
Current Opinion in Biotechnology 2015, 33:119–124 This review comes from a themed issue on Environmental biotechnology Edited by Spiros N Agathos and Nico Boon
http://dx.doi.org/10.1016/j.copbio.2015.02.005 0958-1669/# 2015 Elsevier Ltd. All rights reserved.
Introduction Natural and man-made hypersaline environments (here defined as having salt concentrations more than twice that of seawater) such as salt lakes and evaporation ponds for the production of salt from seawater are found on all continents. They are inhabited by a great diversity of microorganisms adapted to life at high salt concentrations. Growth of representatives of all three domains of life — Archaea, Bacteria and Eukarya — is possible even in NaCl-saturated brines [1,2] (Figure 1). In the past few years our insight in the microbial processes in high-salt environments and the possibilities and limitations of the biota to adapt to extreme salinities has greatly improved. Application of state-of-the-art cultureindependent techniques of high-throughput sequencing www.sciencedirect.com
Great Salt Lake was the object of a number of cultureindependent studies of both the less saline south arm and the north arm approaching NaCl saturation. The primary producers are cyanobacteria in the lower salinity areas and Dunaliella spp. at the highest salt concentrations. Microcosm experiments showed salinity as a strong determinant of phytoplankton diversity. Species richness decreased with increasing salinity and increased with nutrient enrichment [3]. Inventories of bacterial and archaeal diversity at sites of different salinities and at different depths along the vertical salinity gradient in the south arm showed the majority of 16S rRNA gene sequences within the clone libraries to be distantly related to previously described halophilic archaeal and bacterial taxa, and they represent novel phylotypes [4,5]. A 16S rRNA oligonucleotide microarray (PhyloChip) and a functional gene array (GeoChip) were used to study the correlation between the phylogenetic diversity within the community and increased salt concentration [6,7]. The lake is also an excellent model to study horizontal gene transfer, as shown by monitoring chromium resistance genes within the microbial community [7]. Understanding the Dead Sea ecosystem is important in view of the planned implementation of a water conveyor from the Red Sea that will raise the water level and dilute the upper water layers [8]. Because of the extreme salinity (35%) and divalent cation concentrations (2 M Mg2+, 0.5 M Ca2+) the lake does not currently support dense Current Opinion in Biotechnology 2015, 33:119–124
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Figure 1
Current Opinion in Biotechnology
Examples of hypersaline environments and their biota discussed in this review: the Dead Sea (upper left panel), a saltern evaporation pond populated by different eukaryotic algae and cyanobacteria (Atlit, Israel) (upper middle panel), a saltern crystallizer pond (Eilat, Israel) colored red by dense communities of Archaea and Dunaliella cells (upper left panel), a benthic cyanobacterial mat from a saltern pond (Secˇovlje, Slovenia; lower left panel), and a bundle of filaments of the cyanobacterium Coleofasciculus chthonoplastes from this mat, enclosed in a common sheath (lower right panel). Photographs by the author.
microbial communities. The last archaeal bloom was observed in 1992–1995, triggered by dilution by rain floods. Culture-independent studies showed that that bloom was dominated by a single, yet-uncultured phylotype, while the small residual population 15 years later was highly diverse [9,10]. Large amounts of bacteriorhodopsin were present in a bloom of Archaea that developed in the lake in 1980–1981, but no rhodopsin genes were detected in the 1992 bloom. Thus, bacteriorhodopsin probably does not play a role during that bloom; however, novel bacteriorhodopsin and sensory rhodopsin genes were found in samples collected in 2007 and 2010 [11]. Underwater freshwater to brackish springs are inhabited by interesting microbial communities including chemolithotrophs, phototrophs, sulfate reducers, nitrifiers, iron oxidizers, iron reducers, and others. The springs supply nitrogen, phosphorus and organic matter to the Dead Sea microbial communities. Due to frequent fluctuations in the freshwater flow and local salinity, microorganisms that inhabit these springs must be capable of withstanding large and rapid salinity fluctuations [12,13]. Microbial communities of a number of hypersaline lakes not or poorly explored in the past were recently studied. Current Opinion in Biotechnology 2015, 33:119–124
Metagenomic assembly of DNA libraries from Lake Tyrrell, Victoria, Australia enabled reconstruction of two unusual archaeal genomes. These belong to an uncultured but abundant type of very small halophiles (0.6 mm diameter). These ‘Nanohaloarchaea’ form a distinct euryarchaeotal lineage, distantly related to the Halobacteriaceae [14]. Sequences related to this group were first reported from Lake Magadi, Kenya, in 1999, and they were since detected in hypersaline environments elsewhere. Seasonal succession was examined over a twoyear period, using a metagenomic approach [15]. Eukaryotic diversity was investigated based on 18S rRNA gene sequences; a novel cluster of the Alveolate Colpodella spp. were the dominant planktonic eukaryotes, while the eukaryotic phototroph Dunaliella was abundant in the salt crusts [16]. The hypersaline stratified and heliothermal Lake Ursu, Romania, was explored using culture-based and cultivation-independent techniques [17], and the archaeal community of Ocnei Lake, a salt mine-associated Romanian salt lake, was assessed from 16S rRNA libraries. Archaeal diversity increased with water depth and salinity [18]. Hot Lake, Washington, is an unusual MgSO4-dominated meromictic lake, unexplored since the 1950s [19]. www.sciencedirect.com
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Renewed study of the site demonstrated complex seasonal phototrophic microbial mats that are subject to >10-fold variation in salinity. 16S RNA gene clone libraries yielded different types of Cyanobacteria, Actinobacteria, and groups of Proteobacteria involved in the sulfur cycle. A great diversity of Bacteria growing in either 1.7 M NaCl or 2 M MgSO4 was isolated from the lake. Archaea are scarce, despite nearsaturated salinities [20,21]. Metagenomic sequencing was also reported for the Salton Sea, California [22].
Studies in saltern ponds Understanding the microbiology of saltern ponds is important as the biota directly influence the salt production process [23]. From the early days of molecular microbial ecology, saltern crystallizer ponds have been popular study objects for culture-independent studies [24,25]. In a 19% salinity pond of a Spanish saltern, novel microbial groups were abundantly found, including a group of Actinobacteria and Euryarchaeota related to the recently described Nanohaloarchaea [14], which probably lead a photoheterotrophic and polysaccharide-degrading lifestyle [26]. More than half of the 16S rRNA genes retrieved from a 13% salinity pond could not be assigned to previously described genera, and they belonged to many groups, mainly of Euryarchaeota, Gammaproteobacteria, Alphaproteobacteria, Actinobacteria and Bacteroidetes. The abundance of genes for bacteriorhodopsins and other retinal proteins suggests that light absorbed by retinal proteins may contribute to the energy supply of the prokaryote community also at these relatively low salinities [27]. A novel gammaproteobacterium, present in high numbers and first detected by its 16S rRNA sequence in a culture-independent study [27], was brought into culture and characterized as Spiribacter salinus. It is an aerobic heterotroph growing optimally at 2.6 M NaCl [28]. Comparative metagenomic studies were performed in Mediterranean and Atlantic saltern ponds in Spain [29] and along the salinity gradient of the Guerrero Negro, Baja California salterns [30]. Saltern evaporation ponds typically contain benthic mats with colorful layers of cyanobacteria and anoxygenic phototrophs, stratified due to sharp gradients of light, oxygen, and sulfide. The most intensively investigated mat system is that at Guerrero Negro (salinity 9%) [31]. High-throughput 16S rRNA sequencing of the different layers led to the recognition of several new phylum-level groups of Bacteria and many novel lower-level taxa [32]. Reconstruction of the proteins encoded by the metagenomes showed near-identical, acid-shifted isoelectric point profiles in all layers. This was interpreted as molecular convergence of amino-acid usage dictated by hypersalinity [33]; however, similar isoelectric point profiles are common in marine Proteobacteria [34]. Trace metal concentrations vary on a millimeter-scale, ascribed to different geochemical processes and bacterial population distributions [35]. During daytime the cyanobacteria www.sciencedirect.com
(Coleofasciculus, Lyngbya) perform oxygenic photosynthesis, but at nighttime they produce molecular hydrogen and organic fermentation products from photosynthetic storage products. Most hydrogenase transcripts were attributed to cyanobacterial [NiFe]-hydrogenases. Chloroflexi and sulfate reducing bacteria are the main consumers of the cyanobacterial fermentation products [36]. A study of lipids in a higher-salinity, gypsum-containing mat at Guerrero Negro provided a wealth of biomarkers witnessing the vertical stratification of different prokaryotes [37]. A metagenomic study of the benthic community of a crystallizer pond on Mallorca, Spain, was carried out with special emphasis on the detection of yet-uncultured methanogenic Archaea adapted to high salt [38].
Antarctic lakes and deep-sea brines Metagenomic analysis of the microbial communities in Organic Lake (Vestfold Hills, Antarctica) was made in view of its high dimethylsulfide concentration. Bacteria were dominated by Marinobacter, Roseovarius and Psychroflexus. Abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, and rhodopsins indicate the use of phototrophy and lithoheterotrophy. Dimethylsulfoniopropionate lyase genes were abundant [39]. A metagenomic study of the cold and hypersaline Deep Lake, Antarctica, showed an ecosystem dominated by four halophilic Archaea whose complete genomes could be reconstructed: Halorubrum lacusprofundi (10% of the community) and the types designated tADL (isolated and described as Halohasta litchfieldiae) (44%), DL31 (18%), and DL1 (0.3%). The organisms differ in their abilities to degrade organic substrates [40]. Below the permanent ice cover of the Antarctic Lake Vida a diverse Bacteria-dominated aphotic ecosystem was found at 13 8C and 20% salt. Abiotic interactions between the brine and the rocks may be responsible for the generation of hydrogen as an energy source for the biota [41]. The anoxic brine pools on the bottom of the Red Sea and the Eastern Mediterranean have been objects of intensive studies lately [42]. The Red Sea Atlantis II and Discovery brines are characterized by steep salinity as well as temperature gradients. Alphaproteobacteria dominated the water above the brines, Planctomycetaceae and Deferribacteres abounded at the interfaces, and Gammaproteobacteria and several Euryarchaeota groups increased in the brine [43]. Metagenomic study of the Atlantis II brine (>2000 m depth, pH 5.3, 68 8C, salinity 26%, high heavy metal concentrations) led to the identification of a novel mercuric reductase, thermostable, functional in high salt, and efficiently detoxifying Hg2+ in vivo [44]. The main components of the biota of the recently discovered Lake Medee in the Eastern Mediterranean belong to the euryarchaeal MSBL1 and the bacterial KB1 candidate divisions. These organisms at least partially rely on reductive cleavage of the osmoprotectant glycine Current Opinion in Biotechnology 2015, 33:119–124
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betaine [45], but many other phylotypes are present [46]. Anaerobic ammonium oxidation (anammox) is functional in the chemoclines of the Bannock and L’Atalante brines in the eastern Mediterranean [47]. Mediterranean deepsea brines are also inhabited by a surprising diversity of ciliates, stramenopiles, and other eukaryotes [48].
Metabolic diversity at the highest salt salinities Aerobic heterotrophic halophiles are very versatile with respect to the organic substrates catabolized. Even the Archaea of the family Halobacteriaceae have a surprisingly large range of metabolizable substrates [49]. A thus far poorly explored substrate is chitin, often abundant in hypersaline environments inhabited by brine shrimp and brine flies. Chitin degrading aerobic (Saccharospirillum and Arhodomonas spp.) and anaerobic bacteria (Orenia chitinitropha) were enriched from hypersaline neutral-pH lakes of the Kulunda Steppe (Altai, Russia) in 2 M NaCl media [50]. In alkaline hypersaline lakes chitin degradation was demonstrated up to 3.5–4 M Na+, and a number of alkaliphilic chitin-degrading aerobes were isolated from soda lake sediments and surrounding soils [51]. To live at high salt is energetically expensive, and the salt concentration limit supporting different dissimilatory processes may be determined by bioenergetic constraints. Further studies of the dynamics of different processes in hypersaline environments are needed to test and refine current insights [52,53]. Sulfate reduction by ‘complete oxidizers’ using acetate as electron donor is not known above 12% salt, while ‘incomplete oxidizers’ can oxidize organic substrates to acetate + CO2 at much higher salinities [52,53]. However, a recent study of coastal salt pans in South Africa, based on amplification of dissimilatory sulfite reductase genes, suggested activity of Desulfobacteraceae (complete oxidizers) and Desulfohalobiaceae (incomplete oxidizers) at salinities as high as 300–400% [54]. In anoxic sediments of hypersaline lakes in the Kulunda Steppe (Altai, Russia), thiosulfate and elemental sulfur were better electron acceptors than sulfate. No growth of sulfate reducing bacteria was observed at 4 M NaCl. Enrichments with lactate, propionate, acetate, or butyrate using sulfate or thiosulfate as electron acceptors yielded isolates related to Desulfosalsimonas propionicica, Desulfohalobium utahense, and Desulfocella halophila. Sulfur-reducing enrichments at 4 M NaCl with acetate yielded Archaea closest related to the genus Halobacterium [55]. Another interesting novel type of metabolism detected in haloarchaea is oxidation of arsenite to arsenate, observed in samples from Salar de Punta Negra, Chile [56].
Bioremediation of polluted hypersaline environments A great diversity of hydrocarbonoclastic Bacteria and Archaea were found in oil-contaminated hypersaline (3–4 M NaCl ambient salinity) coastal areas in Kuwait, Current Opinion in Biotechnology 2015, 33:119–124
using culture-dependent methods [57,58]. Amino acids, vitamins and light enhanced hydrocarbon removal in laboratory microcosms [58,59]. Two Marinobacter spp. isolated from Kuwait grew optimally at 1–1.5 M NaCl, but maintained hydrocarbonoclastic activity up to 5 M NaCl. Both use aliphatic hydrocarbons (C9–C40), benzene, biphenyl, phenanthrene, anthracene and naphthalene as sole carbon and energy sources, and both fix nitrogen. Their use in bioremediation was successfully tested in hypersaline microcosms [60].
Concluding remarks Culture-independent studies, using the rapidly developing methods for ever faster and higher-throughput sequencing, and the ever-improving tools to evaluate the results and to make sense of the wealth of data obtained, have clearly dominated the field of hypersaline environmental microbiology in the past years. This situation is similar to what has happened in other areas of microbial ecology. It is encouraging to note that culture-dependent studies were not completely neglected [50,51,60]. Studying the relevant organisms in culture is indispensable to obtain an insight into the functioning of the ecosystems and the interactions between its components. The isolation in the first years of this century of Haloquadratum and Salinibacter has greatly increased our understanding of saltern crystallizer ecosystems. The case of Spiribacter salinus gives an excellent recent example of a novel type of halophile isolated after its importance in the ecosystem was ascertained by culture-independent techniques [27,28]. The earlier isolation of Salinibacter ruber represents a similar case [61]. We must hope that representatives of the nanohaloarchaea, a group discovered by sequencing of environmental DNA [14,26], will soon become available in culture.
Acknowledgement This study was supported by Grants no. 1103/10 and 343/13 from the Israel Science Foundation.
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Current Opinion in Biotechnology 2015, 33:119–124
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