Cyanobacteria in Hypersaline Environments: Biodiversity and Physiological Properties

Home , Gloeocapsa, Halotolerance, Hypersaline lake, Oscillatoria limnetica, Stenohaline

Biodivers Conserv (2015) 24:781–798 DOI 10.1007/s10531-015-0882-z

REVIEW PAPER

Cyanobacteria in hypersaline environments: biodiversity and physiological properties

Aharon Oren

Received: 7 July 2014 / Revised: 3 February 2015 / Accepted: 12 February 2015 / Published online: 3 March 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Within the cyanobacterial world there are many species adapted to life in hypersaline environments. Some can even grow at salt concentrations approaching NaCl saturation. Halophilic cyanobacteria often form dense mats in salt lakes, and on the bottom of solar saltern ponds, hypersaline lagoons, and saline sulfur springs, and they may be found in evaporite crusts of gypsum and halite. A wide range of species were reported to live at high salinities. These include unicellular types (Aphanothece halophytica and similar morphotypes described as Euhalothece and Halothece), as well as non-heterocystous ﬁlamentous species (Coleofasciculus chthonoplastes, species of Phormidium, Halospirulina tapeticola, Halomicronema excentricum, and others). Cyanobacterial diversity in high-salt environments has been explored using both classic, morphology-based taxonomy and molecular, small subunit rRNA sequence-based techniques. This paper reviews the diversity of the cyanobacterial communities in hypersaline environments worldwide, as well as the physiological adaptations that enable these cyanobacteria to grow at high salt concentrations. To withstand the high osmotic pressure of their surrounding medium, halophilic cyanobacteria accumulate organic solutes: glycine betaine is the pre- ferred solute in the most salt-tolerant types; Coleofasciculus produces the heteroside glucosylglycerol, and the less salt-tolerant cyanobacteria generally accumulate the disaccharides sucrose and trehalose under salt stress. Some cyanobacteria growing in benthic mats in hypersaline environments are adapted to life under anoxic conditions and they can use sulﬁde as an alternative electron donor in an anoxygenic type of photosynthesis through a process which involves photosystem I only.

Keywords Cyanobacteria Hypersaline salterns Osmotic adaptation Anoxygenic photosynthesis

Communicated by Anurag Chaurasia.

A. Oren (&) Department of Plant and Environmental Sciences, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, 91904 Jerusalem, Israel e-mail: [email protected] 123 782 Biodivers Conserv (2015) 24:781–798

Introduction

Cyanobacteria are the main primary producers in inland hypersaline lakes, coastal hypersaline lagoons, saltern evaporation ponds for the production of salt from seawater, saline springs, and other environments with salt concentrations exceeding that of seawater (3.5 %). In contrast to most groups of eukaryotic micro- and macroalgae, the cyanobacteria contain many members adapted to life at elevated salt concentrations. Green algae, diatoms, dinoflagellates and other eukaryotic phototrophic microorganisms generally perform very poorly at salt concentrations above 10 %. But in shallow hypersaline lakes, in saltern evaporation ponds, and in other light- exposed environments with high salt concentrations cyanobacteria often form dense benthic mats with high photosynthetic activities (Caumette et al. 1994;Cohen1984; Corneéetal. 1992; Des Marais 1995, Ionescu et al. 2007;Krumbeinetal.1977;Orenetal.1995;Rˇ eha´kova´ et al. 2009; Schneider et al. 2013). Both unicellular and filamentous types participate in the formation of these mats. Although generally less conspicuous than the benthic mat-forming relatives, planktonic halophilic cyanobacteria also exist. At the highest salt concentrations—from about 25 % up to NaCl saturation— cyanobacteria are rarely found, and primary production is mainly due to activity of eukaryotic green algae of the genus Dunaliella. However, there are exceptions. For ex- ample, halite evaporites in the Atacama Desert, Chile, contain viable and active unicellular cyanobacteria morphologically resembling Chroococcidiopsis (Wierzchos et al. 2006) but phylogenetically closer to Halothece (de los Rios et al. 2010). There are more such reports of the occurrence of cyanobacteria in evaporite crusts (Rothschild et al. 1994). The first treatise on cyanobacteria living in hypersaline environments is probably the 1933 paper by Hof and Fre´my titled ‘‘On Myxophyceae living in strong brines’’. In this paper the authors described Aphanothece halophytica as a new species of unicellular cyanobacteria. Aphanothece and similar forms described as Halothece and Euhalothece spp. are among the most prominent inhabitants of hypersaline environments, planktonic as well as benthic (Garcia-Pichel et al. 1998). Another key paper on cyanobacterial life at high salt concentrations is that by Golubic (1980). Realizing that cyanobacteria live over a wide range of salt concentrations and that many species can adapt to changing salinity, Golubic divided the organisms into euryhaline species that live in a wide salinity range and stenohaline types whose distribution is restricted to a narrow range of salt concentrations. He further classified the species as oligohaline, mesohaline, and polyhaline, for types growing optimally at low, intermediate and high salt concentrations, without proposing the boundaries separating these categories. Indeed, it is not feasible to divide the cyanobacteria into well-defined groups based on their salt tolerance and salt requirement. This paper reviews the biodiversity and the physiology of halophilic/halotolerant cyanobacteria, a group operationally defined here as requiring (halophilic) or tolerating (halotolerant) salt concentrations above *5 %, i.e. one and a half times that of seawater. The biology of cyanobacteria living in high-salt environments has been reviewed several times in the past, most recently in a comprehensive review by Oren (2012). Therefore special emphasis in this short review will be on the most recent advances in the field.

Diversity of cyanobacteria in hypersaline environments as assessed by microscopy and culture-dependent approaches

Many surveys have been published of the cyanobacterial diversity in hypersaline environments. In older studies, as well as in many newer ones, identification of the taxa is 123 Biodivers Conserv (2015) 24:781–798 783 based on morphological characters. In recent years, molecular characterization based on 16S rRNA gene sequencing became increasingly used for the taxonomic characterization of cyanobacteria, in cultures as well as in natural samples. A recurrent problem when comparing data from different studies is the confusing state of the cyanobacterial nomenclature and taxonomy. Different classification systems have been devised over the years, and organisms are often known under more than one name. The situation is further complicated by the fact that the nomenclature is covered by two Codes of Nomenclature: the International Code of Nomenclature for algae, fungi, and plants (the ‘Botanical Code’) and the International Code of Nomenclature of Prokaryotes (the ‘Bacteriological Code’). Only very few cyanobacteria genera and species were named under the provisions of the International Code of Nomenclature of Prokaryotes, including one halophilic representative: Halospirulina tapeticola (Nu¨bel et al. 2000b). In most cases the names listed in this paper are those used in the original publications. An exception is made for Microcoleus chthonoplastes, which was renamed C. chthonoplastes based on morphological and molecular studies of Microcoleus species from salty and from freshwater environments (Siegesmund et al. 2008). Table 1 summarizes the distribution of different types of cyanobacteria (morphological identification to the genus level) in a number of hypersaline environments and other high-salt habitats of special interest in which cyanobacteria are an important part of the biota: Great Salt Lake (Utah), saltern evaporation ponds, the lagoons of Guerrero Negro (Baja California) Solar Lake (Sinai, Egypt), Hot Lake (Washington), hypersaline lagoonal mats on San Salvador Island (Bahamas), the stromatolites of Shark Bay (Western Aus- tralia), and the Great Salt Plains (Oklahoma). When dividing the environments according to the salinity range (5–15, 15–22 and [22 % total dissolved salts), it is obvious that the higher the salt concentration, the smaller the diversity of cyanobacteria encountered. At the highest salinities filamentous cyanobacteria such as Coleofasciculus, Phormidium and Halospirulina that dominate in the intermediate salt concentration range are rarely seen. The only form adapted to life at salt concentrations approaching NaCl saturation is the unicellular type represented by the Aphanothece–Halothece–Euhalothece cluster. The environments studies and listed in Table 1 include: – Great Salt Lake, Utah, which is divided by a causeway into the less saline south arm (salinity *6–10 %) and the northern part which is currently approaching NaCl saturation. A. halophytica is a characteristic inhabitant of the higher-salinity sites (Brock 1976; Roney et al. 2009). The most important cyanobacterium living in the less- saline southern basin is the nitrogen-fixing heterocystous Nodularia spumigena (Roney et al. 2009). Heterocystous cyanobacteria are seldom encountered anywhere at salt concentrations exceeding 10 %. – Solar Lake (Sinai, Egypt), on the shore of the Gulf of Aqaba, Red Sea, is a small hypersaline heliothermal lake. In summer the lake is mixed down to the bottom (*4.5–5 m) and has a high salinity of *18 %. In winter a less saline (*7 % salt) layer floating on the denser deep waters causes heliothermal heating of the bottom layers, which may reach temperatures up to *60 °C (Cohen et al. 1975a). In-depth studies in the 1970s and 1980s showed a diversity of cyanobacteria, both planktonic types (Dactylococcopsis salina (Myxobactron salinum), Aphanothece) and filamentous species (Coleofasciculus, Phormidium), in benthic mats accompanied by unicellular types (Aphanothece, Aphanocapsa, Synechococcus) (Campbell and Golubic 1985; Cohen 1984; Jørgensen et al. 1983; Krumbein et al. 1977; van Rijn and Cohen 1983; Walsby et al. 1983). A filamentous isolate from Solar Lake named Oscillatoria

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Table 1 The major genera of cyanobacteria identiﬁed by microscopy and/or by culture-dependent techniques in selected hypersaline environments

Solar Salterns Great Solar Lake Hot Lake, Mono Hypersaline Lake Salada Shark Bay, Great Salt worldwide, including Salt and coastal Washington Lake, lagoonal lakes, San de Chiprana, Western Plains, Guerrero Negro, Baja Lake, sabkhas, California Salvador Island, Spain Australia Oklahoma California, Mexico Utah Sinai, Egypt Bahamas Salt concentration range 5-15% Unicellular types:

Aphanothece / Halothece /Euhalothece Synechococcus Myxobactron / Dactylococcopsis Gloeothece / Gloeocapsa Chroococcidiopsis Pleurocapsa

Filamentous, non- heterocystous types:

Oscillatoria Coleofasciculus Phormidium /Geitlerinema Lyngbya Leptolyngbya Johannesbaptistia Halospirulina / Spirulina Halomicronema Plectonema

Filamentous, heterocystous types:

Nodularia

limnetica, renamed as Phormidium hypolimneticum and again renamed as Leptolyngbya hypolimnetica (Anagnostidis 2001; Campbell and Golubic 1985) is of special interest, as in this organism facultative anoxygenic photosynthesis with sulfide as an electron donor was first identified (Cohen et al. 1975a). Anoxygenic photosynthesis by cyanobacteria in hypersaline habitats is discussed in further depth below. – Coastal salterns for the production of salt by evaporation of seawater are rich habitats for cyanobacteria adapted to life at high salt concentrations. Salterns consist of series of ponds, each kept at appoximately constant salinity, enabling the sequential precipita- tion of calcium carbonate, gypsum and halite. Benthic cyanobacterial mats develop up to a brine salinity of *20–22 % and sometimes even higher. Development of cyanobacterial mats is considered advantageous to the salt production process. Radiation absorption by the photosynthetic pigments raises the temperature of the brine and stimulates evaporation, and extracellular polysaccharides produced by the cyanobacteria seal off the bottom of the ponds and prevent brine leakage (Davis 1993; Javor 2002). Especially unicellular Aphanothece-type cells can excrete large amounts of polysaccharide slime (De Philippis et al. 1993, 1998). However, excessive development of slime-forming cyanobacteria in the saltern ponds is detrimental as the polysaccharides interfere with the proper crystallization of halite in the crystallizer ponds (Davis 1993; Davis and Giordano 1996). 123 Biodivers Conserv (2015) 24:781–798 785

Table 1 continued

Aphanothece / Halothece / Euhalothece Synechococcus

Filamentous, non- heterocystous types:

Oscillatoria Coleofasciculus Phormidium /Geitlerinema Leptolyngbya Halospirulina / Spirulina Plectonema

Salt concentration >22% Unicellular types:

Aphanothece / Halothece / Euhalothece

The table lists the most important genera of cyanobacteria reported from different hypersaline environments. Light-grey fields: only occasionally encountered. More exhaustive lists and information to the species level (in selected studies only) can be found in Oren (2012) and in the original publications. It must be taken into account that synonyms abound, that there is often considerable confusion about the correct names of certain types of cyanobacteria, and that different studies may have used different nomenclature systems. The table is based on data presented by Brock (1976), Roney et al. (2009) (Great Salt Lake, Utah); Bebout et al. (2002), Caumette et al. (1994), Corneé et al. (1992), Davis and Giordano (1996), Des Marais et al. (1995); Fourçans et al. (2004, 2006), Golubic (1980), Rˇ eha´kova´ et al., 2009; Sørensen et al. 2009; Wieland and Ku¨hl (2006) (saltern evaporation ponds, including Guerrero Negro, Baja California); Campbell and Golubic (1985), Cohen (1984), Cohen et al. (1975a), Jørgensen et al. (1983), Krumbein et al. (1977), van Rijn and Cohen (1983), Walsby et al. (1983) (Solar Lake); Anderson (1958), Lindemann et al. (2013) (Hot Lake, Wash- ington); Budinoff and Hollibaugh (2007) (Mono Lake, California); Paerl et al. (2003) (San Salvador Island, Bahamas); de Wit et al. (2005) (La Salada de Chiprana, Spain); Allen et al. (2009) (Shark Bay, W. Australia); and Kirkwood et al. (2008) (Great Salt Plains, Oklahoma)

The cyanobacterial mats of the world’s largest salt production facility in the lagoons of Guerrero Negro (Baja California, Mexico) have been the object of in-depth studies (e.g. Des Marais 1995; Lee et al. 2014;Nu¨bel et al. 1999, 2000a; Rothschild et al. 1994). Coleofasciculus mats from Guerrero Negro were used by NASA in a long-term greenhouse experiment. Increasing salinity from 9 to 12 % led to an increase in carotenoid content of Coleofasciculus as response to oxidative stress. Increased salinity also led to an increase in abundance of Aphanothece-type cells (Bebout et al. 2002). Colorful layers of unicellular and ﬁlamentous cyanobacteria can be found in evaporite crusts of gypsum and halite in the Guerrero Negro area. These cyanobacteria can remain viable and active for many months after they were trapped within the mineral deposits (Rothschild et al. 1994). At higher salinities (*15–20 %), benthic gypsum crusts with stratiﬁed endoevaporitic cyanobacterial communities are often found in the salterns. Typically, Aphanothece-like cells with a high content of carotenoids cause an orange coloration of the upper layer which

123 786 Biodivers Conserv (2015) 24:781–798 can be several centimeters thick, and overlies a dark-green layer of Phormidium-like filaments. The biological properties of such gypsum crusts were well documented in the salterns of Salins-de-Giraud on the Mediterranean coast of France (Caumette et al. 1994; Corneé et al. 1992; Fourçans et al. 2004) and of Eilat on the Red Sea coast of Israel (Ionescu et al. 2007; Oren et al. 1995, 2009). – Cyanobacteria are present in brine pools ([10 % salt) and in dry salt crusts in the Great Salt Plains, Oklahoma. These pools of dissolved Permian salt can reach salt saturation in dry periods. In this interesting environment with its often rapidly changing salinities, Phormidium/Geiterinema species were most often encountered. A Geitlerinema isolate from the site could adapt to a very wide range of NaCl concentrations, from 1 to 15 %. Another isolate with a wide salt range for growth (5–15 %) was identified as a Cyanodictyon sp. Other types of cyanobacteria were isolated, including Chroococcales (Aphanothece/Aphanocapsa), Chlorogloeopsis sp. and also the heterocystous Nodu- laria sp. Some isolates grew up to 15 % salt, but most did not tolerate concentrations about 5 % (Kirkwood et al. 2008). – San Salvador Island, Bahamas has sites with hypersaline lagoonal mats with salt concentrations of up to *11 %, inhabited by a variety of unicellular and filamentous cyanobacteria, including the heterocystous Scytonema sp. (Paerl et al. 2000). Photosynthetic activity of the mats increases after dilution of the water by hurricane rain floods, which cause growth stimulation especially of Coleofasciculus and Lyngbya (Paerl et al. 2003). – The inland Lake Salada de Chiprana in northeastern Spain (7.8 % total dissolved solids; maximum depth 5.5 m) has benthic Coleofasciculus-dominated mats (de Wit et al. 2005). – Hot Lake, Washington, a stratified lake with heliothermal heating, with magnesium sulfate (epsomite) as the main salt, is an interesting, but little studied hypersaline lake with unusual water chemistry. The MgSO4 concentration varies from *10 % in the surface layers to *40 % near the bottom (maximum depth *3.5 m) and the pH ranges from 8.1 to 8.6. The shallow sediments are covered by microbial mats. A study performed more than half a century ago identified Anacystis thermalis, Gomphosphaeria aponina, Oscillatoria chlorina, and Plectonema nostocorum as the cyanobacteria present (Anderson 1958). A recent study of the mats reported a Leptolyngbya sp. to be the main cyanobacterium present. Coleofasciculus, a typically dominant species of microbial mats in NaCl-dominated brines, is absent (Lindemann et al. 2013). – Another interesting lake with an unusual water chemistry is alkaline Mono Lake, California (pH * 9.7–10; *9 % total dissolved solids). A picocyanobacterium, phylogenetically related to marine Synechococcus and Prochlorococcus, forms blooms of up to *5 9 107 cells per liter in late summer. The organism was brought into culture. It grows up to 10 % salt (optimum at 0–6 %), its cells are rich in phycoerythrin, but do not contain phycourobilin, and do not show complementary chromatic adaptation (Budinoff and Hollibaugh 2007). – The smooth and pustular microbial mat communities in the hypersaline lagoon of Hamelin Pool, Shark Bay, Western Australia (average salt concentration of the waters *6 %) yielded isolates with high 16S rRNA similarity to Halomicronema, Euhalothece, Halothece, Chroococcus, Microcoleus, and Lyngbya, and further isolates with \96 % similarity to Chondrocystis, Spirulina, Myxosarcina, Stanieria, Cyanothece, Halothece, and Chroococcidiopsis (Allen et al. 2009).

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Diversity of cyanobacteria in hypersaline environments as assessed by culture-independent, 16S rRNA-based approaches

The 16S rRNA genes of many isolates of halophilic cyanobacteria have been sequenced, and the 16S rRNA-based phylogeny is increasingly used in taxonomic studies of the group. Comparative studied were reported for Coleofasciculus isolates (Garcia-Pichel et al. 1996), for the Aphanothece–Halothece–Euhalothece group (Garcia-Pichel et al. 1998; Margheri et al. 1999, 2008) and for halotolerant Chroococcidiopsis isolates (Cumbers and Rothschild 2014). Other genes may also serve as suitable markers: the genes rbcL (encoding the large subunit of RuBisCO), desC1 (D9 acyl lipid desaturase) and gltX (glutamyl-tRNA syn- thetase) were used in addition to the 16S rRNA gene to show the polyphyletic nature of the Chroococcidiopsis cluster (Cumbers and Rothschild 2014). These and other studies have established a good data base to enable the assessment of the cyanobacterial diversity in hypersaline environments using culture-independent molecular methods based on the sequencing of relevant genes from DNA isolated from the environment. Such studies were reported for several hypersaline sites: – The Guerrero Negro lagoons, Baja California. Sequencing of cyanobacterial 16S rRNA genes from DNA collected along the salinity gradient (6–21 % salt) showed that the number of phylotypes recovered was more than twice as high as the number of morphological types observed in the microscope (Nu¨bel et al. 1999). At salt concentrations up to 11 %, Coleofasciculus phylotypes were most abundantly found, while at 14 % salt most sequences recovered belonged to Aphanothece and Halospirulina spp. L. hypolimnetica (P. hypolimneticum) was also found (Nu¨beletal.2000a). 16S rRNA-based molecular techniques were used to assess the effect of salinity changes on the species composition of the mats. When mats dominated by Coleofasciculus and phylotypes assigned to different groups of Oscillatoria spp. were equilibrated with 3.5 % salt solution, no great changes in the species composition were observed (Green et al. 2008). – Cyanobacterial phylotypes found in evaporation ponds (7-15 % salt) of the salterns of Salins- de-Giraud in the south of France, based on denaturing gradient gel electrophoresis of cyanobacterial 16S rRNA genes amplified from the site, confirmed presence of Coleofas- ciculus, Oscillatoria, Leptolyngbya (alsorecognizedbymicroscopy)andalsoyielded Phormidium, Pleurocapsa,andCalothrix-related sequences. No sequences were found of Halomicronema, a type observed microscopically in the mats (Fourçans et al. 2004). – 16S rRNA clone libraries of cyanobacteria in the microbial mat communities in the lagoon of Hamelin Pool, Shark Bay, Western Australia (average salt concentration 6 %) indicated massive presence of phylotypes of Chroococcidiopsis, Gloeocapsa, Halothece, and Coleofasciculus, with smaller numbers of Leptolyngbya, Cyanothece, Euhalothece, Spirulina, Phormidium, and Stanieria in the pustular mat. A sample of the smooth mat yielded sequences of Coleofasciculus, Euhalothece, Halothece, Lep- tolyngbya, Stanieria, Chroococcidiopsis, and Cyanothece (Allen et al. 2009). –AHalothece/Euhalothece phylotype was dominantly found in the 16S rRNA gene library of the microbialite-forming microbial mat from a hypersaline (17 % salt) lake of the Kiritimati Atoll, Central Pacific (Schneider et al. 2013). – A salt pond on San Salvador Island, Bahamas (9–11 % salt) yielded 16S rRNA gene sequences affiliated with types of Chroococcales and Oscillatoriales. Pleurocapsales and Nostocales sequences were found as minor components. The nitrogenase gene nifH was also used as a phylogenetic marker; nifH sequences of Oscillatoriales and of unicellular cyanobacteria were detected (Paerl et al. 2003).

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– Among the 16S rRNA genes of cyanobacteria retrieved from the brine pools ([10 % salt) of the Great Salt Plains, Oklahoma, were a number of heterocystous lineages, otherwise rarely found in hypersaline environments (Kirkwood et al. 2008). – Analysis of the 16S rRNA genes ampliﬁed from the halite evaporites collected in the Atacama Desert, Chile, which contain cells microscopically resembling Chroococcidiopsis, showed an afﬁliation with the genus Halothece (de los Rios et al. 2010).

Selected species of halophilic cyanobacteria and their properties

A few types of cyanobacteria occur in hypersaline environments worldwide: the unicellular Aphanothece/Halothece/Euhalothece, the ﬁlamentous C. chthonoplastes, and the tightly coiled H. tapeticola. Here follow some notes (derived in part from Oren 2012) on the properties of these and a few other taxa speciﬁcally found in high-salt habitats.

Aphanothece halophytica

A. halophytica and morphologically similar halophilic unicellular cyanobacteria designated and described by various authors as Halothece, Euhalothece, Coccochloris elabens or Cyanothece, is a cosmopolitic inhabitant of light-exposed hypersaline environments. It can be found from salt concentrations as low as 3 % nearly up to NaCl saturation. Its optimum salt concentration for growth is around 6–15 % (Brock 1976). As already noted by Hof and Fre´my (1933), cell size and shape are highly variable, depending on growth conditions. Colonies of the unicellular Aphanothece (Halothece, Euhalothece) prominently colonize the upper layer of microbial mats and gypsum crusts in saltern evaporation ponds (Caumette et al. 1994; Margheri et al. 1987; Oren et al. 1995) and some natural salt lakes, including Great Salt Lake, Utah (Brock 1976). Planktonic growth was reported as well, e.g. in Solar Lake, Sinai. To provide osmotic balance of the cytoplasm with the surrounding hypersaline medium, the cells accumulate glycine betaine (see below). A conspicuous property of A. halophytica and related halophilic unicellular cyanobacteria is the formation of extracelluar polysaccharide slime (De Philippis et al. 1993, 1998). As stated above, excessive slime production by Aphanothece in saltern evaporation ponds can negatively affect both the quality and the quantity of the harvested salt (Davis 1993; Davis and Giordano 1996). There is probably no other cyanobacterium that is known by so many different names. Organisms morphologically very similar to A. halophytica have been named Aphanocapsa halophytica (Brock 1976), Halothece, Euhalothece (Garcia-Pichel et al. 1998), C. elabens, Synechococcus sp., and Cyanothece (De Philippis et al. 1993; Campbell and Golubic 1985). To what extent these names refer to identical or to different taxa is seldom clear. A. halophytica also contains different morphotypes, ecotypes and phylotypes. The name Euhalothece was created following phylogenetic, 16S rRNA gene-based analysis of a large collection of isolates (Garcia-Pichel et al. 1998).

Coleofasciculus chthonoplastes

The ﬁlamentous cyanobacterium that forms bundles surrounded by a common sheath and found in marine and hypersaline sediments, known in the older literature under the name

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Microcoleus chthonoplastes, was renamed as C. chthonoplastes following an in-depth taxonomic study based on 16S rRNA sequence comparisons (Siegesmund et al. 2008). C. chthonoplastes isolates from different parts of the world are very similar, both pheno- typically and phylogenetically (Garcia-Pichel et al. 1996; Karsten and Garcia-Pichel 1996). Coleofasciculus is a major mat-building organism, and it can grow in a very wide range of salt concentrations, even up to 20 % and higher. It is possible to distinguish different ecotypes adapted to different ranges of salt concentration (Karsten 1996). The osmotic solute accumulated within the cytoplasm in response to the high salinity of the medium is the heteroside glucosylglycerol (see below).

Halospirulina tapeticola

Tightly coiled ﬁlamentous cyanobacteria are commonly observed in benthic microbial mats in saltern evaporation ponds at salt concentrations up to *20 %. In the past such organisms were often named Spirulina sp. The helically coiled forms are a phylogenetically diverse group. One cluster of organisms was described under the name H. tapeticola. The organism can grow from NaCl concentrations as low as 3 % up to 20 %, making this a true euryhaline species (Nu¨bel et al. 2000b).

Halomicronema excentricum

Halomicronema excentricum (a name as yet without standing under either Code of Nomenclature) is a filamentous organism with cells of a diameter of slightly less than 1 lm and a length of 2–8 lm. Gas vesicles are generally present near the cross walls in the filament. Optimal growth is at 28–50 °C and 15 % salt (range: 3.2–12 %) (Abed et al. 2002). It was reported as a major component of the cyanobacterial community of the microbial mat in the saltern evaporation ponds (7–9 % salt) in France (Fourçans et al. 2006).

Myxobactron salinum

The unicellular spindle-shaped and gas-vacuolate cyanobacterium Myxobactron salinum was reported from Solar Lake, Sinai, and from brines from saltern evaporation ponds (van Rijn and Cohen 1983; Walsby et al. 1983). Growth was observed up to 45 °C at salt concentrations between 7.5 and 15 %. In the original publications the organisms was described as Dactylococcopsis salina, but a green algae genus named Dactylococcopsis was already described in 1888.

Organic osmotic solutes of halophilic cyanobacteria and their biosynthesis

Biological membranes are permeable to water, and therefore microorganisms living at elevated salt concentrations must provide osmotic balance of their cytoplasm with the outside medium. Basically there are two strategies used in the microbial world to achieve this goal. One is to balance ‘salt-out’ with ‘salt-in’, by the massive accumulation of ions, mainly K? and Cl- and to adapt the entire intracellular enzymatic machinery to the presence of high ionic concentrations. This strategy is used only by a few specialized groups of microorganisms: the halophilic Archaea of the family Halobacteriaceae and Salinibacter, an extremely halophilic member of the Bacteroidetes (Oren 2006). Cyanobacteria, like most other groups of bacteria, do not tolerate/allow high salt 123 790 Biodivers Conserv (2015) 24:781–798 concentrations to accumulate inside the cells. Indeed, ions (Na?,K?,Cl-) can transiently enter as a response to a sudden increase in medium salinity, but overall the ionic concentrations measured in cyanobacteria, including the most halophilic ones, are low. Thus, intracellular K? concentrations in A. halophytica and other halophilic species were found to be no greater than 170–310 mM, concentrations many times lower than the cation concentrations in the medium (Reed et al. 1984). Table 1 in the review paper by Hagemann (2011) summarizes the available data: measured concentrations of Na? and K? never exceeded 200–300 mM each, not even in cells grown in media containing 2–3 M salt. As yet little known to what extent the salt tolerance is determined by different ion export capacities. Like most other groups of the Bacteria, the cyanobacteria use organic osmotic solutes (‘compatible solutes’): small, generally uncharged or zwitterionic compounds with a high solubility, to osmotically adjust the cytoplasm with the external medium. Since the early 1980s when techniques such as HPLC and NMR spectroscopy were applied to analyze the cellular contents (Mackay et al. 1984), a clear correlation is known to exist between the salinity range in which a certain species grows and the type of solute or solutes synthesized. Species with no more than a slight salt tolerance generally rely on the disaccharides sucrose and/or trehalose, cyanobacteria living at intermediately high salt concentrations often produce glucosylglycerol (2-O-a-D-glucopyranosyl-(1?2)-glycerol), and the most salt-tolerant or salt-requiring types use glycine betaine (N,N,N-trimethylglycine) as an osmotic solute. A few novel compounds were added to this list in recent years, and these are further described at the end of this section: glucosylglycerate (Kla¨hn et al. 2010), trimethylamine-N-oxide (Goh et al. 2010), and mycosporine-like amino acids (MAAs)— pigments that absorb harmful UV radiation but that also may contribute to the osmotic equilibrium (Oren 1997). Osmotic adaptation in cyanobacteria has been reviewed several times in the past (Hagemann 2011; Joset et al. 1996; Kla¨hn and Hagemann 2011; Oren 2012). Recently a comparative assessment of the production of organic compatible solutes and of the bio- chemical pathways involved was made based on the screening of the genomes of more than 60 strains of cyanobacteria with different levels of halotolerance and salt requirement (Hagemann 2013). The biochemistry of the production and degradation of the organic osmotic solutes is now known quite well; however, information on the mechanisms for sensing salt stress and transduction of the signal is still lacking. Our knowledge of the biochemistry of the pathways, the properties of the enzymes involved and their regulation is mainly based on in-depth studies of a few model organisms. One is Synechocystis strain PCC 6803, a glucosylglycerol-accumulating halotolerant and euryhaline organism strain isolated from a freshwater environment, but growing well at salt concentrations up to 2–3 times as high as seawater. This strain can also be manipulated genetically. A. halophytica, Synechococcus WH8102 and Synechococcus PCC 7418 have been the organisms of choice to elucidate glycine betaine metabolism in halophilic cyanobacteria.

Sucrose and trehalose

The disaccharides sucrose (glucose–fructose) and/or trehalose (glucose–glucose) are produced by many freshwater cyanobacteria as a reaction to salt stress. The compounds are little effective as osmotic solutes and they do not provide osmotic protection in the higher salt concentration range. When grown at low salt concentrations, C. chthonoplastes produces trehalose, while at higher salinities glucosylglycerol takes over as the main osmotic stabilizer (Karsten 1996). 123 Biodivers Conserv (2015) 24:781–798 791

Sucrose is synthesized by sequential action of sucrose-phosphate synthase (Sps) and sucrose-phosphate phosphatase (Spp): UDP-glucose þ fructose-6-phosphate ! sucrose-phosphate þ UDP

Sucrose-phosphate ! sucrose þ Pi If necessary, excess sucrose can be degraded by sucrose synthase (Sus): Sucrose þ UDP ! UDP-glucose þ fructose Alternatively, sucrose can be hydrolyzed by invertase (sucrase): Sucrose ! fructose þ glucose

Trehalose biosynthesis in cyanobacteria mostly follows the TreYZ pathway in which a polysaccharide (glycogen) precursor is ﬁrst charged at the terminal end to a-1,1 sugar bound by maltooligosyltrehalose synthase (TreY); then trehalose is cleaved off by maltooligosyltrehalose trehalohydrolase (TreZ): a-1; 4-Polyglucose ! a-1; 1-maltooligosyltrehalose a-1; 1-Maltooligosyltrehalose ! trehalose þ a-1; 4-polyglucoseðÞ n 2

Glucosylglycerol

Glucosylglycerol is widespread as an osmotic solute in filamentous (e.g. Coleofasciculus) and unicellular (e.g. Synechocystis spp.) halophilic cyanobacteria. It was first identified by means of 13C-NMR in a marine Synechococcus isolate from intertidal rocks in Australia (Borowitzka et al. 1980). Its biosynthesis proceeds via glucosylglycerol-phosphate synthase (GgpS) and glucosylglycerol-phosphate phosphatase (GgpP): ADP-glucose þ glycerol-3-phosphate ! glucosylglycerol-phosphate þ ADP

Glucosylglycerol-phosphate ! glucosylglycerol þ Pi Characterization of different salt-sensitive mutants of Synechocystis PCC 6803 enabled an in-depth analysis of the pathway and its regulation (Joset et al. 1996; Hagemann 2011).

Glycine betaine

Cyanobacteria adapted to life at the highest salt concentrations generally produce glycine betaine as their osmotic solute. Examples are A. halophytica and Synechocystis DUN52 isolated from calcareous stromatolites of intertidal ﬂats in Kuwait (Reed et al. 1984). Glycine betaine was found together with trehalose in the halophilic Gloeocapsa strain ACMM N107 (Mackay et al. 1984). The pathway known from higher plants in which glycine betaine is produced from choline is not operative in cyanobacteria. Instead, halophilic cyanobacteria synthesize glycine betaine by stepwise methylation of glycine by glycine/sarcosine-N-methyltransferase (GSMT) and dimethylglycine-N-methyltransferase (DMT), using S-adeno- sylmethionine (SAM) as the methyl donor which is converted to S-adenosylhomoserine (SAH):

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Glycine þ SAM ! N-methylglycineðÞþ sarcosine SAH Sarcosine þ SAM ! N; N-dimethylglycine þ SAH N; N-dimethylglycine þ SAM ! glycine betaine þ SAH

The two N-methyltransferases of A. halophytica have been characterized. Glycine betaine-producing cyanobacteria also possess an active transport system for the compound that enables its uptake from the environment when available. Following salt downshock, intracellular glycine betaine may be released to the outer medium, and degradation of excess glycine betaine is also possible by an inducible betaine-homocysteine methyltransferase with the formation of dimethylglycine and methionine. Further information on the pathways of glycine betaine formation and degradation can be found in a number of review papers (Hagemann 2011; Oren 2012).

Glucosylglycerate, trimethylamine-N-oxide, glutamate betaine and mycosporine-like amino acids as osmotic solutes in cyanobacteria

Glucosylglycerate is a recent addition to the list of compounds that serve as osmotic solutes. Its occurrence was ﬁrst detected in 1979 as a minor compound in Synechococcus sp. PCC 7002, but it is now known to form up to 50 % of the osmotic solute content of the marine picoplankton cyanobacteria Prochlorococcus and Synechococcus PCC 7002. It is found together with glucosylglycerol and sucrose. The negatively charged compound may function as a counterion to cations, and may be preferable to glutamate in the nitrogen- limited marine environment (Kla¨hn et al. 2010). Biosynthesis of glucosylglycerate proceeds via glucosylglycerate-phosphate synthase (GpgS) and a phosphatase (GpgP): NDP-glucose þ glycerate-3-phosphate ! glucosylglycerate-phosphate þ NDP

Glucosylglycerate-phosphate ! glucosylglycerate þ Pi where NDP is a yet-to-be-identiﬁed nucleoside diphosphate carrier. Trimethylamine-N-oxide was identiﬁed as a novel compatible solute in Cyanothece and Gloeothece isolates from the hypersaline stromatolites of Shark Bay, Western Australia. It was found during NMR analysis together with several other novel intracellular solutes: various disaccharides (including trehalose), trisaccharides and pentasaccharides (Goh et al. 2010). Glutamate betaine (N,N,N-trimethyl-L-glutamate), functionally probably equivalent to glycine betaine, was found, in combination with sucrose and/or trehalose, in supralittoral and intertidal Calothrix/Rivularia isolates ACMM N181 and ACMM N201/PCC 7426 (Mackay et al. 1984). No further studies on the occurrence of this compound were reported since. Unicellular cyanobacteria within a gypsum crust on the bottom of a saltern evaporation pond (salt concentration *20 %) in Eilat, Israel (Oren 1997) contained large concentrations of two mycosporine-like amino acids (MAAs) with absorption maxima at 332 and at 365 nm, and their chemical structure has been elucidated (Kedar et al. 2002; Volkmann et al. 2006). The absorption spectrum of an extract of the upper layer of unicellular cyanobacteria in the gypsum crust of a similar pond of the Salins-de-Giraud salterns, France (Caumette et al. 1994) showed a sharp rise in absorbance from 390 to 350 nm (the lowest wavelength measured), indicating that similar pigments may be present there as well. Estimations of the intracellular MAA concentrations within the cyanobacterial cells

123 Biodivers Conserv (2015) 24:781–798 793 in the benthic crust in Eilat yielded values of *0.1 M or possibly higher. MAAs are known to protect the cells against UV radiation damage. However, at such high concentrations they must also contribute to the intracellular osmotic pressure, suggesting a possible role as osmotic solutes. When cell material from the crust was subjected to hypotonic stress, MAAs were rapidly released from the cells, conﬁrming an involvement in osmotic processes (Oren 1997).

Selected physiological properties of cyanobacteria in hypersaline environments: halotaxis, nitrogen ﬁxation, hydrogen production, and anoxygenic photosynthesis

Halotaxis

An interesting observation of what may be a salinity-driven migration, designated ‘halotaxis’, was made during a study of an intertidal hypersaline microbial mat in Abu Dhabi (United Arab Emirates). During low tide the salinity of the overlying water varies from 6 to 20 % and the mats contain a rich diversity of cyanobacteria, including C. chthonoplastes, Lyngbya aestuarii, Entophysalis sp., Chroococcus sp. and others (Abed et al. 2008). Color changes of the surface layer of the mat were observed within several hours following changes in salinity of the overlying water: when the salinity exceeded 15 %, the mat had an orange-reddish appearance, at lower salinities green Coleofasciculus filaments migrated to the upper layer. Light conditions, ionic composition of the water, oxygen concentrations, pH, and sulfide concentration could all be excluded as possible triggers for the migration, so that the only factor considered to be responsible for the phenomenon was salinity (Kohls et al. 2010). The mechanism of the salinity-triggered migration of the Coleofasciculus filaments is unknown.

Nitrogen ﬁxation

Heterocystous cyanobacteria are seldom found at high salt concentrations. A filamentous cyanobacterium identified as Nodularia spumigena occurs in the moderately hypersaline (7 % salt) south arm of Great Salt Lake, Utah, but not in the hypersaline north arm (Roney et al. 2009). Scytonema was found in Storr’s Lake, San Salvador Island, Bahamas at 4.5–9 % salt (Paerl et al. 2000). Some non-heterocystous halophilic cyanobacteria can fix nitrogen, including the unicellular A. halophytica. Light-dependent nitrogen fixation (as assessed by acetylene reduction) by a ‘Synechococcus’, possibly a relative of A. halophytica, was observed in an evaporite crusts from Guerrero Negro, Baja California, both under aerobic and under anaerobic conditions (Rothschild et al. 1994). Nitrogen fixation was sometimes reported from Coleofasciculus mats (de Wit et al. 2005), and nitrogenase gene clusters, including an atypical nifH gene not related to the typical cyanobacteria nifH, were found in a number of isolates (Bolhuis et al. 2010).

Generation of hydrogen and other fermentation products in the dark

A recent study of a permanently submerged Coleofasciculus mat and an intertidal Lyngbya mat at Guerrero Negro (covered by 0.5–1 m of brine with 8–10 % salt) showed that during nighttime the cyanobacteria in the mats fermented storage material such as glycogen accumulated during daytime photosynthesis, and produce hydrogen gas and other

123 794 Biodivers Conserv (2015) 24:781–798 fermentation products, which are then used as electron donors by the sulfate reducing bacteria present. Sequencing of transcripts of type 3b [NiFe]-hydrogenases showed that the majority of the sequences recovered were derived from cyanobacteria, both ﬁlamentous (Oscillatoriales) and unicellular (Pleurocapsales) types (Lee et al. 2014). C. chthonoplastes possesses different mechanisms of energy generation in the dark under anaerobic conditions, including fermentation of carbohydrate reserves to acetate as the main product (Moezelaar et al. 1996); under similar conditions ‘Oscillatoria limnetica’(L. hypolimnetica) (for nomenclatural comments see Anagnostidis (2001), Campbell and Golubic (1985) and Golubic (1980)) from Solar Lake produces mainly lactate (Oren and Shilo 1979). When no oxygen is available during the night, both types can also use elemental sulfur as an electron acceptor for respiration with the formation of sulﬁde (Moezelaar et al. 1996; Oren and Shilo 1979).

Anoxygenic photosynthesis by cyanobacteria in hypersaline environments

Some cyanobacteria can under anaerobic conditions can shift from oxygenic photosynthesis (in which water serves as an electron donor) to anoxygenic photosynthesis in which the electrons for CO2 fixation are supplied by sulfide, which in the process is oxidized to elemental sulfur or to thiosulfate (Padan 1979a, b). Most cyanobacteria in which anoxygenic photosynthesis is known to occur live at high salt concentrations. The process was first discovered in L. hypolimnetica (‘Oscillatoria limnetica’) from the hypersaline Solar Lake, Sinai. This organism develops near the bottom of the lake at a depth of 3–5 m in winter. In this season this heliothermal lake is stratified, with a warm (up to *60 °C) anaerobic hypolimnion (up to *18 % salt), below a less saline aerobic surface layer. Sulfide accumulates in the bottom layers due to the activity of sulfate reducing bacteria (Cohen et al. 1975a). When sulfide serves as the electron donor in anoxygenic photosynthesis, photosystem II does not participate in the process, and electrons from sulfide are transferred directly to photosystem I. Therefore DCMU [3-(3,4-dichlorophenyl-1,1-dimethylurea], a herbicide that blocks electron flow at the acceptor side of photosystem II, does not affect the anoxygenic process (Cohen et al. 1975b). Anoxygenic photosynthesis in L. hypolimnetica functions optimally at sulfide concentrations of 2–3 mM. It is an inducible process, requiring the synthesis of a sulfide-quinone reductase that feeds electrons from sulfide to photosystem I. The organism can grow anaerobically in the light in the presence of sulfide. Special adaptations enabling anaerobic growth are the absence of polyunsaturated fatty acids (present in the membrane lipids of most filamentous cyanobacteria and requiring molecular oxygen for their biosynthesis) and the functioning of an oxygen-independent biosynthetic pathway for the synthesis of monounsaturated fatty acids. The ability to perform anoxygenic photosynthesis was also detected in an Aphanothece isolate from Solar Lake. However, it does not tolerate as high sulfide concentrations as L. hypolimnetica (Garlick et al. 1977). Coleofasciculus strains also can use sulfide in an anoxygenic type of photosynthesis, but sulfide tolerance is low, and thiosulfate rather than elemental sulfur is formed as the oxidation product. Oxygenic and anoxygenic photosynthesis can proceed simultaneously in Coleofasciculus mats (Cohen 1984; Cohen et al. 1986; de Wit et al. 1988). Other hypersaline environments in which sulfide-dependent anoxygenic photosynthesis by cyanobacteria, not inhibited by DCMU, was demonstrated are the Phormidium layer in the gypsum crust in the salterns in Eilat, where the cells are exposed to sulfide and anaerobic conditions during many hours each day (Ionescu et al. 2007), and the Phormidium-like cyanobacteria found on the bottom of the hypersaline 123 Biodivers Conserv (2015) 24:781–798 795

(*16 % salt) sulfur springs and their outﬂow channels that were present in the past at Hamei Mazor on the shore of the Dead Sea (Oren 1989), but have now dried up.

Final comments

Only few types of cyanobacteria are adapted to grow at salt concentrations approaching saturation, but much of the primary production in salt lakes, hypersaline lagoons, and saltern ponds at salinities exceeding that of seawater is due to the activity of diverse communities of halophilic or halotolerant cyanobacteria belonging to different taxonomic groups. Unfortunately the present state of cyanobacterial taxonomy makes it difficult to compare results from different studies, especially when different identification methods (classical morphology-based or sequencing methods) were applied. Different mechanisms of salt adaptation have evolved in these groups, as shown e.g. by the diversity in organic solutes used for osmotic adaptation. The phenomenon of facultative anoxygenic photosynthesis in cyanobacteria was first discovered in a halophilic cyanobacterium from a hypersaline lake. The study of cyanobacterial life at high salt concentrations has thus contributed much important information, not only about the functioning of hypersaline ecosystems, but also about the different modes of adaptation of this versatile group of prokaryotes to life under extreme conditions.

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