Diatoms are one of the most important organisms contributing to aquatic primary productivity and their sterols are frequently used as markers for their presence and abundance. In this study, the sterol composition of >100 diatom cultures was analyzed and its distribution was compared to the diatom phylogeny to identify typical diatom biomarkers. Forty-four sterols were detected, 11 of them being commonly present as major sterols (contributing >10% to the total sterols). 24-Methylcholesta-5,24(28)-dien-3β-ol is the most common sterol in diatoms, being present in 67% of all cultures analyzed, followed by the △5 sterols, cholest-5-en-3β-ol (cholesterol), 24-methylcholest-5-en-3β-ol, and 24-ethylcholest-5-en-3β-ol. 24-Methylcholesta-5,22E-dien-3β-ol, previously suggested to be specific for diatoms, was only the fifth most common sterol; this sterol was absent in some of the major diatom groups, and high relative concentrations seem to be restricted to pennate diatoms. No sterols are restricted to specific phylogenetic groups of diatoms. Cluster analyses, however, do reveal distinct sterol distributions: Thalassiosirales typically contain high relative abundances of 24-methylcholesta-5,24(28)-dien-3βol, high relative abundances of cholesta-5,22E-dien-3β-ol are typical for Cymatosirales, high relative abundances of 24-ethylcholesta-5,22E-dien-3β-ol are characteristic for related Amphora, Amphiprora, and Entomoneis species, and a combination of high relative abundances of 24-methylcholest-5-en-3β-ol, 24-methylcholesta-5,24(28)-dien-3β-ol, and 24-ethylcholest-5-en-3β-ol is typical for Attheya species. High contributions of 24-methylcholesta-5,22E- dien-3β-ol (>50% of all sterols) seem to be restricted to pennate diatoms. None of the major sterols found in diatoms can be used as an unambiguous diatom biomarker because all of them have been reported as common sterols in other algal groups.@en

Nine pure cultures of neutrophilic haloaloarchaea capable of anaerobic growth by carbohydrate dependent sulfur respiration were isolated from hypersaline lakes in southwestern Siberia and southern Russia. According to phylogenomic analysis the isolates were closely related to each other and formed a new species within the genus Halapricum (family Haloarculaceae). They have three types of catabolism: fermentative, resulting in H2 formation; anaerobic respiration using sulfur compounds as e-acceptors and aerobic respiration. Apart from elemental sulfur, all isolates can also use three different sulfoxides as acceptors and the type strain also grows with thiosulfate, reducing it partially to sulfide and sulfite. All strains utilized sugars and glycerol as the e-donors and C source for anaerobic growth and some can also grow with alpha-glucans, such as starch and dextrins. The major respiratory menaquinones are MK-8:8 and MK-8:7, but 5–19% consists of ‘‘thermoplasmata” quinones (MMK-8:8 and MMK-8:7), whose occurrence in haloarchaea is unprecedented. On the basis of their unique physiological properties and results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum desulfuricans sp. nov. (type strain HSR12-2T = JCM 34032T = UNIQEM U1001T).@en

Methanogenic enrichments from hypersaline lakes at moderate thermophilic conditions have resulted in the cultivation of an unknown deep lineage of euryarchaeota related to the class Halobacteria. Eleven soda lake isolates and three salt lake enrichment cultures were methyl-reducing methanogens that utilize C1 methylated compounds as electron acceptors and H2 or formate as electron donors, but they were unable to grow on either substrates alone or to form methane from acetate. They are extreme halophiles, growing optimally at 4 M total Na+ and the first representatives of methanogens employing the ‘salt-in’ osmoprotective mechanism. The salt lake subgroup is neutrophilic, whereas the soda lake isolates are obligate alkaliphiles, with an optimum around pH 9.5. Both grow optimally at 50°C. The genetic diversity inside the two subgroups is very low, indicating that the soda and salt lake clusters consist of a single genetic species each. The phylogenetic distance between the two subgroups is in the range of distant genera, whereas the distance to other euryarchaea is below 83 % identity of the 16S rRNA gene. These isolates and enriched methanogens, together with closely related environmental clones from hypersaline habitats (the SA1 group), form a novel class-level clade in the phylum Euryarchaeota. On the basis of distinct phenotypic and genetic properties, the soda lake isolates are classified into a new genus and species, Methanonatronarchaeum thermophilum, with the type strain AMET1T (DSM 28684T=NBRC 110805T=UNIQEM U982T), and the salt lake methanogens into a candidate genus and species ‘Candidatus Methanohalarchaeum thermophilum’. These organisms are proposed to form novel family, order and class Methanonatronarchaeaceae fam. nov., Methanonatronarchaeales ord. nov. and Methanonatronarchaeia classis nov., within the phylum Euryarchaeota.@en

Two groups of alkaliphilic haloarchaea from hypersaline alkaline lakes in Central Asia, Egypt and North America were enriched and isolated in pure culture using chitin as growth substrate. These cultures, termed AArcht, were divided into two groups: group 1 which includes eleven isolates from highly alkaline soda lakes and group 2 which contains a single isolate obtained from the alkaline hypersaline Searles Lake. The colonies of chitin-utilizing natronoarchaea were red-pigmented and surrounded by large zones of chitin hydrolysis. The free cells of both groups were mostly flat nonmotile rods, while the cells that attached to chitin or formed colonies on chitin plates were mostly coccoid. The isolates are obligate aerobic saccharolytic archaea utilizing chitin and chitosane (less actively) as the only sugar polymers as well as a few hexoses as their carbon and energy source. Both groups are extremely halophilic, growing optimally at 3.5–4 M total Na+, but they differ in their pH profiles: the main group 1 isolates are obligately alkaliphilic, while the single group 2 strain (AArcht-SlT) is alkalitolerant. The core archaeal lipids in both groups are dominated by C20–C20 and C20–C25 dialkyl glycerol ethers (DGE) in approximately equal proportion. Phylogenetic analysis indicated that the isolates form an independent genus-level lineage within the family Natrialbaceae with 3 species-level subgroups. The available genomes of the closest cultured relatives of the AArcht strains, belonging to the genera Natrialba and Halopiger, do not encode any chitinase-related genes. On the basis of their unique phenotypic properties and distinct phylogeny, we suggest that the obligate alkaliphilic AArcht isolates (group 1) with an identical phenotype are classified into a new genus and species Natrarchaeobius chitinivorans gen. nov., sp. nov., with strain AArcht4T as the type strain (JCM 32476T = UNIQEM U966T), while the facultatively alkaliphilic strain AArcht-SlT (group 2) — as a new species Natrarchaeobius halalkaliphilus sp. nov. (JCM 32477T = UNIQEM U969T).@en

Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (MSAO) and sulfate-reducing (S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that H2 and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was 'Candidatus Syntrophonatronum acetioxidans,' and a near-complete draft genome of this SAOB was reconstructed. 'Ca. S. acetioxidans' has all genes necessary for operating the Wood-Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na+dependent ATP synthase and marker genes for 'salt-out'strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of 'Ca. S. acetioxidans' showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA gene amplicon datasets of the soda lake sediments showed a high similarity of reads to 'Ca. S. acetioxidans' with abundance as high as 1.3% of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers were not abundant (≤0.1%) or could not be detected. These combined results indicate that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline conditions performed by haloalkaliphilic syntrophic consortia.@en

The genus Thioalkalivibrio comprises sulfur-oxidizing bacteria thriving in soda lakes at high pH and salinity. Depending on the geographical location and the season, these lakes can strongly vary in temperature. To obtain a comprehensive understanding of the molecular and physiological adaptations to low temperature, we compared the responses of two Thioalkalivibrio strains to low (10 C) and high (30 C) temperatures. For this, the strains were grown under controlled conditions in chemostats and analyzed for their gene expression (RNA sequencing [RNA-Seq]), membrane lipid composition, and glycine betaine content. The strain Thioalkalivibrio versutus AL2T originated from a soda lake in southeast Siberia that is exposed to strong seasonal temperature differences, including freezing winters, whereas Thioalkalivibrio nitratis ALJ2 was isolated from an East African Rift Valley soda lake with a constant warm temperature the year round. The strain AL2T grew faster than ALJ2 at 10 C, likely due to its 3-fold-higher concentration of the osmolyte glycine betaine. Moreover, significant changes in the membrane lipid composition were observed for both strains, leading to an increase in their unsaturated fatty acid content via the Fab pathway to avoid membrane stiffness. Genes for the transcriptional and translational machinery, as well as for counteracting cold-induced hampering of nucleotides and proteins, were upregulated. Oxidative stress was reduced by induction of vitamin B12 biosynthesis genes, and growth at 10 C provoked downregulation of genes involved in the second half of the sulfur oxidation pathway. Genes for intracellular signal transduction were differentially expressed, and interestingly, AL2T upregulated flagellin expression, whereas ALJ2 downregulated it.@en

Anaerobic enrichment from sediments of hypersaline alkaline lakes in Wadi el Natrun (Egypt) with chitin resulted in the isolation of a fermentative haloalkaliphilic bacterium, strain ACht6-1, growing exclusively with insoluble chitin as the substrate in a sodium carbonate-based medium at pH 8.5-10.5 and total Na+ concentrations from 0.4 to 1.75 M. The isolate had a Gram-negative cell wall and formed lipid cysts in old cultures. The chitinolytic activity was associated with cells. Analysis of the 4.4 Mb draft genome identified pathways for chitin utilization, particularly, secreted chitinases linked to the cell surface, as well as genes for the hydrolysis of other polysaccharides and fermentation of sugars, while the genes needed for aerobic and anaerobic respiration were absent. Adaptation to a haloalkaliphilic lifestyle was reflected by the gene repertoire encoding sodium rather than proton-dependent membrane-bound ion pumps, including the Rnf-type complex, oxaloacetate decarboxylase, V-type ATPase, and pyrophosphatase. The phylogenetic analysis using 16S rRNA gene and ribosomal proteins indicated that ACht6-1 forms a novel deep lineage at the class level within the bacterial candidate division TG3. Based on phylogenetic, phenotypic and genomic analyses, the novel chitinolytic bacterium is described as Chitinispirillum alkaliphilum gen. nov., sp. nov., within a novel class Chitinispirillia that could be included into the phylum Fibrobacteres.@en

Anaerobic enrichments with acetate as electron donor and carbon source, and elemental sulfur as electron acceptor at 4 M NaCl using anaerobic sediments and brines from several hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in isolation in pure culture of four strains of obligately anaerobic haloarchae growing exclusively by sulfur respiration. Such metabolism has not yet been demonstrated in any known species of Halobacteria, and in the whole archaeal kingdom, acetate oxidation with sulfur as acceptor was not previously demonstrated. The four isolates had nearly identical 16S rRNA gene sequences and formed a novel genus-level branch within the family Halobacteriaceae. The strains had a restricted substrate range limited to acetate and pyruvate as electron donors and elemental sulfur as electron acceptor. In contrast to aerobic haloarchaea, the biomass of anaerobic isolates completely lacked the typical red pigments. Growth with acetate+sulfur was observed between 3–5 M NaCl and at a pH range from 6.7 to 8.0. The membrane core lipids were dominated by archaeols. On the basis of distinct physiological and phylogenetic data, the sulfur-respiring isolates represent a novel species of a new genus in the family Halobacteriaceae, for which the name Halanaeroarchaeaum sulfurireducens gen. nov., sp. nov. is proposed. The type strain of the type species is HSR2T (=JCM 30661T=UNIQEM U935T).@en

We analyzed the polar membrane lipids of 13 strains of halo(alkali)philic euryarchaea from hypersaline lakes. Nine belong to the class Halobacteria, representing two functional groups: aerobic polysaccharide utilizers and sulfur-respiring anaerobes. The other four strains represent halo(alkali)philic methanogens from the class Methanomicrobia and a recently discovered class Methanonatronarchaeia. A wide range of polar lipids were detected across the 13 strains including dialkyl glycerol diethers (archaeols), membrane-spanning glycerol tetraethers and diether-based cardiolipins. The archaeols contained a range of core lipid structures, including combinations of C20 and C25 isoprenoidal alkyl chains, unsaturations, and hydroxy moieties. Several diether lipids were novel, including: (a) a phosphatidylglycerolhexose (PG-Gly) headgroup, (b) a N,N,N-trimethyl aminopentanetetrol (APT)-like lipid with a methoxy group in place of a hydroxy group on the pentanetetrol, (c) a series of polar lipids with a headgroup with elemental composition of either C12H25NO13S or C12H25NO16S2, and (d) novel cardiolipins containing a putative phosphatidylglycerolphosphate glycerophosphate (PGPGP) polar moiety. We found that the lipid distribution of the 13 strains could be generally separated into two groups, the methanogens (group) and the Halobacteria (class) based on the presence of specific core lipids. Within the methanogens, adaption to a high or more moderate salt concentration resulted in different ratios of glycerol dialkyl glycerol tetraethers (GDGTs) to archaeol. The methanogen Methanosalsum natronophilum AME2T had the most complex diether lipid composition of any of the 13 strains, including hydroxy archaeol and macrocyclic archaeol which we surmise is an order-specific membrane adaption. The zwitterionic headgroups APT and APT-Me were detected only in the Methanomicrobiales member Methanocalculus alkaliphilus AMF2T which also contained the highest level of unsaturated lipids. Only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four analyzed neutrophilic members of the Halobacteria were characterized by the presence of sulfur-containing headgroups and glycolipids. The presence of cardiolipins with one or more i-C25 alkyl chains, generally termed extended archaeol (EXT-AR), in one of the Methanonatronarchaeia strains was unexpected as only one other order of methanogenic archaea has been reported to produce EXT-AR. We examined this further by looking into the genomic potential of various archaea to produce EXT-AR.@en

An extremely halophilic euryarchaeon, strain HArcel1T, was enriched and isolated in pure culture from the surface brines and sediments of hypersaline athalassic lakes in the Kulunda Steppe (Altai region, Russia) using amorphous cellulose as the growth substrate. The colonies of HArcel1T are pale-orange, and form large zones of cellulose hydrolysis around them. The cells are non-motile cocci of variable size with a thin monolayer cell wall. The isolate is an obligate aerobic heterotroph capable of growth with only three substrates: various forms of insoluble cellulose, xylan and cellobiose. Strain HArcel1T is an extremely halophilic neutrophile, growing within the salinity range from 2.5 to 5 M NaCl (optimum at 3.5-4 M). The core archaeal lipids are dominated by C20-C20 and C25-C20 dialkyl glycerol ethers, in approximately 6:1 proportion. The 16S rRNA and rpoB' gene analysis indicated that HArcel1T forms a separate lineage within the family Haloarculaceae, order Halobacteriales, with the genera Halorhabdus and Halopricus as closest relatives. On the basis of the unique phenotypic properties and distinct phylogeny of the 16S rRNA and rpoB' genes, it is suggested that strain HArcel1T is classified into a new genus and species Halococcoides cellulosivorans gen. nov., sp. nov. (JCM 31941T=UNIQEM U975T).@en

Six strains of extremely halophilic and alkaliphilic euryarchaea were enriched and isolated in pure culture from surface brines and sediments of hypersaline alkaline lakes in various geographical locations with various forms of insoluble cellulose as growth substrate. The cells are mostly flat motile rods with a thin monolayer cell wall while growing on cellobiose. In contrast, the cells growing with cellulose are mostly nonmotile cocci covered with a thick external EPS layer. The isolates, designated AArcel, are obligate aerobic heterotrophs with a narrow substrate spectrum. All strains can use insoluble celluloses, cellobiose, a few soluble glucans and xylan as their carbon and energy source. They are extreme halophiles, growing within the range from 2.5 to 4.8 M total Na+ (optimum at 4 M) and obligate alkaliphiles, with the pH range for growth from 7.5 to 9.9 (optimum at 8.5–9). The core archaeal lipids of strain AArcel5T were dominated by C20–C20 dialkyl glycerol ether (DGE) (i.e. archaeol) and C20–C25 DGE in nearly equal proportion. The 16S rRNA gene analysis indicated that all six isolates belong to a single genomic species mostly related to the genera Saliphagus-Natribaculum-Halovarius. Taking together a substantial phenotypic difference of the new isolates from the closest relatives and the phylogenetic distance, it is concluded that the AArcel group represents a novel genus-level branch within the family Natrialbaceae for which the name Natronobiforma cellulositropha gen. nov., sp. nov. is proposed with AArcel5T as the type strain (JCM 31939T = UNIQEM U972T).@en

Eight pure cultures of alkaliphilic haloaloarchaea capable of growth by dissimilatory sulfur reduction (previously only shown for neutrophilic haloarchaea) were isolated from hypersaline alkaline lakes in different geographic locations. These anaerobic enrichments, inoculated with sediments and brines, used formate, butyrate and peptone as electron donors and elemental sulfur as an electron acceptor 4 M total Na+ and at pH 9-10. According to 16S rRNA gene sequencing, the isolates fell into two distinct groups. A major group, comprising seven obligate alkaliphilic isolates from highly alkaline soda lakes, represents a new species-level branch within the genus Natronolimnobius (order Natrialbales), while a single moderately alkaliphilic isolate from the less alkaline Searles Lake forms a novel genus-level lineage within the order Haloferacales. The cells of the isolates are either flat rods or coccoid. They are facultative anaerobes using formate or H2 (in the presence of acetate or yeast extract as carbon source), C4-C9 fatty acids or peptone (the major group) as electron donors and either sulfur or DMSO (the major group) as electron acceptors. Aerobic growth is only possible with organic acids and peptone-yeast extract. All isolates are extreme halophiles, growing optimally at 4 M total Na+. On the basis of their unique physiological properties and distinct phylogeny, we propose that the seven isolates from the soda lakes are placed into a novel species, Natronolimnobiussulfurireducens sp. nov. (type strain AArc1T=JCM 30663T=UNIQEM U932T), and the Searles Lake isolate, AArc-SlT, into a new genus and species Halalkaliarchaeum desulfuricum (=JCM 30664T=UNIQEM U999T).@en

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.@en

An anaerobic enrichment with CO from sediments of hypersaline soda lakes resulted in a methane-forming binary culture, whereby CO was utilized by a bacterium and not the methanogenic partner. The bacterial isolate ANCO1 forms a deep-branching phylogenetic lineage at the level of a new family within the class ‘Natranaerobiia’. It is an extreme haloalkaliphilic and moderate thermophilic acetogen utilizing CO, formate, pyruvate and lactate as electron donors and thiosulfate, nitrate (reduced to ammonia) and fumarate as electron acceptors. The genome of ANCO1 encodes a full Wood–Ljungdahl pathway allowing for CO oxidation and acetogenic conversion of pyruvate. A locus encoding Nap nitrate reductase/NrfA ammonifying nitrite reductase is also present. Thiosulfate respiration is encoded by a Phs/Psr-like operon. The organism obviously relies on Na-based bioenergetics, since the genome encodes for the Na+-Rnf complex, Na+-F1F0 ATPase and Na+-translocating decarboxylase. Glycine betaine serves as a compatible solute. ANCO1 has an unusual membrane polar lipid composition dominated by diethers, more common among archaea, probably a result of adaptation to multiple extremophilic conditions. Overall, ANCO1 represents a unique example of a triple extremophilic CO-oxidizing anaerobe and is classified as a novel genus and species Natranaerofaba carboxydovora in a novel family Natranaerofabacea.@en

The presence of the C30 sterol gorgosterol (22,23-methylene-23,24-dimethylcholest-5-en-3ß-ol) and its analogues in some marine and freshwater environments is generally associated with invertebrate animals or dinoflagellates since there have been no reports of them in other microalgal classes. Here we show that two unialgal cultures of different species of the marine diatom Delphineis contain gorgosterol in addition to sterols more commonly found in diatoms. Our findings suggest that some reports of gorgosterol in seawater and marine sediments may well have an origin, at least in part, from diatoms.@en

Protologue Table 4 describing properties of Natrachaeobius chitinivorans gen. nov., sp. nov., and Natrarchaeobius haloalkaliphilus sp. nov. has been amended with an extra line designating Natrarchaeobius chitinivoransas the type species of the genus Natrarchaeobius, in accordance to the Rule 20a of the ICNPA.@en

This is a corrigendum to the protologue Table 3 describing properties of Natronobiforma cellulositropha gen. nov sp. nov. 1. The species name “cellulotropha” was corrected to “cellulositropha” in the (SPNA), (SPEP) and TITL lines.2. The author name “Damstéd” in the (AUT) was corrected to “Damsté”.3. The word “neutral” in the (GETY/SPTY) was corrected to “neut”.4. Some of the relevant values have been added to both genus and species columns.@en