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Inland athalassohaline solar salterns provide unique opportunity to study microbial successions along salinity gradients that resemble transition in natural hypersaline lakes. We analyzed for the first time 16S rRNA gene amplicon sequences of bacteria (V1–V2) and archaea (V4–V5) in saltern brines of India’s largest inland hypersaline Sambhar Lake. Brines of the salterns (S1–S4) are alkaline (pH 9.5–10.5) with salinities of 130, 170, 280 and 350 gL−1 respectively. 16S rRNA gene copy-number of archaea outnumbered that of bacteria in all salterns. Their diversity also increased along S1 through S4, while that of bacteria decreased. Brines of S3 and S4 were dominated by specialized extreme halophilic bacterial (Halanaerobiales, Rhodothermaceae) and archaeal (Halobacteriales, Haloferacales) members with recognized salt-in strategy for osmoadaptation. Microbial assemblages positively correlated to saltern pH, total salinity, and ionic composition. Archaea in S1 and S2 were unprecedentedly represented by poorly known as-yet uncultivated groups, Woesearchaeota (90.35–93.51%) and Nanohaloarchaeota that belong to the newly proposed nano-sized superphylum DPANN. In fact, these taxa were identified in archaeal datasets of other athalassohaline salterns after re-analysis using latest RDP database. Thus, microbial compositions in hypersaline lakes are complex and need revisit particularly for their archaeal diversity to understand their hitherto unknown ecological function in extreme environments.

Phylogenomic analysis supports a diderm ancestor of the Firmicutes and points to an early origin of two-membraned cells in Bacteria and the derived nature of the Gram-positive envelope following multiple outer membrane losses. The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales. When several copies of 5S rRNA gene are present in a genome, the expanded versions may co-exist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Microbial Enhanced Oil Recovery (MEOR) represents a promising economic and sustainable EOR approache but is still scarcely employed due to its image as a “black-box” technology. Within the framework of this dissertation, opportunities for in situ MEOR in a low-temperature (37 °C) and high-salinity (180 g*L-1) oilfield by stimulating the growth of anaerobic fermentative bacteria of the order Halanaerobiales was assessed using dynamic sandpacks operated anaerobically under 5 bar with different materials including crushed reservoir rock and resulting in reservoir-representative permeabilities of 10,000 – 800 mD. Wettability alteration, mineral dissolution and potentially selective plugging were the main contributors for oil mobilization, but were not fully accounted for in water-wet sandpacks with quartz sand. However, incremental recovery in sandpacks with reservoir rock was 23.2 ± 6.4 %, with respect to the Original Oil In Place (OOIP). Sucrose consumption rates in sandpacks with oil-wet crushed reservoir rock with a cumulative content of 1.2 % carbonates and sulfides was six-fold higher compared to inert quartz sand, i.e. 3.7 mM*d-1. The dissolution of surface-coating minerals was confirmed by both XPS measurements and a decrease in specific surface area and is assumed to have induced the observed wettability change towards more water-wet conditions which was concurrent with significant incremental oil recovery. Besides promoting oil recovery, microorganisms can also have detrimental effects in oil reservoirs, most notably by the activity of Sulfate Reducing Microorganisms (SRM). Molybdate was tested as a SRM-specific souring control agent not interfering with the chosen MEOR approach. 0.5 mM molybdate resulted in the sustained and complete inhibition of SRM over twenty days in batch experiments, but significant adsorption of ≥ 34 µmolMolybdate*g-1Rock to reservoir rock was observed, mainly controlled by accessory amounts of pyrite and iron (hydr)oxides. In summary, this thesis combined reservoir engineering, microbiological and geological aspects for an interdisciplinary approach on MEOR. It was shown for the first time that MEOR is applicable to reservoirs with a salinity of up to 180 g*L-1, thus extending previous recommendations for a maximum salinity of 10 %. In addition, these results indicate that in situ, fermentation based MEOR has potential for supporting the transition phase between the age of fossil fuels to alternative energy sources as an economic and sustainable tertiary recovering technique for enhancing oil recovery from mature fields., Microbial Enhanced Oil Recovery (MEOR) kann maßgeblich zu einer effizienteren Ausbeutung von sich bereits lange in Produktion befindlichen Erdöllagerstätten beitragen, findet bisher allerdings auf Grund der vorherrschenden Reputation als „Black Box“-Technologie nur selten Anwendung. Im Rahmen dieser Dissertation wurde das Potential für in situ MEOR in einer Onshore-Lagerstätte mit einer Salinität von 180 g*L-1 und einer Temperatur von 37 °C mittels fermentativen Wachstum anaerober, halophiler Bakterien der Ordnung Halanaerobiales, das durch die Injektion von Saccharose in situ angeregt wurde, erkundet. Hierzu wurden sogenannte Sandpacks mit verschiedenen Materialien und einer lagerstättenspezifischen Permeabilität von 10,000 – 800 mD angefertigt, die unter 5 bar mit verschiedenen, dem Testfeld entnommenen Fluiden geflutet wurden. Die Änderung der Benetzbarkeit, Mineralauflösung und sowie potentiell selektives Plugging wurden als Hauptmechanismen für MEOR identifiziert, allerdings konnten nicht all diese Effekte in Sandpacks mit reinem Quarzsand beobachtet werden, sodass die inkrementelle Mehrentölung lediglich ungefähr ein Drittel im Vergleich zu Sandpacks mit Lagerstättengestein, die eine Mehrentölung von 23,2 ± 6,4 % aufwiesen, betrug. Die Saccharose-Abbaurate in Sandpacks mit Lagerstättengestein mit einem kumulativen Gehalt von 1.2 % Karbonaten und Sulfiden betrug im Vergleich zu Sandpacks mit inertem Quarzsand etwa das sechsfache, 3,7 mM*d-1. Zeitgleich wurden eine Abnahme der spezifischen Oberfläche sowie eine Auflösung von oberflächlichen Eisenmineralen mittels XPS registriert. Die beobachteten Änderungen der relativen Permeabilitäten deuten zudem darauf hin, dass dieser hauptsächlich oberflächliche Prozess die Benetzbarkeitsänderung in Richtung einer hydrophileren Oberfläche des porösen Mediums ausgelöst hat. Mikroorganismen können eine Erdöllagerstätte auch nachteilig beeinflussen, maßgeblich durch die Aktivität von Sulfat reduzierenden Mikroorganismen (SRM). Molybdat wurde hier als ein SRM-spezifisches und den MEOR-Ansatz nicht nachteilig beeinflussendes Inhibierungsmittel getestet. Die Zugabe von 0.5 mM Molybdat führte in Batchversuchen zu einer Hemmung der Aktivität von SRM über den gesamten Versuchszeitraum von 20 Tagen, allerdings wurde mit einer geschätzten Langmuir-Sättigung von ≥ 34 µmolMoO4*g-1Gestein eine beträchtliche Adsorption am Lagerstättengestein festgestellt. Die vorliegende Dissertation vereint unter Berücksichtigung geologischer Aspekte sowohl Aspekte des Lagerstätteningenieurwesens als auch der Mikrobiologie. Es wurde erstmals gezeigt, dass MEOR auch in Ölfeldern mit einer Salinität von 180 g*L-1 Anwendung finden kann und die Ergebnisse lassen darauf schließen, dass in situ MEOR eine vielversprechende und gleichzeitig ökologische Methode darstellt, um während der bevorstehenden Übergangsphase zwischen dem Ölzeitalter hin zu einer wie auch immer gearteten alternativen Energieversorgung einen bedeutsamen Beitrag zu leisten.

Anoxybacter fermentans DY22613T is a novel piezophilic dissimilatory iron(III)-reducing bacterium isolated from East Pacific Rise (EPR) hydrothermal sulfides. The strain shows very low 16S rRNA gene similarity (86.92%) with the Halanaerobiales, and represents at least a novel genus Anoxybacter within the class Clostridia. Here, we describe the first complete genome of strain DY22613T in this genus. The genome contains 3,557,532 bp with a G + C content of 35.88%. Genome sequence analysis of strain DY22613T reveals the presence of genes encoding riboflavin (ribBA,D,A,F,H), FAD‑hydrogenases, etc. are involved in dissimilatory iron(III)-reducing process. The genome will provide insights into the mechanism of dissimilatory iron(III)-reducing in strain DY22613T, and contribute to better understand the role of this group in the biogeochemistry cycling of iron in deep-sea hydrothermal fields.

The class Clostridia in the phylum Firmicutes includes a very heterogeneous assemblage of bacteria. Their evolutionary relationships are not well established; revisions of their phylogenetic placements based on comparative studies of 16S rRNA gene sequences are in progress as genome sequence information accumulates. In this work, phylogenetic trees were reconstructed based on 21 concatenated ribosomal protein sequences using Bayesian and maximum-likelihood methods. Both trees consistently indicate that the Halanaerobiales is a deeply branching order among the class Clostridia. The rest of the Clostridia species are grouped into 10 monophyletic clusters, most of which are comprised of two or three orders and families according to the current Clostridial taxonomy. The maximum-likelihood tree placed Coprothermobacter proteolyticus and Thermodesulfobium narugense in the class Clostridia in accordance with the current taxonomy, in which these two bacteria are assigned to the family Thermodesulfobiaceae. However, the Bayesian tree placed these two bacteria at the boundary between the Firmicutes and Actinobacteria. A gene arrangement that is present uniquely in the Firmicutes species was identified. Both Coprothermobacter proteolyticus and Thermodesulfobium narugense do not have this arrangement characteristic of the Firmicutes. On the basis of the Bayesian tree and gene arrangement comparison, it is suggested that Coprothermobacter proteolyticus and Thermodesulfobium narugense should be placed outside the phylum Firmicutes.

One of the major unanswered questions in evolutionary biology is when and how the transition between diderm (two membranes) and monoderm (one membrane) cell envelopes occurred in Bacteria. The Negativicutes and the Halanaerobiales belong to the classically monoderm Firmicutes, but possess outer membranes with lipopolysaccharide (LPS-OM). Here, we show that they form two phylogenetically distinct lineages, each close to different monoderm relatives. In contrast, their core LPS biosynthesis enzymes were inherited vertically, as in the majority of bacterial phyla. Finally, annotation of key OM systems in the Halanaerobiales and the Negativicutes shows a puzzling combination of monoderm and diderm features. Together, these results support the hypothesis that the LPS-OMs of Negativicutes and Halanaerobiales are remnants of an ancient diderm cell envelope that was present in the ancestor of the Firmicutes, and that the monoderm phenotype in this phylum is a derived character that arose multiple times independently through OM loss.

One of the major unanswered questions in evolutionary biology is when and how the transition between diderm (two membranes) and monoderm (one membrane) cell envelopes occurred in Bacteria. The Negativicutes and the Halanaerobiales belong to the classically monoderm Firmicutes, but possess outer membranes with lipopolysaccharide (LPS-OM). Here, we show that they form two phylogenetically distinct lineages, each close to different monoderm relatives. In contrast, their core LPS biosynthesis enzymes were inherited vertically, as in the majority of bacterial phyla. Finally, annotation of key OM systems in the Halanaerobiales and the Negativicutes shows a puzzling combination of monoderm and diderm features. Together, these results support the hypothesis that the LPS-OMs of Negativicutes and Halanaerobiales are remnants of an ancient diderm cell envelope that was present in the ancestor of the Firmicutes, and that the monoderm phenotype in this phylum is a derived character that arose multiple times independently through OM loss. DOI: http://dx.doi.org/10.7554/eLife.14589.001, eLife digest The cell envelope is one of the evolutionarily oldest parts of a bacterium. This structure – made up of a cell wall and either one or two cell membranes – surrounds the bacterial cell, maintaining the cell’s structure and providing an interface through which bacteria can sense their environment and communicate. Bacteria can be broadly classed based on the number of cell membranes that their envelope consists of. Bacteria that have a single cell membrane are known as “monoderm”, whereas those with two membranes are termed “diderm”. The number of membranes that bacteria have can affect how well they resist antibacterial compounds. When, how and why bacteria switched between monoderm and diderm cell envelopes are some of the major unanswered questions in evolutionary biology. The textbook example of a monoderm cell envelope can be found in bacteria called Firmicutes. This group includes some notoriously harmful bacteria such as Staphylococcus, which can cause conditions ranging from abscesses to pneumonia. However, some Firmicutes possess two cell membranes. It was unclear how these unusual diderm Firmicutes developed a second membrane, and how they are related to their monoderm relatives. Antunes, Poppleton et al. set out to answer these questions by analyzing the information contained in the thousands of bacterial genomes that have already been described. The results indicate that Firmicutes originally had diderm envelopes, and that species with monoderm envelopes arose independently several times through the loss of their outermost membrane. Future work is needed to investigate the driving forces and the precise mechanism that led most Firmicutes to lose their outer membrane. Also, further characterization of diderm Firmicutes will provide key information about the biology of these poorly understood bacteria. DOI: http://dx.doi.org/10.7554/eLife.14589.002

We performed a comparative analysis of the genome sequences of three anaerobic halophilic fermentative bacteria belonging to the order Halanaerobiales: Halanaerobium praevalens, the alkaliphilic "Halanaerobium hydrogeniformans", and the thermophilic Halothermothrix orenii to assess the amino acid composition of their proteins. Members of the Halanaerobiales were earlier shown to accumulate KCl rather than organic compatible solutes for osmotic balance, and therefore the presence of a dominantly acidic proteome was predicted. Past reports indeed showed a large excess of acidic over basic amino acids in whole-cell hydrolysates of selected members of the order. However, the genomic analysis did not show unusually high contents of acidic amino acids or low contents of basic amino acids. The apparent excess of acidic amino acids in these anaerobic halophiles reported earlier is due to the high content in their proteins of glutamine and asparagine, which yield glutamate and aspartate upon acid hydrolysis. It is thus suggested that the proteins of the Halanaerobiales, which are active in the presence of high intracellular KCl concentrations, do not possess the typical acidic signature of the 'halophilic' proteins of the Archaea of the order Halobacteriales or of the extremely halophilic bacterium Salinibacter.

Acetohalobium arabaticum Zhilina and Zavarzin 1990 is of special interest because of its physiology and its participation in the anaerobic C(1)-trophic chain in hypersaline environments. This is the first completed genome sequence of the family Halobacteroidaceae and only the second genome sequence in the order Halanaerobiales. The 2,469,596 bp long genome with its 2,353 protein-coding and 90 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Halothermothirx orenii is a strictly anaerobic thermohalophilic bacterium isolated from sediment of a Tunisian salt lake. It belongs to the order Halanaerobiales in the phylum Firmicutes. The complete sequence revealed that the genome consists of one circular chromosome of 2578146 bps encoding 2451 predicted genes. This is the first genome sequence of an organism belonging to the Haloanaerobiales. Features of both Gram positive and Gram negative bacteria were identified with the presence of both a sporulating mechanism typical of Firmicutes and a characteristic Gram negative lipopolysaccharide being the most prominent. Protein sequence analyses and metabolic reconstruction reveal a unique combination of strategies for thermophilic and halophilic adaptation. H. orenii can serve as a model organism for the study of the evolution of the Gram negative phenotype as well as the adaptation under thermohalophilic conditions and the development of biotechnological applications under conditions that require high temperatures and high salt concentrations.

A formal proposal is made to rename the order Haloanaerobiales as Halanaerobiales, the family Haloanaerobiaceae as Halanaerobiaceae, the genera Haloanaerobium and Haloanaerobacter as Halanaerobium and Halanaerobacter, respectively, and to change the specific epithets of Halanaerobium acetoethylicum, Halanaerobacter lacunaris, Halocella cellulolytica and Halanaerobium chitinovorans to acetethylicum, lacunarum, cellulosilytica and chitinivorans, respectively. These changes are required in view of Rule 61, Appendix 9, of the Bacteriological Code.

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.