Description of two cultivated and two uncultivated new Salinibacter species, one named following the rules of the bacteriological code: Salinibacter grassmerensis sp. nov.; and three named following the rules of the SeqCode: Salinibacter pepae sp. nov., Salinibacter abyssi sp. nov., and Salinibacter pampae sp. nov.

Current -omics methods allow the collection of a large amount of information that helps in describing the microbial diversity in nature. Here, and as a result of a culturomic approach that rendered the collection of thousands of isolates from 5 different hypersaline sites (in Spain, USA and New Zealand), we obtained 21 strains that represent two new Salinibacter species. For these species we propose the names Salinibacter pepae sp. nov. and Salinibacter grassmerensis sp. nov. (showing average nucleotide identity (ANI) values < 95.09% and 87.08% with Sal. ruber M31T, respectively). Metabolomics revealed species-specific discriminative profiles. Sal. ruber strains were distinguished by a higher percentage of polyunsaturated fatty acids and specific N-functionalized fatty acids; and Sal. altiplanensis was distinguished by an increased number of glycosylated molecules. Based on sequence characteristics and inferred phenotype of metagenome-assembled genomes (MAGs), we describe two new members of the genus Salinibacter. These species dominated in different sites and always coexisted with Sal. ruber and Sal. pepae. Based on the MAGs from three Argentinian lakes in the Pampa region of Argentina and the MAG of the Romanian lake Fara Fund, we describe the species Salinibacter pampae sp. nov. and Salinibacter abyssi sp. nov. respectively (showing ANI values 90.94% and 91.48% with Sal. ruber M31T, respectively). Sal. grassmerensis sp. nov. name was formed according to the rules of the International Code for Nomenclature of Prokaryotes (ICNP), and Sal. pepae, Sal. pampae sp. nov. and Sal. abyssi sp. nov. are proposed following the rules of the newly published Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). This work constitutes an example on how classification under ICNP and SeqCode can coexist, and how the official naming a cultivated organism for which the deposit in public repositories is difficult finds an intermediate solution.

Sediment cooling triggers germination and sulfate reduction by heat-resistant thermophilic spore-forming bacteria.

Thermophilic endospores are widespread in cold marine sediments where the temperature is too low to support growth and activity of thermophiles in situ. These endospores are likely expelled from warm subsurface environments and subsequently dispersed by ocean currents. The endospore upper temperature limit for survival is 140°C, which can be tolerated in repeated short exposures, potentially enabling transit through hot crustal fluids. Longer-term thermal tolerance of endospores, and how long they could persist in an environment hotter than their maximum growth temperature, is less understood. To test whether thermophilic endospores can survive prolonged exposure to high temperatures, sediments were incubated at 80-90°C for 6, 12 or 463 days. Sediments were then cooled by 10-40°C, mimicking the cooling in subsurface oil reservoirs subjected to seawater injection. Cooling the sediments induced sulfate reduction, coinciding with an enrichment of endospore-forming Clostridia. Different Desulfofundulus, Desulfohalotomaculum, Desulfallas, Desulfotomaculum and Desulfofarcimen demonstrated different thermal tolerances, with some Desulfofundulus strains surviving for >1 year at 80°C. In an oil reservoir context, heat-resistant endospore-forming sulfate-reducing bacteria have a survival advantage if they are introduced to, or are resident in, an oil reservoir normally too hot for germination and growth, explaining observations of reservoir souring following cold seawater injection.

Electrobioremediation of oil spills.

Annually, thousands of oil spills occur across the globe. As a result, petroleum substances and petrochemical compounds are widespread contaminants causing concern due to their toxicity and recalcitrance. Many remediation strategies have been developed using both physicochemical and biological approaches. Biological strategies are most benign, aiming to enhance microbial metabolic activities by supplying limiting inorganic nutrients, electron acceptors or donors, thus stimulating oxidation or reduction of contaminants. A key issue is controlling the supply of electron donors/acceptors. Bioelectrochemical systems (BES) have emerged, in which an electrical current serves as either electron donor or acceptor for oil spill bioremediation. BES are highly controllable and can possibly also serve as biosensors for real time monitoring of the degradation process. Despite being promising, multiple aspects need to be considered to make BES suitable for field applications including system design, electrode materials, operational parameters, mode of action and radius of influence. The microbiological processes, involved in bioelectrochemical contaminant degradation, are currently not fully understood, particularly in relation to electron transfer mechanisms. Especially in sulfate rich environments, the sulfur cycle appears pivotal during hydrocarbon oxidation. This review provides a comprehensive analysis of the research on bioelectrochemical remediation of oil spills and of the key parameters involved in the process.

Anodes Stimulate Anaerobic Toluene Degradation via Sulfur Cycling in Marine Sediments.

Hydrocarbons released during oil spills are persistent in marine sediments due to the absence of suitable electron acceptors below the oxic zone. Here, we investigated an alternative bioremediation strategy to remove toluene, a model monoaromatic hydrocarbon, using a bioanode. Bioelectrochemical reactors were inoculated with sediment collected from a hydrocarbon-contaminated marine site, and anodes were polarized at 0 mV and +300 mV (versus an Ag/AgCl [3 M KCl] reference electrode). The degradation of toluene was directly linked to current generation of up to 301 mA m(-2) and 431 mA m(-2) for the bioanodes polarized at 0 mV and +300 mV, respectively. Peak currents decreased over time even after periodic spiking with toluene. The monitoring of sulfate concentrations during bioelectrochemical experiments suggested that sulfur metabolism was involved in toluene degradation at bioanodes. 16S rRNA gene-based Illumina sequencing of the bulk anolyte and anode samples revealed enrichment with electrocatalytically active microorganisms, toluene degraders, and sulfate-reducing microorganisms. Quantitative PCR targeting the α-subunit of the dissimilatory sulfite reductase (encoded by dsrA) and the α-subunit of the benzylsuccinate synthase (encoded by bssA) confirmed these findings. In particular, members of the family Desulfobulbaceae were enriched concomitantly with current production and toluene degradation. Based on these observations, we propose two mechanisms for bioelectrochemical toluene degradation: (i) direct electron transfer to the anode and/or (ii) sulfide-mediated electron transfer.

Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, Central Spain).

The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4) × 2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.

Determination of cobalamins (hydroxo-, cyano-, adenosyl- and methyl-cobalamins) in seawater using reversed-phase liquid chromatography with diode-array detection.

A high-performance liquid chromatography method was developed for the separation and determination of four cobalamins in seawater. Chromatographic separation was performed on a reversed-phase discovery RP-amide C(16) column with buffer potassium dihydrogenphosphate and acetonitrile as the mobile phases in linear gradients elution mode. Cobalamins were previously preconcentrated in C(18) resins cartridges. Detection was performed using UV-diode array detector in a range of λ of 200-400 nm. The method showed to be linear over a range of 1-300 ng mL(-1) with acceptable precision and accuracy. The detection limits ranged between 0.07 pg mL(-1) for 5'-deoxyadenosylcobalamin and 0.5 pg mL(-1) for hydroxocobalamin. The mean cobalamins recoveries for direct determination ranged between 76 and 93% for hydroxo-, cyano- and methylcobalamin, while the recovery for 5'-deoxyadenosylcobalamin was only 31% suggesting that the preconcentration method was not valid for this cobalamin. The method was successfully applied to coastal seawater where the concentrations ranged from 4.2 to 7.3 ng L(-1) for hydroxo-, 1.4-3.9 ng L(-1) for cyano-, 2.1-4.6 ng L(-1) for 5'-deoxyadenosyl- and 33-83.5 ng L(-1) for methylcobalamin.

Response of sulfate-reducing bacteria to an artificial oil-spill in a coastal marine sediment.

In situ mesocosm experiments using a calcareous sand flat from a coastal area of the island of Mallorca in the Mediterranean Sea were performed in order to study the response of sulfate-reducing bacteria (SRB) to controlled crude oil contamination, or heavy contamination with naphthalene. Changes in the microbial community caused by the contamination were monitored by a combination of comparative sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, cultivation approaches and metabolic activity rates. Our results showed that crude oil and naphthalene negatively influenced the total microbial community as the natural increase in cell numbers due to the seasonal dynamics was attenuated. However, both contaminants enhanced the sulfate reduction rates, as well as the culturability of SRB. Our results suggested the presence of autochthonous deltaproteobacterial SRBs that were able to degrade crude oil or polycyclic aromatic hydrocarbons such as naphthalene in anaerobic sediment layers.

Extremely halophilic microbial communities in anaerobic sediments from a solar saltern.

The prokaryotic communities inhabiting hypersaline sediments underlying a crystallizer pond of a Mediterranean solar saltern have been studied in a polyphasic approach including 16S rRNA and dsrAB gene libraries analysis [the last encoding for dissimilatory (bi)sulfite reductase], most probable number of cultivable counts, and metabolic measurements of sulfate reduction. The samples studied here represent one of the most hypersaline anoxic environments sampled worldwide that harbour a highly diverse microbial community different from those previously reported in other hypersaline sediments. Both bacterial and archaeal types are present but, contrarily to the overlying brine system, the former dominates. Molecular analyses indicated that the bacterial fraction is highly diverse and mostly composed by groups related to sulfate-reducing bacteria (SRB). In good agreement with this, sulfate-reducing activity was detected in the sediment, as well as the metabolic diversity within SRB (as indicated by the use of different electron donors in enrichments). On the other hand, the archaeal fraction was phylogenetically homogeneous and, surprisingly, strongly affiliated with the MBSl-1 candidate division, an euryarchaeotal group only reported in deep-sea hypersaline anoxic basins of the Western Mediterranean, for which a methanogenic metabolism was hypothesized. The hypersaline studied samples constitute a valuable source of new prokaryotic types with metabolisms adapted to the prevalent in situ extreme conditions.