Redox gradient shapes the abundance and diversity of mercury-methylating microorganisms along the water column of the Black Sea.

In the global context of seawater deoxygenation triggered by climate change and anthropogenic activities, changes in redox gradients impacting biogeochemical transformations of pollutants, such as mercury, become more likely. Being the largest anoxic basin worldwide, with high concentrations of the potent neurotoxic methylmercury (MeHg), the Black Sea is an ideal natural laboratory to provide new insights about the link between dissolved oxygen concentration and hgcAB gene-carrying (hgc+) microorganisms involved in the formation of MeHg. We combined geochemical and microbial approaches to assess the effect of vertical redox gradients on abundance, diversity, and metabolic potential of hgc+ microorganisms in the Black Sea water column. The abundance of hgcA genes [congruently estimated by quantitative PCR (qPCR) and metagenomics] correlated with MeHg concentration, both maximal in the upper part of the anoxic water. Besides the predominant Desulfobacterales, hgc+ microorganisms belonged to a unique assemblage of diverse-previously underappreciated-anaerobic fermenters from Anaerolineales, Phycisphaerae (characteristic of the anoxic and sulfidic zone), Kiritimatiellales, and Bacteroidales (characteristic of the suboxic zone). The metabolic versatility of Desulfobacterota differed from strict sulfate reduction in the anoxic water to reduction of various electron acceptors in the suboxic water. Linking microbial activity and contaminant concentration in environmental studies is rare due to the complexity of biological pathways. In this study, we disentangle the role of oxygen in shaping the distribution of Hg-methylating microorganisms consistently with MeHg concentration, and we highlight their taxonomic and metabolic niche partitioning across redox gradients, improving the prediction of the response of marine communities to the expansion of oxygen-deficient zones. IMPORTANCE Methylmercury (MeHg) is a neurotoxin detected at high concentrations in certain marine ecosystems, posing a threat to human health. MeHg production is mainly mediated by hgcAB gene-carrying (hgc+) microorganisms. Oxygen is one of the main factors controlling Hg methylation; however, its effect on the diversity and ecology of hgc+ microorganisms remains unknown. Under the current context of seawater deoxygenation, mercury cycling is expected to be disturbed. Here, we show the strong effect of oxygen gradients on the distribution of potential Hg methylators. In addition, we show for the first time the significant contribution of a unique assemblage of potential fermenters from Anaerolineales, Phycisphaerae, and Kiritimatiellales to Hg methylation, stratified in different redox niches along the Black Sea gradient. Our results considerably expand the known taxonomic diversity and ecological niches prone to the formation of MeHg and contribute to better apprehend the consequences of oxygen depletion in seawater.

Organic Matter Type Defines the Composition of Active Microbial Communities Originating From Anoxic Baltic Sea Sediments.

Carbon cycling in anoxic marine sediments is dependent on uncultured microbial communities. Niches of heterotrophic microorganisms are defined by organic matter (OM) type and the different phases in OM degradation. We investigated how OM type defines microbial communities originating from organic-rich, anoxic sediments from the Baltic Sea. We compared changes in the sediment microbial community, after incubation with different stable isotope labeled OM types [i.e., particulate algal organic matter (PAOM), protein, and acetate], by using DNA stable isotope probing (DNA-SIP). Incorporation of 13C and/or 15N label was predominantly detected in members of the phyla Planctomycetes and Chloroflexi, which also formed the majority (>50%) of the original sediment community. While these phylum-level lineages incorporated label from all OM types, phylogenetic analyses revealed a niche separation at the order level. Members of the MSBL9 (Planctomycetes), the Anaerolineales (Chloroflexi), and the class Bathyarchaeota, were identified as initial degraders of carbohydrate-rich OM, while other uncultured orders, like the CCM11a and Phycisphaerales (Planctomycetes), Dehalococcoidia, and JG30-KF-CM66 (Chloroflexi), incorporated label also from protein and acetate. Our study highlights the importance of initial fermentation of complex carbon pools in shaping anoxic sediment microbial communities and reveals niche specialization at the order level for the most important initial degraders in anoxic sediments.

The bacterial sulfur cycle in expanding dysoxic and euxinic marine waters.

Dysoxic marine waters (DMW, < 1 µM oxygen) are currently expanding in volume in the oceans, which has biogeochemical, ecological and societal consequences on a global scale. In these environments, distinct bacteria drive an active sulfur cycle, which has only recently been recognized for open-ocean DMW. This review summarizes the current knowledge on these sulfur-cycling bacteria. Critical bottlenecks and questions for future research are specifically addressed. Sulfate-reducing bacteria (SRB) are core members of DMW. However, their roles are not entirely clear, and they remain largely uncultured. We found support for their remarkable diversity and taxonomic novelty by mining metagenome-assembled genomes from the Black Sea as model ecosystem. We highlight recent insights into the metabolism of key sulfur-oxidizing SUP05 and Sulfurimonas bacteria, and discuss the probable involvement of uncultivated SAR324 and BS-GSO2 bacteria in sulfur oxidation. Uncultivated Marinimicrobia bacteria with a presumed organoheterotrophic metabolism are abundant in DMW. Like SRB, they may use specific molybdoenzymes to conserve energy from the oxidation, reduction or disproportionation of sulfur cycle intermediates such as S0 and thiosulfate, produced from the oxidation of sulfide. We expect that tailored sampling methods and a renewed focus on cultivation will yield deeper insight into sulfur-cycling bacteria in DMW.

Pontiella desulfatans gen. nov., sp. nov., and Pontiella sulfatireligans sp. nov., Two Marine Anaerobes of the Pontiellaceae fam. nov. Producing Sulfated Glycosaminoglycan-like Exopolymers.

Recently, we isolated two marine strains, F1T and F21T, which together with Kiritimatiella glycovorans L21-Fru-ABT are the only pure cultures of the class Kiritimatiellae within the phylum Verrucomicrobiota. Here, we present an in-depth genome-guided characterization of both isolates with emphasis on their exopolysaccharide synthesis. The strains only grew fermentatively on simple carbohydrates and sulfated polysaccharides. Strains F1T, F21T and K. glycovorans reduced elemental sulfur, ferric citrate and anthraquinone-2,6-disulfonate during anaerobic growth on sugars. Both strains produced exopolysaccharides during stationary phase, probably with intracellularly stored glycogen as energy and carbon source. Exopolysaccharides included N-sulfated polysaccharides probably containing hexosamines and thus resembling glycosaminoglycans. This implies that the isolates can both degrade and produce sulfated polysaccharides. Both strains encoded an unprecedently high number of glycoside hydrolase genes (422 and 388, respectively), including prevalent alpha-L-fucosidase genes, which may be necessary for degrading complex sulfated polysaccharides such as fucoidan. Strain F21T encoded three putative glycosaminoglycan sulfotransferases and a putative sulfate glycosaminoglycan biosynthesis gene cluster. Based on phylogenetic and chemotaxonomic analyses, we propose the taxa Pontiella desulfatans F1T gen. nov., sp. nov. and Pontiella sulfatireligans F21T sp. nov. as representatives of the Pontiellaceae fam. nov. within the class Kiritimatiellae.

Anaerobic Degradation of Non-Methane Alkanes by "Candidatus Methanoliparia" in Hydrocarbon Seeps of the Gulf of Mexico.

Crude oil and gases in the seabed provide an important energy source for subsurface microorganisms. We investigated the role of archaea in the anaerobic degradation of non-methane alkanes in deep-sea oil seeps from the Gulf of Mexico. We identified microscopically the ethane and short-chain alkane oxidizers "Candidatus Argoarchaeum" and "Candidatus Syntrophoarchaeum" forming consortia with bacteria. Moreover, we found that the sediments contain large numbers of cells from the archaeal clade "Candidatus Methanoliparia," which was previously proposed to perform methanogenic alkane degradation. "Ca. Methanoliparia" occurred abundantly as single cells attached to oil droplets in sediments without apparent bacterial or archaeal partners. Metagenome-assembled genomes of "Ca. Methanoliparia" encode a complete methanogenesis pathway including a canonical methyl-coenzyme M reductase (MCR) but also a highly divergent MCR related to those of alkane-degrading archaea and pathways for the oxidation of long-chain alkyl units. Its metabolic genomic potential and its global detection in hydrocarbon reservoirs suggest that "Ca. Methanoliparia" is an important methanogenic alkane degrader in subsurface environments, producing methane by alkane disproportionation as a single organism.IMPORTANCE Oil-rich sediments from the Gulf of Mexico were found to contain diverse alkane-degrading groups of archaea. The symbiotic, consortium-forming "Candidatus Argoarchaeum" and "Candidatus Syntrophoarchaeum" are likely responsible for the degradation of ethane and short-chain alkanes, with the help of sulfate-reducing bacteria. "Ca. Methanoliparia" occurs as single cells associated with oil droplets. These archaea encode two phylogenetically different methyl-coenzyme M reductases that may allow this organism to thrive as a methanogen on a substrate of long-chain alkanes. Based on a library survey, we show that "Ca. Methanoliparia" is frequently detected in oil reservoirs and may be a key agent in the transformation of long-chain alkanes to methane. Our findings provide evidence for the important and diverse roles of archaea in alkane-rich marine habitats and support the notion of a significant functional versatility of the methyl coenzyme M reductase.

Anaerobic Degradation of Sulfated Polysaccharides by Two Novel Kiritimatiellales Strains Isolated From Black Sea Sediment.

The marine environment contains a large diversity of sulfated polysaccharides and other glycopolymers. Saccharolytic microorganisms degrade these compounds through hydrolysis, which includes the hydrolysis of sulfate groups from sugars by sulfatases. Various marine bacteria of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum have exceptionally high numbers of sulfatase genes associated with the degradation of sulfated polysaccharides. However, thus far no sulfatase-rich marine anaerobes are known. In this study, we aimed to isolate marine anaerobes using sulfated polysaccharides as substrate. Anoxic enrichment cultures were set up with a mineral brackish marine medium, inoculated with anoxic Black Sea sediment sampled at 2,100 m water depth water and incubated at 15°C (in situ T = 8°C) for several weeks. Community analysis by 16S rRNA gene amplicon sequencing revealed the enrichment of Kiritimatiellaeota clade R76-B128 bacteria in the enrichments with the sulfated polysaccharides fucoidan and iota-carrageenan as substrate. We isolated two strains, F1 and F21, which represent a novel family within the order of the Kiritimatiellales. They were capable of growth on various mono-, di-, and polysaccharides, including fucoidan. The desulfation of iota-carrageenan by strain F21 was confirmed quantitatively by an increase in free sulfate concentration. Strains F1 and F21 represent the first marine sulfatase-rich anaerobes, encoding more sulfatases (521 and 480, 8.0 and 8.4% of all coding sequences, respectively) than any other microorganism currently known. Specific encoded sulfatase subfamilies could be involved in desulfating fucoidan (S1_15, S1_17 and S1_25) and iota-carrageenan (S1_19). Strains F1 and F21 had a sulfatase gene classification profile more similar to aerobic than anaerobic sulfatase-rich PVC bacteria, including Kiritimatiella glycovorans, the only other cultured representative within the Kiritimatiellaeota. Both strains encoded a single anaerobic sulfatase-maturating enzyme which could be responsible for post-translational modification of formylglycine-dependent sulfatases. Strains F1 and F21 are potential anaerobic platforms for future studies on sulfatases and their maturation enzymes.

Microbial Diversity and Organic Acid Production of Guinea Pig Faecal Samples.

The guinea pig (Cavia porcellus) or cavy is a grass-eating rodent. Its main diet consists of grass or hay, which comprises cellulose, hemicellulose, lignin and their derivatives. Here, the microbial diversity of faecal samples of two guinea pigs and microbial enrichments made with substrates, including starch waste and dried grass, were investigated along with organic acid production profiles. The microbial communities of the faecal samples were dominated by the phyla Bacteroidetes (40%) and Firmicutes (36%). Bacteroidales S24-7 (11% in Cavy 1 and 21% in Cavy 2) was the most abundant order. At genus level, many microorganisms remained unclassified. Different carbon sources were used for organic acid production in faecal enrichments. The dominant bacterial groups in the secondary enrichments with dried grass, starch waste and xylose were closely related to Prevotella and Blautia. Acetate was the predominant organic acid from all enrichments. The organic acid production profiles corresponded to a mixed acid fermentation but differed depending on the substrate. Eight phylogenetically different isolates were obtained, including a novel Streptococcus species, strain Cavy grass 6. This strain had a low abundance (1%) in one of the faecal samples but was enriched in the dried grass enrichment (3%). Cavy grass 6, a fast-growing heterolactic bacterium, ferments cellobiose to lactate, acetate, formate and ethanol. Our results show that cavy faecal samples can be applied as microbial source for organic acid production from complex organic substrates. The cavy gut contains many as-yet-uncultivated bacteria which may be appropriate targets for future studies.