Cultivation of Diverse Novel Marine Bacteria from Deep Ocean Sediment Using Spent Culture Supernatant of Ca. Bathyarchaeia Enrichment.

Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

Description of the first cultured representative of "Candidatus Synoicihabitans" genus, isolated from deep-sea sediment of South China Sea.

In this study we describe the first cultured representative of Candidatus Synoicihabitans genus, a novel strain designated as LMO-M01T, isolated from deep-sea sediment of South China Sea. This bacterium is a facultative aerobe, Gram-negative, non-motile, and has a globular-shaped morphology, with light greenish, small, and circular colonies. Analysis of the 16S rRNA gene sequences of strain LMO-M01T showed less than 93% similarity to its closest cultured members. Furthermore, employing advanced phylogenomic methods such as comparative genome analysis, average nucleotide identity (ANI), average amino acids identity (AAI), and digital DNA-DNA hybridization (dDDH), placed this novel species within the candidatus genus Synoicihabitans of the family Opitutaceae, Phylum Verrucomicrobiota. The genomic analysis of strain LMO-M01T revealed 175 genes, encoding putative carbohydrate-active enzymes. This suggests its metabolic potential to degrade and utilize complex polysaccharides, indicating a significant role in carbon cycling and nutrient turnover in deep-sea sediment. In addition, the strain's physiological capacity to utilize diverse biopolymers such as lignin, xylan, starch, and agar as sole carbon source opens up possibilities for sustainable energy production and environmental remediation. Moreover, the genome sequence of this newly isolated strain has been identified across diverse ecosystems, including marine sediment, fresh water, coral, soil, plants, and activated sludge highlighting its ecological significance and adaptability to various environments. The recovery of strain LMO-M01T holds promise for taxonomical, ecological and biotechnological applications. Based on the polyphasic data, we propose that this ecologically important strain LMO-M01T represents a novel genus (previously Candidatus) within the family Opitutaceae of phylum Verrucomicrobiota, for which the name Synoicihabitans lomoniglobus gen. nov., sp. nov. was proposed. The type of strain is LMO-M01T (= CGMCC 1.61593T = KCTC 92913T).

Mariluticola halotolerans gen. nov., sp. nov., a novel member of the family Devosiaceae isolated from South China Sea sediment.

A novel member of class Alphaproteobacteria was isolated from marine sediment of the South China Sea. Cells of strain LMO-2T were Gram-stain negative, greyish in colour, motile, with a single lateral flagellum and short rod in shape with a slight curve. Strain LMO-2T was positive for oxidase and negative for catalase. The bacterium grew aerobically at 10-40 °C (optimum, 25-30 °C), pH 5.5-10.0 (optimum, pH 7.0) and 0-9 % NaCl (w/v; optimum, 2-3 %). Phylogenetic analysis of the 16S rRNA gene sequence and phylogenomic analysis of the whole genome sequence indicated that strain LMO-2T represents a new genus and a new species within the family Devosiaceae, class Alphaproteobacteria, phylum Pseudomonadota. Comparisons of the 16S rRNA gene sequences of strain LMO-2T showed 94.8 % similarity to its closest relative. The genome size is ~3.45 Mbp with a DNA G+C content of 58.17 mol%. The strain possesses potential capability for the degradation of complex organic matter, i.e. fatty acid and benzoate. The predominant cellular fatty acids (>10 %) were C16 : 0 and C18 : 1 ω7c 11-methyl. The sole respiratory quinone was ubiquinone-10. The major identified polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phospholipid. Based on the polyphasic taxonomic data, strain LMO-2T represents a novel genus and a novel species for which the name Mariluticola halotolerans gen. nov., sp. nov., was proposed in the family Devosiaceae. The type strain is LMO-2T (=CGMCC 1.19273T=JCM 34934T).

Taxonomic and carbon metabolic diversification of Bathyarchaeia during its coevolution history with early Earth surface environment.

Bathyarchaeia, as one of the most abundant microorganisms on Earth, play vital roles in the global carbon cycle. However, our understanding of their origin, evolution, and ecological functions remains poorly constrained. Here, we present the largest dataset of Bathyarchaeia metagenome assembled genome to date and reclassify Bathyarchaeia into eight order-level units corresponding to the former subgroup system. Highly diversified and versatile carbon metabolisms were found among different orders, particularly atypical C1 metabolic pathways, indicating that Bathyarchaeia represent overlooked important methylotrophs. Molecular dating results indicate that Bathyarchaeia diverged at ~3.3 billion years, followed by three major diversifications at ~3.0, ~2.5, and ~1.8 to 1.7 billion years, likely driven by continental emergence, growth, and intensive submarine volcanism, respectively. The lignin-degrading Bathyarchaeia clade emerged at ~300 million years perhaps contributed to the sharply decreased carbon sequestration rate during the Late Carboniferous period. The evolutionary history of Bathyarchaeia potentially has been shaped by geological forces, which, in turn, affected Earth's surface environment.

Widespread Bathyarchaeia encode a novel methyltransferase utilizing lignin-derived aromatics.

Lignin degradation is a major process in the global carbon cycle across both terrestrial and marine ecosystems. Bathyarchaeia, which are among the most abundant microorganisms in marine sediment, have been proposed to mediate anaerobic lignin degradation. However, the mechanism of bathyarchaeial lignin degradation remains unclear. Here, we report an enrichment culture of Bathyarchaeia, named Candidatus Baizosediminiarchaeum ligniniphilus DL1YTT001 (Ca. B. ligniniphilus), from coastal sediments that can grow with lignin as the sole organic carbon source under mesophilic anoxic conditions. Ca. B. ligniniphilus possesses and highly expresses novel methyltransferase 1 (MT1, mtgB) for transferring methoxyl groups from lignin monomers to cob(I)alamin. MtgBs have no homology with known microbial methyltransferases and are present only in bathyarchaeial lineages. Heterologous expression of the mtgB gene confirmed O-demethylation activity. The mtgB genes were identified in metagenomic data sets from a wide range of coastal sediments, and they were highly expressed in coastal sediments from the East China Sea. These findings suggest that Bathyarchaeia, capable of O-demethylation via their novel and specific methyltransferases, are ubiquitous in coastal sediments.

Isolation of a Novel Thermophilic Methanogen and the Evolutionary History of the Class Methanobacteria.

Methanogens can produce methane in anaerobic environments via the methanogenesis pathway, and are regarded as one of the most ancient life forms on Earth. They are ubiquitously distributed across distinct ecosystems and are considered to have a thermophilic origin. In this study, we isolated, pure cultured, and completely sequenced a single methanogen strain DL9LZB001, from a hot spring at Tengchong in Southwest China. DL9LZB001 is a thermophilic and hydrogenotrophic methanogen with an optimum growth temperature of 65 °C. It is a putative novel species, which has been named Methanothermobacter tengchongensis-a Class I methanogen belonging to the class Methanobacteria. Comparative genomic and ancestral analyses indicate that the class Methanobacteria originated in a hyperthermal environment and then evolved to adapt to ambient temperatures. This study extends the understanding of methanogens living in geothermal niches, as well as the origin and evolutionary history of these organisms in ecosystems with different temperatures.

Iron (oxyhydr)oxides shift the methanogenic community in deep sea methanic sediment - insights from long-term high-pressure incubations.

Intermittent increases of dissolved ferrous iron concentrations have been observed in deep marine methanic sediments which is different from the traditional diagenetic electron acceptor cascade, where iron reduction precedes methanogenesis. Here we aimed to gain insight into the mechanism of iron reduction and the associated microbial processes in deep sea methanic sediment by setting up long-term high-pressure incubation experiments supplemented with ferrihydrite and methane. Continuous iron reduction was observed during the entire incubation period. Intriguingly, ferrihydrite addition shifted the archaeal community from the dominance of hydrogenotrophic methanogens (Methanogenium) to methylotrophic methanogens (Methanococcoides). The enriched samples were then amended with 13C-labeled methane and different iron (oxyhydr)oxides in batch slurries to test the mechanism of iron reduction. Intensive iron reduction was observed, the highest rates with ferrihydrite, followed by hematite and then magnetite, however, no anaerobic oxidation of methane (AOM) was observed in any treatment. Further tests on the enriched slurry showed that the addition of molybdate decreased iron reduction, suggesting a link between iron reduction with sulfur cycling. This was accompanied by the enrichment of microbes capable of dissimilatory sulfate reduction and sulfur/thiosulfate oxidation, which indicates the presence of a cryptic sulfur cycle in the incubation system with the addition of iron (oxyhydr)oxides. Our work suggests that under low sulfate conditions, the presence of iron (oxyhydr)oxides would trigger a cascade of microbial reactions, and iron reduction could link with the microbial sulfur cycle, changing the kinetics of the methanogenesis process in methanic sediment.

Methanococcoides orientis sp. nov., a methylotrophic methanogen isolated from sediment of the East China Sea.

A novel methylotrophic methanogen Methanococcoides orientis sp. nov. was isolated from East China Sea sediment. Type strain LMO-1T of Methanococcoides orientis sp. nov. was irregular 1-2 µm cocci without flagella. Strain LMO-1T could utilize a variety of methylated compounds including methanol, methylamine, dimethylamine and trimethylamine for growth and methanogenesis, while H2/CO2 or acetate could not be used for growth or methanogenesis. Optimum growth temperature was 30-35 °C, optimum pH range for growth was 7.0-7.5, while the optimum salinity spectrum for growth was 1.0%-5.0% NaCl. Based on 16S rRNA gene similarity, strain LMO-1T belongs to Methanococcoides, with the highest sequence similarity to Methanococcoides methylutens DSM 2657T (99.8 %), Methanococcoides vulcani SLH33T(99.4 %), followed by Methanococcoides alaskense AK-5T(98.1 %), Methanococcoides burtonii DSM 6242T (98.0 %). Digital DNA-DNA hybridization also showed highest similarity with Methanococcoides methylutens DSM 2657T, with the value of 58.4 %. The average nucleotide identity between strain LMO-1T and Methanococcoides methylutens DSM 2657T was 94.06 %. In summary, LMO-1T represents a novel species of the genus Methaococcoides, for which the name Methanococcoides orientis sp. nov. is proposed. The type strain is LMO-1T (=MCCC 4K00106T=JCM 39195T).

Draft Genome Sequences of Four Bacterial Strains Isolated from Sediment of the South China Sea.

Here, we report the draft genome sequences of four bacterial isolates from sediment of the South China Sea. Three of the isolates belong to the class Alphaproteobacteria and encode complete SoxXAYZBCD gene clusters, related to thiosulfate oxidation, while one isolate belongs to the class Opitutae and possesses a total of 397 carbohydrate active enzymes (CAZymes), related to predicted polysaccharide degradation.

Metal-dependent anaerobic methane oxidation in marine sediment: Insights from marine settings and other systems.

Anaerobic oxidation of methane (AOM) plays a crucial role in controlling global methane emission. This is a microbial process that relies on the reduction of external electron acceptors such as sulfate, nitrate/nitrite, and transient metal ions. In marine settings, the dominant electron acceptor for AOM is sulfate, while other known electron acceptors are transient metal ions such as iron and manganese oxides. Despite the AOM process coupled with sulfate reduction being relatively well characterized, researches on metal-dependent AOM process are few, and no microorganism has to date been identified as being responsible for this reaction in natural marine environments. In this review, geochemical evidences of metal-dependent AOM from sediment cores in various marine environments are summarized. Studies have showed that iron and manganese are reduced in accordance with methane oxidation in seeps or diffusive profiles below the methanogenesis zone. The potential biochemical basis and mechanisms for metal-dependent AOM processes are here presented and discussed. Future research will shed light on the microbes involved in this process and also on the molecular basis of the electron transfer between these microbes and metals in natural marine environments.