Traditional cultivation methods with defined growth media can only isolate and cultivate a small number of microbes. However, much higher microbial diversity has been detected by cultivation-independent tools from a range of natural ecosystems. These represent a large unexplored pool of potentially novel taxa. In this study, a diffusion-based integrative cultivation approach (DICA) was developed to efficiently isolate novel taxonomic candidates from marine sediment. DICA combined a newly designed diffusion-based apparatus called a "microbial aquarium" with modified low-nutrient media. To determine the efficiency of DICA, cultivation results were compared with traditional cultivation approach (TCA). Both cultivation approaches resulted in the isolation of numerous representatives from the phyla Pseudomonadota, Actinomycetota, Bacteroidota, and Bacillota. However, the newly developed DICA also led to the successful cultivation of species from rarely cultivated phyla such as Verrucomicrobiota and Balneolota. Based on 16S rRNA analyses, the application of DICA resulted in the successful cultivation of 115 previously uncultured taxa out of a total of 196 isolates. Among these, 39 were identified at the genus level and 4 at the family level, showcasing a novelty ratio of 58%. Conversely, the TCA cultivated 12% (20/165) of novel isolates, with all at species level only. The isolated microbial diversity showed that species recovered by DICA belong to 12 different classes, twice the number produced by TCA. Overall, these results demonstrate that the newly designed DICA produces a high recovery of diverse and previously uncultured bacteria.

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.