Sub-seafloor sediment is the largest microbial habitat on Earth. The study of microbes in sub-seafloor sediment is largely limited by the technical challenge of acquiring ambient microbial DNA because of sediment heterogeneity. Changes in the extraction method, even just by one step, can affect the extraction yields for complicated sediment samples. In this work, sub-seafloor sediment samples from the Baltic Sea with high organic carbon content were used to evaluate the influence of different grinding beads on DNA extraction. We found that the grinding beads can affect the DNA extraction from the organic-matter- and biosiliceous-clay-rich samples. A mixture of 0.5-mm and 0.1-mm grinding beads exhibited higher DNA yields and recovered more unique taxa than other bead combinations, such as Stenotrophomonas from Gammaproteobacteria and Leptotrichia from Fusobacteria; therefore, these beads are more suitable than the others for DNA extraction from the samples used in this study. This advantage might be magnified in samples with high biomass. On the contrary, the use of only small beads might lead to underestimation for certain Gram-positive strains. Overall, the discovery of abundant widespread deep biosphere clades in our samples indicated that our optimized DNA extraction method successfully recovered the in situ microbial community.

Mangrove forests are intertidal ecosystems that harbor diverse microbial communities essential for biogeochemical cycles and energy flow. This study investigated the seasonal and spatial patterns of bacterial communities in the artificially introduced mangrove sediments of the Ao River estuary using 16S rRNA gene amplicon high-throughput sequencing. Alpha diversity analyses indicated that the bacterial community diversity in the mangrove sediments of the Ao River estuary was similar to those of natural-formed mangroves, with the Shannon index ranging from 5.16 to 6.54, which was significantly higher in winter compared to other seasons. The dominant bacterial phyla included Proteobacteria (43.65%), Actinobacteria (11.55%), Desulfobacterota (11.16%), and Bacteroidetes (5.52%), while beta diversity analysis revealed substantial differences in bacterial community structure across different seasons and regions. For instance, the relative abundance of Woeseiaceae and Bacteroidota during the summer was significantly higher than that observed in other seasons. And the relative abundance of Bacillaceae in autumn and winter increased by one order of magnitude compared to spring and summer. Woeseiaceae, Desulfobulbaceae, Thermoanaerobaculaceae, and Sva1033 (family of Desulfobacterota) exhibited significantly higher relative abundance in the unvegetated area, whereas Bacillaceae and S085 (family of Chloroflexi) demonstrated greater abundance in the mangrove area. Seasonal variations in bacterial community structure are primarily attributed to changes in environmental factors, including temperature and salinity. Regional differences in bacterial community structure are primarily associated with environmental stressors, such as wave action, fluctuations in salinity, and organic matter content, which are further complicated by seasonal changes. This study is significant for understanding the microbial diversity and seasonal dynamics of estuarine mangrove wetlands, and it contributes to the assessment of mangrove wetland restoration efforts in Zhejiang Province, providing important guidance for the development of strategies to maintain the health of mangrove ecosystems in the future.