[Objective] To obtain the enriched groups of target microorganisms from the natural environment of the study area and establish the pure culture, improve the key link of the basic research on bio-geochemistry in methane leakage areas, and provide ideas and references for the enrichment and culture of other unknown microorganisms. [Methods] Microorganisms in marine sediments from methane leakage areas were isolated and cultured, and a sound experimental methodology for marine microorganisms was refined, including on-site treatment of microbial samples, preparation and sterilization of anaerobic culture media, and enrichment, culture, and isolation of microorganisms. High-throughput sequencing of microorganisms was conducted for gene sequencing and microbial identification, and different experimental conditions and experimental cycles were designed for in vivo microbial culture experiments. The short-term experiment (1.5 d) was conducted with the single factor method to study the effects of environmental factors (light, medium concentration, temperature, and pH) on microorganisms. The medium-term experiment (22 d) verified the results of the short-term experiment and determined the more suitable culture conditions. The long-term experiment (more than 250 d) was carried out to further study the growth status and activity of enriched microbial groups under specific conditions by real-time quantitative tracking of microorganisms. [Results] In the short-term culture, the activity of anaerobic sludge microbial suspension significantly increased under natural light (reached the peak at the time point of 17 h, with the iron concentration of 16.7 mg/L, four times the initial value), while the buffer period was prolonged in the dark environment (0-15 h). The activity of marine sediment microbial suspension was better in the dark environment (with high values at time points of 14 h/35 h), and the activity in the acid/base environment (pH 5.0/9.0) was higher than that in the neutral environment (iron concentrations of 4.3 mg/L vs. 11.1 mg/L, respectively). In the medium-term culture, the activity of anaerobic sludge microbial suspension was stable under 4 ℃ and acid conditions (with the decrease of only 20% in iron concentration), while the marine sediment microbial suspension preferred higher temperature and alkaline environment (with the activity increased after adaptation to pH 9.0). The global analysis showed that the first 15 days were the temperature adaptation period, and then temperature became the key regulatory factor. In the long-term culture, the activity of anaerobic sludge microbial suspension fluctuated periodically (first decreasing and then increasing after change of the culture medium every 50 days), while it declined irreversibly after 150 d. The marine sediment microbial suspension showed strong adaptability (with the activity peaked on days 117-145 under high pressure and the estimated doubling cycle of about 130 d) and maintained serrated stable activity under room pressure (iron concentration of 3.0-10.4 mg/L). [Conclusion] Anaerobic sludge microorganisms are sensitive to light and medium concentration. Their activity is improved by short-term light but inhibited by long-term light. Dark environment and 100% concentration medium are more suitable for growth of anaerobic sludge microorganisms (4 ℃, acidic environment, doubling cycle of 15 d). However, marine sediment microorganisms under the dark+100% medium and high temperature+alkaline environment conditions demonstrate stronger adaptability. Although their short-term activity is less affected by light, it takes about 130 days to double, and the adaptability to the high pressure environment significantly affects the growth process.