环境对甲烷渗漏区海洋沉积物微生物生长特性的影响

Environmental factors influence the microbial growth characteristics in marine sediments of methane leakage areas

摘要:

目的 从研究区域自然环境中获得目标微生物富集体并建立纯培养，完善甲烷渗漏区生物地球化学基础研究的关键环节，为其他未知微生物的富集与培养提供思路和借鉴。方法 对甲烷渗漏区域海洋沉积物微生物进行分离和培养，提炼一套较为完善的海洋微生物实验方法，包括微生物样品现场处理、厌氧培养基配制与灭菌、微生物富集培养与分离等。利用微生物高通量测序技术进行基因测序与菌群鉴定，并设置不同的实验条件和实验周期，开展微生物活体培养实验。短期实验(1.5 d)采用单因子变量方法研究环境因素(光照、培养基浓度、温度、酸碱度)对微生物的影响；中期实验(22 d)验证短期实验结果，并确定更适宜的培养条件；长期实验(250 d以上)通过对微生物实时定量追踪，进一步研究微生物富集体在特定条件下的生长状况和活性表达。结果 短期培养中，厌氧污泥菌液在自然光照下活性显著提升(17 h达峰值，铁浓度16.7 mg/L，约为初始浓度4倍)，黑暗环境则延长其缓冲期(0-15 h)，而海洋底泥菌液在黑暗环境中活性更优(14 h/35 h出现高值)，且在酸碱环境(pH 5.0/9.0)后期活性高于中性(铁浓度分别约为4.3 mg/L vs. 11.1 mg/L)。中期培养阶段，厌氧污泥菌液在4 ℃+酸性条件下活性稳定(铁浓度降幅仅为20%)，而海洋底泥菌液偏好较高温度及碱性环境(pH 9.0适应后活性提升)。全局分析显示，前15天为温度适应期，此后温度成为关键调控因子。长期培养中，厌氧污泥菌液活性呈周期性波动(每50 d更换培养基引发先降后升)，但150 d后出现不可逆衰减；海洋底泥菌液则表现出强适应性(高压下117-145 d活性达峰值，推测生长周期约为130 d)，且在室压下维持锯齿状稳定活性(铁浓度3.0-10.4 mg/L)。结论 厌氧污泥微生物对光照和培养基浓度敏感，短期光照促进活性，但长期抑制，黑暗及100%浓度培养基更适其生长(4 ℃酸性环境，15 d生长周期)；而海洋底泥微生物在黑暗+100%培养基、高温碱性环境下适应性更强，虽短期活性受光照影响小，但需约130 d生长周期，其高压环境适应性显著影响生长进程。

关键词:甲烷渗漏区;厌氧微生物;微生物培养实验

Abstract:

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.

Key words:methane leakage areas;anaerobic microorganisms;microbial culture experiments