黄铁矿生物浸出菌群的驯化及其优势物种与功能

Pyrite-bioleaching microbial community: domestication, identification of dominant species, and characterization of functions

摘要:

【背景】生物浸矿效果与浸矿过程中微生物群落的结构变化密切相关，解析浸矿过程中的微生物群落结构和功能对于进一步认识微生物在浸矿过程中的作用、构建高效菌群具有重要意义。【目的】通过驯化获得稳定高效浸出黄铁矿的微生物菌群，并解析其菌群结构和功能。【方法】以黄铁矿为能源来源将微生物菌群在9K培养基中长期传代转接，对比驯化前后菌群的铁硫氧化能力，利用宏基因组测序结合16S rRNA基因克隆文库对驯化后的菌群进行群落结构解析，通过分化培养实验，探究驯化后菌群中优势微生物在浸出黄铁矿过程中的功能，结合宏基因组功能注释结果分析驯化前后菌群功能基因的变化。【结果】通过为期10代的转接驯化，获得了组成稳定、浸矿功能提升，更加适应黄铁矿浸出环境的微生物菌群。基于宏基因组的结果，驯化后的菌群相对于驯化前，嗜酸硫杆状菌属(Acidithiobacillus)和硫化杆菌属(Sulfobacillus)的比例均上升；16S rRNA基因克隆文库的检测结果显示，驯化后菌群主要由氧化硫硫杆菌(Acidithiobacillus thiooxidans)和耐热硫化芽胞杆菌(Sulfobacillus thermotolerans)这2种微生物组成。驯化后菌群的铁氧化能力提升15.15%，硫氧化能力提升70.86%。对菌群的浸矿效果和组成它的优势种属单独的浸矿效果进行比较，以浸出黄铁矿中总铁的含量计算，驯化后5Biol菌群16 d的浸出率可达81.18%，氧化硫硫杆菌(A.thiooxidans)和耐热硫化芽胞杆菌(S.thermotolerans)单独的浸出率分别为51.86%和37.65%，5Bio菌群的浸矿能力优于组成它的优势种属的单独浸矿能力，说明菌群内优势种属之间可能存在协同作用，使得其浸矿效果达到了单物种不能达到的效果。通过分化培养后菌群组成的变化，判断氧化硫硫杆菌(A.thiooxidans)的主要作用是硫的氧化，而耐热硫化芽胞杆菌(S.thermotolerans)的主要作用是铁的氧化，耐热硫化芽胞杆菌(S.thermotolerans)需要依赖氧化硫硫杆菌(A.thiooxidans)的存在才能发挥铁氧化能力，说明菌群中不同物种可能通过协同合作共同提高了浸出黄铁矿的能力，这与5Biol菌群驯化后嗜酸硫杆状菌属(Acidithiobacillus)和硫化杆菌属(Sulfobacillus)的比例提高、浸矿能力得到增强是一致的。对驯化前后与铁硫代谢相关的功能基因进行分析，发现驯化后菌群铁的氧化主要通过sulfocyanin-like蛋白实现，硫的氧化主要通过sox系统、sor系统和非sox子系统实现。【结论】通过长期驯化传代的方法，可以获得组成稳定、功能强化的高效浸矿菌群。由于长期驯化菌群更加稳定，更容易解析其中不同菌种在执行浸矿功能中的作用。这对高效浸矿菌群的构建具有重要的指导意义。

关键词:黄铁矿浸出;驯化;浸矿菌群;氧化硫硫杆菌;耐热硫化芽胞杆菌;协同作用

Abstract:

[Background]The bioleaching performance is closely related to the microbial community structure and its dynamics during the bioleaching process. Understanding the changes in microbial community structure and function during the bioleaching process is crucial for elucidating the role of microorganisms in this process and building efficient microbial combinations.[Objective] To obtain the microbial community with stable and high pyrite-leaching efficiency by domestication, and analyze the structure and function of the microbial community. [Methods]The microbial community was subcultured in the 9K medium with pyrite as the energy source. The iron and sulfur oxidation capacity of the microbial community was measured before and after domestication. The structure of the domesticated microbial community was analyzed by metagenomic sequencing combined with 16S rRNA gene clone library. Differentiation culture experiments were carried out to study the functions of dominant microorganisms in the domesticated microbial community during pyrite bioleaching. The functional annotation of metagenomic sequencing results was carried out to analyze the functional changes of the microbial community before and after domestication.[Results]The microbial community with a stable structure, improved bioleaching ability, and adaptability to the pyrite leaching environment was obtained after domestication. The metagenomic sequencing results showed that the relative abundance of the genus Acidithiobacillusand Sulfobacillus were both increased. Detection results from the 16S rRNA gene clone library showed that the domesticated microbial community mainly consisted of Acidithiobacillus thiooxidans and Sulfobacillus thermotolerans, with the iron and sulfur oxidation capacity increasing by 15.15% and 70.86%, respectively. The leaching effects were compared between the bacterial community and the dominant species alone. In terms of total iron leached from pyrite, the 16-day leaching rate of the domesticated community 5Biol reached 81.18%, while those of A.thiooxidans and S.thermotolerans were 51.86% and 37.65%, respectively. The better leaching ability of 5Biol than that of single dominant species indicated that the existence of synergistic effect between dominant species in the community, which endowed 5Biol with the leaching effect that a single species cannot achieve. The differentiation culture experiments showcased that A.thiooxidans primarily contributed to sulfur oxidation, while S.thermotolerans was mainly responsible for iron oxidation. S.thermotolerans was dependent on A.thiooxidans for iron oxidation. This indicated that different strains within the community may collaborate synergistically, leveraging their respective strengths to enhance the overall leaching efficiency. This is consistent with the increases in the relative abundance of Acidithiobacillus and Sulfobacillus and the enhancement of leaching ability of 5Biol after domestication. After domestication, sulfocyanin-like proteins were the key proteins involved in iron oxidation, while sulfur oxidation included the sox system, sor system, and non-sox subsystems. [Conclusion] Long-term domestication by passages with a specific ore can yield a stable microbial community with enhanced bioleaching performance. The simplified stable microbial community makes it easy to elucidate the functional roles of different microbial species. The findings provide insights into the construction of efficient microbial communities for bioleaching.

Key words:pyrite leaching;domestication;microbial community for bioleaching;Acidithiobacillus thiooxidans;Sulfobacillus thermotolerans;synergistic effect