【摘要】：通過厭氧微生物作用,將油藏殘余油或重質(zhì)原油降解并轉(zhuǎn)化為甲烷(“油變氣”)進行氣態(tài)開采,是一項極具發(fā)展?jié)摿Φ奈⑸锊捎托录夹g,這個過程需要通過細菌和古菌的互營代謝作用完成。我們小組通過多年努力,富集獲得了正十六烷烴降解產(chǎn)甲烷菌系M82,利用穩(wěn)定同位素探針技術,證實其中一類未培養(yǎng)Syntrophaceae是產(chǎn)甲烷條件下互營降解正十六烷烴的關鍵細菌類群。但是這類互營菌生長緩慢,一個培養(yǎng)周期需要數(shù)月甚數(shù)年的時間,其分離難度極大,迄今尚未見到互營烴降解菌的分離報道,這限制了人們開展互營烴降解菌的分子機理研究。本研究通過454高通量測序擴增細菌和古菌16S rRNA基因,發(fā)現(xiàn)中溫正十六烷烴降解產(chǎn)甲烷菌系M82中的古菌主要屬于甲烷袋狀菌屬(82%),互營菌科下的Smithella(55%)在細菌域中的豐度最高。利用高通量測序技術擴增菌系全基因組DNA,獲得40 Gb的短片段序列,通過序列拼接獲得144,759條scaffolds,N50 scaffold長度為4619 bp。其中片段豐度大于30000,GC摩爾含量約43%的高豐度片段聚集成團(cluster),根據(jù)序列相似性分析,發(fā)現(xiàn)這些序列大部分屬于Proteobacteria,進一步通過16S rRNA基因相似性分析,發(fā)現(xiàn)這個cluster代表的是未培養(yǎng)互營烴降解菌Syntrophaceae(Clone HB1_11,GenBank No.of JX088314,相似性99.7%)。去掉該cluster低豐度以及分類水平上為古菌的序列后,該cluster中存在104個單拷貝基因(SCG),表明其基因組序列的完整度大于97%�；谒暮塑账岫鄳B(tài)性(TNF)的主成成分分析(PCA)顯示聚類出來為一個物種。該基因組大小為2.75 Mb,GC含量為42.8%。該菌株代表一個新的未培養(yǎng)物種,命名為‘Candidatus Smithella cisternae’,菌株名定為M82_1。通過基因注釋發(fā)現(xiàn)菌株M82-1有2815個ORFs,47個tRNA和3個rRNA。其中50%以上的基因能與KEGG、COG、GO和Pfam數(shù)據(jù)庫中的序列相匹配。菌株M82_1具有編碼延胡索酸還原酶、甲基丙二酰輔酶A變位酶和羧基轉(zhuǎn)移酶的基因序列,完整的降解長鏈脂肪酸和丁酸的β-氧化途徑的基因序列,表明該菌可能通過延胡索酸激活啟動烷烴的降解。該菌編碼代謝丙酸和乙酸的途徑的基因不完整,以及羧酸循環(huán)、糖酵解和磷酸戊糖途徑相關基因的不完整,這表明菌株M82_1可利用的底物范圍有限。注釋出Nif基因簇推測該菌可能具有固氮能力。我們獲得未培養(yǎng)互營烷烴降解菌的基因組草圖,并推測分析其遺傳代謝潛力,這將為深入研究互營烴降解的分子機理研究提供基礎。
[Abstract]:The degradation of residual oil or heavy crude oil into methane ("oil to gas") is a new microbial oil recovery technology with great potential for development through the degradation of residual oil or heavy crude oil into methane ("oil to gas"). This process needs to be completed by the mutual metabolism of bacteria and archaea. Through many years of efforts, our team has enriched the methanogenic strain M82 of n-hexadecane. By using stable isotopic probe technique, it is confirmed that one of the uncultured Syntrophaceae is the key bacterial group for the mutual degradation of n-hexadecane under methanogenic conditions. However, the growth of this kind of mutual bacteria is slow, and a culture cycle takes several months or several years, and its isolation is very difficult. Up to now, there have been no reports of the isolation of mutual hydrocarbon degradation bacteria, which limits the study of the molecular mechanism of mutual hydrocarbon degradation bacteria. In this study, the 16s rRNA gene of bacteria and archaea was amplified by high throughput sequencing. It was found that the archaea in methanogenic strain M82 were mainly belong to the genus methane bagged bacteria (82%), and the abundance of Smithella (55%) in the bacterial domain was the highest. The short fragment sequence of 40 Gb was obtained by amplification of the whole genome DNA, of the strain by high throughput sequencing technique, and 144759 scaffolds,N50 scaffold with a length of 4619 bp. were obtained by sequence splicing. According to the sequence similarity analysis, it was found that most of these sequences belonged to Proteobacteria, and further analyzed the similarity of 16s rRNA gene, and it was found that this cluster represented 99.7% similarity of uncultured mutual hydrocarbon degradation bacteria Syntrophaceae (Clone HB1_11,GenBank No.of JX088314,). The abundance of fragments was more than 3000, and the high abundance fragments with GC molar content of about 43% were aggregated into clusters. According to the sequence similarity analysis, it was found that most of these sequences belonged to Proteobacteria, and further analyzed the similarity of 16s DNA gene. After removing the sequence of archaea with low abundance and classification level of the cluster, there were 104 single copy gene (SCG), in the cluster, indicating that the integrity of the genome sequence was more than 97%. Principal component analysis (PCA) based on tetra-nucleotides polymorphism (TNF) showed that clustering came out as a species. The genome size was 2.75 Mb,GC and the content was 42.8%. The strain represents a new uncultured species named 'Candidatus Smithella cisternae', strain M82 鈮，

本文編號：2503465