Microbial diversity of alkane- and plastic-degrading microbiome in offshore sediments of Ross Sea, Southern Ocean
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摘要: 石油污染以及塑料垃圾对海洋生态安全具有严重威胁，甚至在南大洋的罗斯海地区也发现石油污染和微塑料的存在。本研究为了获得该地区的低温烷烃降解菌和塑料降解菌，通过采集自南大洋罗斯海地区的12个沉积物样品用于富集分离南大洋低温烷烃降解菌株，结果表明十四烷富集菌群优势属主要包括假单胞菌属（Pseudomonas）、食烷菌属（Alcanivorax）、海单胞菌属（Marinomonas）、假交替单胞菌属（Pseudoalteromonas）等。进一步利用分离获得的烃类富集菌对聚对苯二甲酸乙二醇酯（PET）和聚乙烯（PE）进行降解验证，扫描电子显微镜（SEM）以及傅里叶变换红外光谱技术（ATR-FTIR）证明了Pseudomonas pelagia R1-05-CR3、Pseudomonas taeanensis A11-04-CA4、Halomonas titanicae A11-02-7C2和Rhodococcus cerastii R1-05-7C3这4株细菌对PE可进行有效降解。高效液相色谱质谱技术（UPLC-MS）和SEM结果表明R. cerastii R1-05-7C3、Microbacterium maritypicum RA1-00-CA1、H. titanicae A11-02-7C2对PET塑料具有降解能力。研究结果表明南大洋罗斯海近海沉积物中存在多样性的低温烃类及塑料降解菌，在原位环境污染中发挥自净作用，同时也为低温下烃与塑料污染生物降解提供了菌种资源。Abstract: Oil and plastic pollutants are a serious threat to marine ecosystems and have even been found in the Ross Sea of the Southern Ocean. In order to obtain low-temperature alkane degrading bacteria and plastic-degrading bacteria in the region, a total of twelve sediment samples were collected in the Ross Sea area for enrichment and isolation of alkane-degrading bacteria at low-temperature, and the diversity analyses of the tetradecane-enriched communities showed that the most dominant genera were Pseudomonas, Alcanivorax, Marinomonas, Pseudoalteromonas. The polyethylene terephthalate(PET)and polyethylene(PE)was further validated using the dominant alkane-degrading bacteria. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (ATR-FTIR) demonstrated that the four pure cultures of Pseudomonas pelagia R1-05-CR3, Pseudomonas taeanensis A11-04-CA4, Halomonas titanicae A11-02-7C2 and Rhodococcus cerastii R1-05-7C3 could degrade PE effectively. The results of UPLC-MS and SEM confirmed the PET degradation by isolates of R. cerastii R1-05-7C3, Microbacterium maritypicum RA1-00-CA1, and H. titanicae A11-02-7C2. In conclusion, this study reports the diversity of the tetradecane-enriched consortia at low-temperature and plastic degrading bacteria in the offshore sediments of the Ross Sea in the Southern Ocean, which play a selfpurifying role in in-situ environmental contamination, and also provided strain resources for the biodegradation of hydrocarbon and plastic contaminants at low temperature.
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Key words:
- Ross Sea /
- alkanes /
- polyethylene /
- polyethylene terephthalate /
- biodegradation /
- cold-adapted microorganisms
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图 1 罗斯海沉积物样品采样站位图
左侧图片来自维基百科
https://www.163.com/dy/article/GHUO74P105526RB5.html Fig. 1 Location map of sediment sampling stations from the Ross Sea
Left image from Wikipedia
https://www.163.com/dy/article/GHUO74P105526RB5.html 
图 2 罗斯海烷烃富集菌群细菌Alpha多样性分析
OTU稀释曲线（a）OTU水平Venn图分析（C0/C1/C2）（b）Shannon指数和Chao指数组间差异分析，Student-t检验（C0/C1/C2）（c1, c2）
Fig. 2 Alpha diversity analysis of tetradecane-enriched consortia from Ross Sea sediments
Rarefaction curves for Sobs index on OTU level (a), Venn diagram analysis on OTU level to depict the number of exclusive and shared OTUs (C0/C1/C2) (b), analysis of intergroup differences in Shannon index and Chao index on OTU level, Student-t test (C0/C1/C2) (c1, c2)
图 3 罗斯海十四烷富集菌群C0/C1/C2组门水平（a），科水平（b）和属水平（c）细菌组成柱形图
Fig. 3 Column diagram of bacterial percent of community abundance atphylum level (a), family level (b), and genus level (c) of tetradecane-enriched consortia of C0/C1/C2 groups from Ross Sea
图 4 罗斯海十四烷富集菌群属水平细菌组成柱形图（a: C0；b: C1, C2）
Fig. 4 Column diagram of bacterial percent of community abundance at genus level of tetradecane-enriched consortia (a: C0; b: C1, C2) from Ross Sea
图 5 罗斯海烷烃富集菌群属水平细菌物种组成差异分析（C0/C1/C2），Kruskal-Wallis秩和检验（a）及OTU水平系统进化分析（C1/C2）（b）
左边为系统发生进化树，进化树中每条树枝代表一类物种，树枝长度为两个物种间的进化距离，右边柱状图展示的是物种在不同分组中的的Reads占比
Fig. 5 Analysis of intergroup differences of community abundance on genus level (C0/C1/C2)，Kruskal-Wallis H test (a) and phylogenetic analysis at OTU level of tetradecane-enriched consortia from Ross Sea (C1/C2) (b)
Each branch of the evolutionary tree on the left represents a OTU, and the length of the branch is the evolutionary distance between two OTUs，the right bar chart shows the proportion of Reads of OTUs in different groups
图 6 细菌Hallmonas titanicae A11-02-7C2，Rhodococcus cerastii R1-05-7C3，Pseudomonas pelagia R1-05-CR3和Pseudomonas taeanensis A11-04-CA4解聚PE的表征特性
左侧图片：菌株生物降解后的PE膜和未接种的PE膜（CK）相比有生物膜附着（a, c）和破损（b）形成。右侧图像：细菌降解后塑料和未接种的PE膜（CK）的 FTIR 分析（d）
Fig. 6 Depolymerization characteristics of the biodegraded PE by the isolated bacterium Hallmonas titanicae A11-02-7C2, Rhodococcus cerastii R1-05-7C3, Pseudomonas pelagia R1-05-CR3 and Pseudomonas taeanensis A11-04-CA4
Left image: the inoculated bacterium attached to PE to form the biofilms (a, c) and visible pores (b) compared with the non-inoculated PE (CK). Right image: FTIR analysis of incubated PE by corresponding strains and non-inoculated PE (CK) (d)
图 7 细菌解聚PET的表征特性
左侧图片：菌株Rhodococcus cerastii R1-05-7C3, Microbacterium maritypicum RA1-00-CA1和H. alomonas titanicae A11-02-7C2生物降解后的PET膜和未接种的PET膜（CK）相比出现生物膜和明显的破损（a）。右侧图像：Rcerastii R1-05-7C3和Maritypicum RA1-00-CA1处理PET 30 d和无细菌处理组（CK）的代谢产物（预测为MHET）和MHET标准品的UPLC出峰时间图（b）
Fig. 7 Depolymerization characteristics of the biodegraded PET by the isolated bacterium
Left image: the inoculated bacterium Rhodococcus cerastii R1-05-7C3, Microbacterium maritypicum RA1-00-CA1 and Halomonas titanicae A11-02-7C2 attached to PET to form the biofilms and visible pores compared with the non-inoculated PET (a). Right image: UPLC spectrum (b) of the standard mono-(2-hydroxyethyl) terephthalate (MHET) and no bacteria-treat group (CK) and the metabolic products (prediction as MHET) released from PET films treated by the pure culture R. cerastii R1-05-7C3 and M. maritypicum RA1-00-CA1 after 30 days
表 1 具体采样点站位信息
Tab. 1 Location information of specific sampling stations
站位名称 取样时间 纬度 经度 水深/m R1-02 2020年1月4日 74º59'S 164º59'E 893.4 R1-05 2020年1月4日 74º59'S 170º24'E 330.5 R1-07 2020年1月5日 75º00'S 175º16'E 285.5 RA1-00 2020年1月10日 75º27'S 149º57'W 3241.5 RA2-01A 2020年1月13日 74º23'S 141º34'W 503.3 RA3-02 2020年1月13日 74º28'S 140º18'W 482 RA3-03 2020年1月14日 74º50'S 139º38'W 2560.8 A3-01 2020年1月29日 73º00'S 119º50'W 405.8 A11-04 2020年1月25日 72º10'S 117º50'W 502.1 A11-02 2020年1月26日 72º58'S 115º03'W 659.5 A11-01 2020年1月26日 73º26'S 113º31'W 622.6 A4-03 2020年1月27日 72º42'S 112º24'W 438.2 
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表 2 验证纯菌株对烷烃，PET和PE降解能力
Tab. 2 Verify the degradation ability of pure strains on alkanes, PET and PE
烷烃降解 PET 降解 PE降解 菌株名称 热门分类单元 所属科 w - + A11-04-CA4 Pseudomonas taeanensis MS-3T Pseudomonadaceae + + - RA1-00-CA1 Microbacterium maritypicum DSM 20578 TMicrobacteriaceae + + + A11-02-7C2 Halomonas titanicae BH1T Halomonadaceae + + + R1-05-CR3 Pseudomonas pelagia CL-AP6T Pseudomonadaceae + + w A11-02-CR2 Pseudomonas xanthomarina DSM 18231 TPseudomonadaceae + + + A3-01-CR8 Psychrobacter okhotskensis MD17T Moraxellaceae + + + R1-05-7C3 Rhodococcus cerastii C5T Nocardiaceae - - - A11-02-CA2 Marinomonas rhizomae IVIA-Po-145T Oceanospirillaceae - - - A11-04-CA2 Pseudoalteromonas neustonica PAMC 28425 TPseudoalteromonadaceae - - - A4-03-CR4 Pseudomonas neustonica SSM26T Pseudomonadaceae + - - A3-01-7C5 Rhodococcus erythropolis NBRC 15567 TNocardiaceae - - - A11-02-CA1 Shewanella livingstonensis LMG 19866 TShewanellaceae + + + A11-02-7C4 Alcanivorax borkumensis SK2T Alcanivoracaceae w + w R1-05-7C2 Pseudomonas zhaodongensis NEAU-ST5-21T Pseudomonadaceae 注：-, 不降解；w，降解效果微弱；+，降解效果良好。
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