舟山群島海域沉積物厭氧氨氧化細菌多樣性

張東聲, 劉鎮盛, 張海峰, 王小谷, 王春生,2

1 國家海洋局海洋生態系統與生物地球化學重點實驗室, 杭州 310012 2 衛星海洋環境動力學國家重點實驗室, 杭州 310012

舟山群島海域沉積物厭氧氨氧化細菌多樣性

張東聲1,*, 劉鎮盛1, 張海峰1, 王小谷1, 王春生1,2

1 國家海洋局海洋生態系統與生物地球化學重點實驗室, 杭州 310012 2 衛星海洋環境動力學國家重點實驗室, 杭州 310012

通過構建16S rRNA基因文庫和克隆測序研究了舟山海域沉積物中厭氧氨氧化細菌(AAOB)的多樣性。從5個克隆文庫中共獲得297條16S rRNA基因序列，包含16個操作分類單元(OTUs)。離岸距離較近的3個站具有相似的群落結構，且與離岸較遠的2個站具有明顯差異。系統發育結果顯示，Scalindua屬是該海域AAOB的優勢類群，95.3%的序列與Scalindua屬AAOB具有較近的親緣關系；1條序列與Kuenenia屬具有較近的親緣關系；此外還有15條序列與已知的AAOB相似性較低。相關性分析表明沉積物有機碳含量與多樣性指數具有顯著正相關，可能是該海域AAOB多樣性變化的重要影響因子。

厭氧氨氧化細菌(AAOB); 舟山群島; 海洋沉積物; 多樣性

微生物的硝化作用能夠將環境中的銨鹽和亞硝酸鹽轉化成硝酸鹽[1]，因而對保持河口等生境的水質和生態健康具有重要作用。然而，近年來河口生態系統的季節性缺氧現象越發嚴重，其出現的頻率、范圍、持續時間和強度都呈現持續上升的趨勢[2]。低氧容易導致好氧的硝化作用中斷，使具有生物毒性的亞硝酸鹽大量積聚，進而引發一系列生態和環境問題[3]。

厭氧氨氧化(Anaerobic ammonium oxidation, anammox)是由一類微生物主導，在缺氧條件下以亞硝酸鹽為底物進行氨氧化并產生氮氣的過程[4]。當硝化作用受到阻礙時，厭氧氨氧化能夠有效地降低亞硝酸鹽的濃度，有助于保持水質，維持生態系統的健康。厭氧氨氧化作用最初在廢水處理裝置中發現[5]，主要受浮霉菌門的一類化能自養細菌調控，這類細菌很難從自然環境中分離純化[6]，目前已有描述的包括5個屬(Brocadia,Kuenenia,Scalindua,Anammoxoglobus和Jettenia)[7-9]。厭氧氨氧化細菌(AAOB)在自然環境中的分布非常廣泛，在淡水沉積物[10-11]、海水沉積物[12]和低氧海水[13]都有發現，厭氧氨氧化作用是水生環境中維持氮平衡的一個重要途徑。

舟山群島位于長江口外海域，受海洋和長江徑流的影響，屬于咸淡水生態系統的交錯群落，具有獨特的氮元素生物地球化學循環特征，是厭氧氨氧化作用的重要發生場所。近年來長江口外夏季低氧現象愈加嚴重，在夏季整個舟山漁場幾乎都被低氧區所覆蓋[14-15]。低氧現象嚴重危害該海域的漁業資源和生態系統健康、破壞該海域的海洋經濟[2]。Dang等人曾對該海域沉積物中的氨氧化古菌進行過報道[16]，但目前還未有關于舟山群島海域低氧區AAOB的研究。本文采用16S rRNA基因文庫構建和克隆測序法，研究夏季舟山海域沉積物中的AAOB多樣性，旨在初步了解其種類組成和分布情況，為揭示海洋沉積環境中AAOB對低氧的響應機制提供科學依據。

1 材料和方法

1.1 樣品采集

2012年6月在舟山以東海域采集沉積物表層泥樣和底層水樣，采樣站位信息見表1。沉積物泥樣使用抓斗采泥器采集，取少量沉積物樣品裝入預滅菌過的微生物采樣杯，-20℃冷凍保存，帶回實驗室分析；底層水樣使用Niskin采水器采集，水樣經GF/F濾膜過濾，于-20℃冷凍保存。

1.2 環境參數分析

水樣溫度和鹽度使用HydroLab多參數分析儀現場測定，水樣營養鹽和溶解氧濃度按照《海洋調查規范》[17]使用分光光度儀測定，沉積物有機碳、總碳和總氮含量參照《海洋監測規范》[18]使用元素分析儀測定。

表1 采樣站位信息

1.3 DNA的提取和擴增

取少量(1—2 g)沉積物樣品，使用Fast DNA@SPIN Kit for soil(MP，美國)提取沉積物總DNA。使用AAOB的16S rRNA基因特異引物[19]進行DNA擴增：Amx368F，5′-TTCGCAATGCCCGAAAGG-3′和Amx820R，5′-AAAACCCCTCTACTTAGTGCCC-3′， 50 μl PCR反應體系包含：5 μL 10×PCR buffer、200nmol/L dNTPs、前后引物各0.25 μmol/L、1 U Taq酶和1 μL DNA提取物。PCR反應條件：94 ℃ 4 min；94 ℃ 30 s，56 ℃ 30 s，72 ℃ 60 s，30個循環；72 ℃ 7 min。1%瓊脂糖凝膠電泳檢測PCR擴增結果，并用QIAquick Gel Extraction Kit(QIAGEN，美國)純化、回收DNA目的擴增產物。

1.4 TA克隆與測序

PCR回收產物與pMD20-T載體(TaKaRa)連接，轉化到感受態細胞E.coliDH5α(TaKaRa)，涂布于LB平板(含Amp、IPTG和X-Gal)，37℃培養過夜，篩選陽性克隆構建基因克隆文庫。將所獲的陽性克隆送生工生物工程(上海)測序。

1.5 AAOB 16S rRNA基因序列分析

測序獲得的16S rRNA 基因序列在NCBI 數據庫中進行BLAST 比對(http://www.ncbi.him.nih.gov)，下載相似性最高的序列作為參比序列，應用Clustal X進行匹配比對。用DOTUR軟件包將相似性≥97%的序列定義為一個分類單元(OTU)[20]，并構建稀釋度曲線。用MEGA4 軟件構建系統發育樹[21]。研究獲得序列已提交GenBank，注冊號為KF029766-KF030062。

1.6 多樣性分析

用DOTUR軟件分析各個克隆文庫的生物多樣性指標(Shannon和Chao 1)，根據OTUs分析結果計算各個克隆文庫的覆蓋度，計算公式如下[22]：

Good =[1-(n/N)]×100

式中，n代表單克隆OUT的數量，N代表文庫中克隆總數量。AAOB群落的生態分布特征使用Fast UniFrac進行分析[23]。

2 結果

2.1 環境特征

研究海域5個采樣站位根據其環境特征可分為河口和海洋兩類(表2)。EZ3-1站距離長江口最近，受長江徑流影響底層海水溫度較高、鹽度較低，硝酸鹽和硅酸鹽濃度是研究站位中最高，沉積物中碳、氮含量也是研究站位中最高。其它4個站位環境特征較為均勻，鹽度較高、營養鹽濃度較低，屬典型的海洋環境特征，但EZ1-3和EZ3-3兩個站營養鹽濃度和沉積物碳、氮含量略高于EZ1-5和EZ3-5，表明長江徑流等對距離較近的站位仍有一定的影響。研究海域底層水的溶解氧濃度較低，其中EZ3-3和EZ3-5站位于長江口外低氧區的核心區內[14]，其溶解氧低于其它3個站(<4 mg/L)。

表2 各采樣站位環境參數

2.2 克隆文庫與多樣性分析

圖1 厭氧氨氧化細菌16S rRNA基因稀釋度曲線Fig.1 Rarefaction curves of AAOB 16S rRNA libraries

針對5個站沉積物樣品分別構建克隆文庫，總共獲得297條AAOB 16S rRNA基因序列。根據基因序列相似性≥97%歸為一個OTU，共獲得16個OTUs，5個文庫共有的OTUs有5個，文庫各自的OUT數在5—8個之間(表3)。稀釋度曲線顯示5個基因文庫用于測序的克隆數量達到或接近飽和(圖1)。5個克隆文庫的覆蓋度較高，均大于80%(表3)，表明本研究所構建的文庫基本涵蓋研究海域沉積物中的AAOB多樣性。各個文庫的香濃指數和Chao 1指數之間有一定差異(表3)，結果顯示EZ1-5、EZ3-1和EZ3-3文庫的多樣性較高，EZ1-3和EZ3-5文庫的多樣性較低，而EZ1-3和EZ1-5文庫的物種數量較高，EZ3-1、EZ3-3和EZ3-5文庫的物種數量較低。

表3 厭氧氨氧化細菌基因文庫的多樣性指數

圖2 基于16S rRNA基因的厭氧氨氧化細菌群落PCoA分析 Fig.2 The Fast UniFrac weighted PCoA analysis of the anammox communities using 16S rRNA gene sequences

使用FastUniFrac PCoA分析了研究海域5個站位AAOB群落組成的空間分布特征。圖2顯示，PCoA的前兩個主坐標軸(PC1和PC2)能夠解釋5個采樣站位之間AAOB群落差異的70.25%，根據AAOB的群落結構可以把5個站分成3類，EZ1-3、EZ3-1和EZ3-3三個站位具有相似的AAOB群落結構，EZ1-5和EZ3-5兩個站AAOB的群落結構與上述3個站具有明顯的差別。

2.3 系統發育分析

對從舟山海域沉積物樣品中構建的5個AAOB的16S rRNA基因文庫進行了系統發育分析，結果顯示，本研究獲得的絕大部份序列(283條)屬于Scalindua屬(圖3)，占總序列數的95.3%。由圖3可以看出，來自Scalindua屬的序列主要聚類于Brodae和Wagneri兩個分支。Brodae分支包含4個OUT，245條序列，占Scalindua屬序列數的86.6%，與CandidatusScalinduabrodae的親緣關系較近。其中，OTU1包含47條序列，與CandidatusS.brodae的相似性在96.0%—97.7%之間，主要與來自河口沉積物[24]、深海熱液噴口沉積物[25]的AAOB非培克隆具有較近的親緣關系；OTU2包含53條序列，與CandidatusS.brodae的相似性在96.8%—97.9%之間，與來自河口沉積物中[24]的AAOB非培克隆具有較近的親緣關系；OTU3包含26條序列，與CandidatusS.brodae(AY254883)的相似性最高(>98%)，與來自秘魯上升流和納米比亞上升流區的氧最小層(OMZ)中[26-27]的AAOB非培克隆具有較近的親緣關系；OTU4包含119條序列，與CandidatusS.brodae的相似性在95.1%—95.6%之間，主要與來自長江口、南海和黃海的AAOB非培克隆[28-30]具有較近的親緣關系。Wagneri分支包含4個OTU，共36條序列，占Scalindua屬序列總數的12.7%，與CandidatusScalinduawagneri的親緣關系較近。其中OTU10的10條序列與CandidatusS.wagneri的相似性較高(>97%)，其他3個OTU的26條序列與CandidatusS.wagneri的相似性在94.5%—96.6%之間，與來自潮間帶沉積物、河口沉積物、和近岸低氧沉積物等環境中的AAOB非培克隆[24,31,32]具有較近的親緣關系。此外，2條來自EZ3-1文庫的序列EZ3-1-8(OTU5)和EZ3-1-49(OTU6)也聚類于Scalindua分支內，但與已知CandidatusScalinduabrodae和CandidatusScalinduawagneri的相似性均較低，分別為91%和93%左右，它們可能代表了該屬未知的AAOB菌株。

圖3 AAOB 16S rRNA基因系統發育樹Fig.3 Neighbor-joining phylogenetic tree of anammox bacteria-related 16S rRNA gene fragments from the study areaClong names include the sample name and the number of times a sequence among all of the sequenced clones of samples; Bootstrap values represent 1000 replications and only values above 50% are shown; Branch lengths correspond to sequence differences as indicated by the scale bar; Numbers in parentheses refer to the number of clones were assigned to an OUT

EZ3-5文庫中有一條序列EZ3-5-46(OTU11)聚類于Kuenenia分支，與Kuenenia屬的其代表種CandidatusKueneniastuttgartiensis的相似性為94.6%，與來自河口、陸源淡水和深海熱液噴口的AAOB[24,33]具有較近的親緣關系。此外，還有5個OTU共15條序列與已知AAOB相似性較低，這些序列可分為2個分支，Unknown I分支包含3個OTU，9條序列，與來自極地海洋沉積物中的非培克隆[34]具有較近的親緣關系；Unknown II分支包含2個OUT，6條序列，與來自紅樹林沉積物中的非培養細菌具有較近的親緣關系(圖3)。

3 討論

厭氧氨氧化細菌(AAOB)廣泛分布在河流、湖泊、海洋等不同的環境中，群落組成在不同環境中具有明顯的差異性。Scalindua屬是海洋環境中AAOB的優勢類群[35]，它們具有較高的鹽耐受性，在多種海洋環境中，特別是上升流區水柱的OMZ和一些次低氧水體中[35-36]都有報道。本文研究結果表明舟山群島海域的AAOB是典型的海洋環境群落，Scalindua屬是研究海域沉積物中AAOB的優勢類群，來自于該屬的序列占總序列數的95.3%。本文研究海域還獲得了1條與Kuenenia屬AAOB 親緣關系較近的16S rRNA基因序列。該屬主要分布在淡水或陸源生境中[36-37]，在河口等咸淡水交匯的低鹽環境中也有分布[24,38]。但本文在距離陸地較遠的EZ3-5站獲得了Kuenenia屬序列，該站位主要受外海水團控制，鹽度較高(34.32)，營養鹽濃度較低，而在鹽度相對較低的EZ3-1站未發現Kuenenia屬序列。近期Byrne等在北大西洋中脊的深海熱液區的貽貝和煙囪體中也獲得了與Kuenenia屬親緣關系較近的序列[33]，表明Kuenenia屬的部分AAOB對鹽度具有較強的耐受能力。本文和Byrne等的研究結果表明來自Kuenenia屬AAOB的分布范圍可能超出我們原來的認識，一方面這可能受到采樣和研究不足的限制，另一方面需要更深水平的二代測序研究證實。此外，除了已知的AAOB類群，本研究還發現了一些與來自海洋環境的浮霉菌門非培養克隆相似性較低(<85%)的序列(Unkown I和Unknown II)。有研究表明，海洋環境中可能存在除浮霉菌門以外的其它厭氧氨氧化微生物，如β-和γ-變形菌中的Nitrosospira、Nitrosomonas和Nitrosococcus[39-40]，甚至在某些海洋環境中存在具有厭氧氨氧化能力的古菌[41]。因此，這些序列可能代表了某些未知門類的厭氧氨氧化微生物。

圖4 有機碳與多樣性指數相關性 Fig.4 Correlation between organic carbon and Shannon index of the five sampling sites

AAOB多樣性和群落結構的分布特征與環境因子有著密不可分的關系。Dale等認為鹽度是影響河口生態系統中AAOB群落結構的重要因子[31]，但本文采樣站位之間鹽度差別不大，除EZ3-1略低外，其它站位的鹽度十分接近。本文中EZ3-1、EZ3-3和EZ1-3三個站的群落結構較為接近，而與EZ1-5和EZ3-5站不同(圖2)，表明離岸距離和水深可能是影響表層沉積物AAOB群落組成的重要因素[30]。另一方面，各站位的多樣性指數卻呈現出不同的分布特征，EZ3-1、EZ3-3和EZ1-5三個站的多樣性指數高于EZ1-3和EZ3-5站(表2)。相關分析顯示沉積物有機碳含量與多樣性指數具有顯著正相關關系(圖4)。Hou等研究認為沉積物有機碳含量是河口生態系統中影響AAOB多樣性的重要環境因子[29]，高有機碳含量能夠促進硝酸鹽還原作用，進而提高亞硝酸鹽濃度，亞硝酸鹽是厭氧氨氧化過程中的電子受體，高濃度的亞硝酸鹽有利于促進厭氧條件下的氨氧化作用[42]。Hu等人在椒江口海域的研究表明AAOB多樣性指數與沉積物有機碳含量呈正比[24]，Li等人在南海北部也有類似結果[30]。

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Diversity of anaerobic ammonium oxidizing bacteria in marine sediments from the Zhoushan Islands

ZHANG Dongsheng1,*, LIU Zhensheng1, ZHANG Haifeng1, WANG Xiaogu1, WANG Chunsheng1,2

1LaboratoryofMarineEcosystemandBiogeochemistry,StateOceanicAdministration,Hangzhou310012,China2StateKeyLaboratoryofSatelliteOceanEnvironmentDynamics,Hangzhou310012,China

Anaerobic ammonium oxidation (anammox) is an important process regulating the balance of marine nitrogen and ecosystem health, particularly under anoxic conditions. The Zhoushan Islands are located east of the Changjiang river estuary, and collect a high load of anthropogenic nitrogen, which leads to severe eutrophication and seasonal hypoxia. Therefore, bacteria that mediate the anammox process are of major interest in this area. Although the importance of anammox-mediating bacteria is known, few studies on these bacteria have been conducted in the East China Sea. To the best of our knowledge, this study is the first to report the diversity, community composition, and distribution of anammox bacteria in the Zhoushan Islands. Field surveys were conducted in June 2012; triplicate surface sediment samples were collected at each site and stored in sterile plastic bags at -80℃ for subsequent DNA extraction and molecular analysis. Total genomic DNA was extracted using the Fast DNA? SPIN Kit for soil. Environmental DNA extracted from sediment samples was used as the template for PCR amplification of anammox 16S rRNA genes using primers Amx368f—Amx820r. The purified fragments were cloned and sequenced for phylogenetic and statistical analyses. In total, 297 sequences belonging to 16 operational taxonomic units (OTUs) were obtained from five 16S rRNA gene libraries. The biodiversity of anammox bacteria was examined using rarefaction analysis of the 16S rRNA genes, the Chao1 estimator, and Shannon index calculations. EZ3-1, EZ3-3, and EZ1-5 exhibited higher diversity than EZ1-3 and EZ3-5. A significant positive correlation between Shannon index and organic carbon content indicate that sediment organic carbon content plays an important role in modulating anammox bacterial diversity in the Zhoushan Island area. Weighted UniFrac PCoA analysis of the 16S rRNA genes demonstrated spatial heterogeneity in the community composition of anammox bacteria; the anammox bacteria in the study area could be divided into three distinct groups. EZ3-1, EZ3-3, and EZ1-3 exhibited similar community composition, while EZ1-5 and EZ3-5 clustered separately. The composition might be affected by distance from land and water depth. Phylogenetic analysis indicated that anammox bacterial communities were dominated by the genusScalindua(283 of 297 sequences). TheScalinduacluster comprised of 245Scalinduasequences with 95.4%—98.5% sequence similarity toCandidatusScalinduabrodae, and 36Scalinduasequences with 94.5%—97.6% sequence identity to the 16S rRNA gene ofCandidatusScalinduawagneri. In addition, twoScalinduasequences that grouped in theScalinduacluster were distantly related to knownScalinduaspecies, indicating that they might represent unidentified species ofScalindua. One sequence recovered from the EZ3-5 library was closely related to genusKuenenia, which is traditionally considered an anammox bacterial genus present in freshwater or low-salinity environments. Our results suggest that members of the genusKueneniamay possess the ability to survive in high-salinity marine environments. Additionally, two clusters of unknown sequences (unknown cluster I and II) were not classified under any known anammox bacterial genus, but were most closely related to 16S rRNA gene sequences recovered from arctic sediment and mangrove sediment, respectively.

anaerobic ammonium oxidation bacteria (AAOB); Zhoushan islands; marine sediments; diversity

浙江省自然科學基金(Y5110185, Y5110171); 國家自然科學基金青年基金項目(41206104, 41203085)

2014-02-21; < class="emphasis_bold">網絡出版日期:

日期:2014-12-04

10.5846/stxb201402210303

*通訊作者Corresponding author.E-mail: dszhang7@163.com

張東聲, 劉鎮盛, 張海峰, 王小谷, 王春生.舟山群島海域沉積物厭氧氨氧化細菌多樣性.生態學報,2015,35(19):6250-6258.

Zhang D S, Liu Z S, Zhang H F, Wang X G, Wang C S.Diversity of anaerobic ammonium oxidizing bacteria in marine sediments from the Zhoushan Islands.Acta Ecologica Sinica,2015,35(19):6250-6258.