太湖富營養(yǎng)化湖區(qū)秋季水體和沉積物中硝化微生物分布特征及控制因素*

吳 玲,秦紅益,朱夢圓,韓 成,朱廣偉,鐘文輝

(1:南京師范大學地理科學學院, 江蘇省物質(zhì)循環(huán)與污染控制重點實驗室,南京 210023)(2:常州工程職業(yè)技術(shù)學院制藥與環(huán)境工程學院,常州 213161)(3:中國科學院南京地理與湖泊研究所湖泊與環(huán)境國家重點實驗室,南京 210008)(4:江蘇省地理信息資源開發(fā)與利用協(xié)同創(chuàng)新中心,南京 210023)

太湖富營養(yǎng)化湖區(qū)秋季水體和沉積物中硝化微生物分布特征及控制因素*

吳 玲1,2,4,秦紅益1,4,朱夢圓3,韓 成1,4,朱廣偉3**,鐘文輝1,4**

(1:南京師范大學地理科學學院, 江蘇省物質(zhì)循環(huán)與污染控制重點實驗室,南京 210023)(2:常州工程職業(yè)技術(shù)學院制藥與環(huán)境工程學院,常州 213161)(3:中國科學院南京地理與湖泊研究所湖泊與環(huán)境國家重點實驗室,南京 210008)(4:江蘇省地理信息資源開發(fā)與利用協(xié)同創(chuàng)新中心,南京 210023)

微生物參與下的氮循環(huán)是富營養(yǎng)化湖泊十分重要的生物地球化學循環(huán)過程. 采用基于amoA功能基因和16S rRNA基因的熒光定量PCR、PCR-DGGE與高通量測序等分子生物學技術(shù), 調(diào)查秋季太湖不同水體和表層沉積物中氨氧化古菌(AOA)、氨氧化細菌(AOB)和亞硝酸鹽氧化菌(NOB)群落豐度和組成, 探討影響硝化微生物分布的關(guān)鍵環(huán)境因子. 結(jié)果表明, 中度富營養(yǎng)化的梅梁灣湖區(qū)水體表層、中層和底層水樣和表層底泥中AOAamoA基因的豐度分別低于輕度富營養(yǎng)化的湖心區(qū), 而不同層水樣中AOBamoA基因的豐度分別高于湖心區(qū). 梅梁灣湖區(qū)和湖心區(qū)水樣中AOA群落組成基本相似, 2個湖區(qū)表層沉積物樣品中AOA群落組成亦基本相似, 水體中AOA群落組成與表層沉積物中AOA群落組成有差異, AOA群落豐度顯著受硝態(tài)氮、pH和DO影響；表層沉積物中AOB群落豐度有明顯差異且顯著受總氮含量影響, 表層沉積物中NOB群落豐度也有明顯差異且顯著受亞硝態(tài)氮含量影響. 太湖梅梁灣湖區(qū)和湖心區(qū)水體與表層沉積物AOA群落包括Nitrosopumilium和Nitrosotalea兩大屬；表層沉積物AOB群落主要包括亞硝化單胞菌(Nitrosomonas)和亞硝化螺菌(Nitrosospira)兩大屬, NOB群落主要包括硝化刺菌(Nitrospina)和硝化螺菌(Nitrospira)兩大屬, 其中硝化螺菌屬是淡水湖泊中比較少見的亞硝酸鹽氧化菌. 影響太湖水體和沉積物中AOA和AOB豐度的最主要環(huán)境因子為總氮、總磷與銨態(tài)氮. 研究表明典型富營養(yǎng)指標(總氮、總磷、銨態(tài)氮、硝態(tài)氮和硝態(tài)氮等)是影響太湖梅梁灣和湖心區(qū)水體和沉積物中AOA或AOB豐度以及硝化微生物群落豐度的重要因素.

氨氧化細菌；氨氧化古菌；亞硝酸鹽氧化菌；富營養(yǎng)化；沉積物；水體；太湖；梅梁灣

太湖是中國五大淡水湖之一. 生活污水排放與過度施用氮肥的農(nóng)田徑流導致太湖水體營養(yǎng)物質(zhì)濃度升高而形成富營養(yǎng)化,大量的有機質(zhì)、氮、磷等營養(yǎng)物質(zhì)在湖泊沉積物中沉降富集[1-3]. 太湖污染物從西北部進入,經(jīng)過水體稀釋,流向東南方向,最終主要從與東太湖相連的太浦河出太湖[4]. 因此,太湖從西北向東南,湖區(qū)富營養(yǎng)化程度逐漸下降,不同湖區(qū)的水質(zhì)狀況不完全相同,主要的環(huán)境因子也呈現(xiàn)一定濃度梯度[5].

太湖北部梅梁灣湖區(qū)和湖心區(qū)水體富營養(yǎng)化程度不同. 梅梁灣是太湖污水的主要入流區(qū)之一,是太湖典型的富營養(yǎng)化湖區(qū),該湖區(qū)具有藻型湖區(qū)特征,水體懸浮物濃度高于湖心區(qū),并頻繁暴發(fā)大面積的藍藻水華[4,8]；而湖心區(qū)水體則輕度富營養(yǎng)化. 太湖梅梁灣與湖心區(qū)水體與沉積物中環(huán)境條件不同,硝化微生物分布、群落組成和豐度也可能不同. 本研究采集梅梁灣和湖心區(qū)內(nèi)不同深度水體樣品和沉積物樣品,分析水體和沉積物樣品中AOA、AOB和NOB的分布和群落組成和豐度,研究太湖不同富營養(yǎng)化水平水體和沉積物中硝化微生物分布和群落組成特征,并探尋驅(qū)動硝化微生物分布與群落豐度的關(guān)鍵環(huán)境因子. 本研究有助于更全面了解太湖生態(tài)系統(tǒng)氮素循環(huán)及相關(guān)功能微生物,為太湖環(huán)境保護和生態(tài)修復提供理論依據(jù).

1 材料與方法

1.1 樣品采集

于2015年11月5日分別在太湖北部梅梁灣和湖心區(qū)各選取1個位點分別標記為HT(31°27′20″N,120°11′23″E)、ET(31°17′22″N,120°10′56″E)(圖1). 水樣采集使用有機玻璃采水器,分別從表層、中層和底層采集3個重復水樣,具體采樣位置為: 梅梁灣湖區(qū)水面下0.53、1.03和2.11 m處水樣(分別記為HT-U、HT-M和HT-B)；湖心區(qū)水面下0.52、1.07和2.18 m處水樣(分別記為ET-U、ET-M和ET-B). 用柱狀采泥器采集30 cm深度柱狀沉積物,在2個采樣區(qū)域各采集3個不同位點的沉積物樣品,沉積物現(xiàn)場分割,取0～2 cm表層樣品保存于滅菌袋中,并沖入氮氣后迅速封口,作為沉積物樣品(分別命名為HT-S和ET-S). 水樣和沉積物樣品采集后于冷藏條件下運回實驗室進行處理和保藏. 水樣采集后24 h內(nèi)通過0.22 μm微孔濾膜(Whatman GmbH,Germany)過濾,濾液用于水體理化指標測定,濾膜殘渣保存在-80℃冰箱中用于分子生物學分析[19]. 每個沉積物樣品分成兩份,一份用于理化指標測定,一份保存在-80℃用于分子生物學分析.

圖1 太湖水體和沉積物樣品采集位置示意Fig.1 Sampling sites of water and sediment in Lake Taihu

1.2 水體和沉積物理化指標的測定

沉積物中的無機氮采用2 mol/L KCl按5∶1(v/m)的水土比浸提,在20℃、200轉(zhuǎn)/min條件下振蕩1 h后過濾,濾液無機氮含量的測定方法同水樣；沉積物用蒸餾水按2.5∶1(v/m)的水土比浸提,振蕩10 min并靜置30 min,采用臺式pH計(Mettler-Toledo)測定懸濁液pH值；沉積物DO值測定采用溶氧微電極(Unisense Company,Denmark)測定；沉積物TP、TN與總有機碳(TOC)的測定方法參見文獻[12,21].

1.3 水體和沉積物總DNA提取

沉積物與濾膜殘渣總DNA采用Fast DNA? SPIN Kit for soil(MP Biomedicals,USA)試劑盒提取,具體操作方法參照說明書,提取后的總DNA溶解于100 μl TE緩沖液中. DNA濃度和純度采用NanoDrop 2000分光光度計(Thermo Scientific, USA)測定,-20℃冰箱保存?zhèn)溆?

1.4 水體和沉積物樣品硝化微生物的實時熒光定量PCR

分別以Arch-amoA F/Arch-amoA R[22]和amoA-1 F/amoA-2 R[23]為引物,采用CFX96 Real-Time PCR System(Bio-rad,USA)實時熒光定量PCR儀測定水樣與沉積物樣品中的AOA和AOBamoA基因豐度. qPCR反應體系為20 μl,包括10 μl SYBR Premix Ex Taq(Takara, Shanghai, China)、上下游引物各0.5 μl及2 μl模板, 最后加入滅菌雙蒸水補足到20 μl. qPCR反應程序為初始變性溫度95℃ 3.0 min；95℃ 30 s,55℃ 20 s,72℃ 20 s循環(huán)40次,83℃讀板；溶解曲線溫度變化范圍為65.0～95.0℃. 以AOA和AOBamoA基因的重組質(zhì)粒作為標準DNA模板,分別以10倍梯度稀釋各模板,標準曲線濃度范圍分別為3.62×102～3.62×109copies/μl(AOA)和7.14×102～7.14×109copies/μl(AOB). AOA和AOB的擴增效率分別為95.4%(R2=0.997)和86.1%(R2=0.986). 水體和沉積物中的NOB均低于檢測限,因此沒有做NOB的定量研究.

1.5 水體和沉積物樣品AOA的PCR-DGGE、克隆測序及系統(tǒng)發(fā)育分析

用于DGGE的AOAamoA基因PCR產(chǎn)物的引物為Arch-amoA F和Arch-amoA R. 50 μl PCR體系包括5 μl含有20 mmol/L Mg2+的10×HotStar-Taqbuffer、2.5 μl dNTP、2.5 U的HotStar-Taqpolymerase(Takara),0.5 μmol/L的上下游引物和1 μl DNA模板,用滅菌雙蒸水補足至50 μl. AOA PCR反應條件: 94℃ 5.0 min；95℃ 30 s,55℃ 45 s,72℃ 45 s,35個循環(huán)；72℃ 7 min. 通過1.2%瓊脂糖凝膠電泳檢測PCR擴增特異性. AOAamoA基因的DGGE分析使用6%的聚丙烯酰胺凝膠[21],變性梯度范圍為20%～50%. 電泳條件為: 0.5×TAE緩沖液,70 V電壓,60℃電泳16 h. 電泳分析通過Dcode Universal Mutation Detection System(Bio-Rad)完成后,采用SYBR Green I染料(1∶10000的稀釋倍數(shù))(Invitrogen,USA)對聚丙烯酰胺凝膠染色30 min,使用Gel Doc XR+凝膠成像系統(tǒng)(Bio-rad)進行紫外成像.

在紫外燈下小心迅速地切割下DGGE膠上的典型條帶,放置于30 μl滅菌雙蒸水中,用槍尖搗碎,4℃過夜. 取1 μl洗脫出來的DNA溶液作為模板連同原始土壤總DNA進行PCR擴增,PCR擴增體系和反應條件如前所述. PCR產(chǎn)物參照pEASYTM-T1 Cloning Kit(TransGen,China)說明書進行克隆. 每個條帶隨機地挑選出5個含有AOAamoA基因片段的白色克隆,并將克隆子進行測序(Genscript,Nanjing,China).

用DNASTAR軟件對測序結(jié)果進行編輯,去除載體序列后用BLAST在GenBank 數(shù)據(jù)庫中搜索相似序列,并與已發(fā)表的相關(guān)細菌和古菌的amoA功能基因序列進行多重序列比對,使用MEGA 4.1軟件以鄰接法(Neighbor-Joining)構(gòu)建系統(tǒng)發(fā)育樹.

1.6 沉積物樣品中AOB和NOB群落豐度分析——基因組Miseq測序

本研究中,水體和沉積物樣品中AOA群落豐度采用基于amoA基因的PCR-DGGE分析獲得,由于部分沉積物樣品中AOB與NOB功能基因豐度低,PCR擴增產(chǎn)物量無法滿足DGGE分析,故本研究采用基于16S rRNA基因的Miseq測序分析沉積物AOB與NOB的群落及豐度. 對細菌16S rRNA基因V3-V4區(qū)進行PCR擴增,引物為338F(5′-ACTCCTACGGGAGGCAGCA-3′)和806R(5′-GGACTACHVGGGTWTCTAAT-3′),每個樣本的引物5′端加barcode序列,不同樣本用不同的barcode序列進行區(qū)分. 每個樣本3個重復,將同一樣本的PCR產(chǎn)物混合后用2%瓊脂糖凝膠電泳檢測,用AxyPrepDNA凝膠回收試劑盒(Axygen,USA)切膠回收PCR產(chǎn)物,再用QuantiFluorTM-ST藍色熒光定量系統(tǒng)(Promega,USA)進行檢測定量,按照每個樣本的測序量要求,進行相應比例的混合. 利用Illumina平臺進行文庫構(gòu)建,由上海美吉生物公司采用Illumina Miseq方法進行測序,Miseq測序基于resample處理來分析細菌各類群相對豐度,resample序列數(shù)為24000. 利用Usearch(Version 7.1)對測序數(shù)據(jù)進行處理,按照97%相似性對非重復序列(不含單序列)進行OTU聚類,在聚類過程中去除嵌合體,得到OTU的代表序列. 利用DNAman將AOB和NOB主要類群序列與97%相似度的OTUs序列進行比對[24].

1.7 數(shù)據(jù)分析

采用雙因素方差分析(Two-way ANOVA,Tukey多重比較)評估不同采樣點與采樣深度分別對太湖水體理化指標的影響和兩因素對理化指標的交互影響；采用單因素方差分析(One-way ANOVA,Tukey多重比較)評估不同數(shù)據(jù)間的差異；采用獨立樣本T檢驗比較HT表層沉積物各理化指標與ET是否存在顯著差異；采用Pearson相關(guān)分析評估水體與表層沉積物AOA和AOB豐度與各環(huán)境因子的關(guān)系,P<0.05表示具有統(tǒng)計顯著性. 利用逐步回歸分析進一步評估水體與表層沉積物AOA和AOB豐度及其比值與環(huán)境因子關(guān)系,保留顯著影響變量(P<0.05). 采用Bio-rad image lab 4.1軟件對水體與表層沉積物中AOAamoA基因DGGE條帶進行數(shù)字化分析,DGGE條帶的位置和相對亮度分別表征微生物種類和數(shù)量. 將DGGE圖譜條帶數(shù)字化結(jié)果、表層沉積物中AOB或NOB各屬16S rRNA基因相對豐度與環(huán)境因子進行逐步回歸分析,篩選顯著影響變量(P<0.05). 所有分析基于 SPSS統(tǒng)計軟件(SPSS,USA).

2 結(jié)果與分析

2.1 梅梁灣與湖心區(qū)水體與沉積物的理化性質(zhì)

表1 太湖水體理化指標及雙因素方差分析

*表示P<0.05, **表示P<0.01, ***表示P<0.001. 水體理化指標數(shù)值為平均值±標準偏差(n=3)，同一列上標不同小寫字母表示使用ANOVA顯示顯著差異(P<0.05).

表2 太湖沉積物理化指標獨立樣本T檢驗結(jié)果*

*同一行上標不同小寫字母表示HT-S和ET-S間有顯著差異,T檢驗P<0.01,N=3.

2.2 梅梁灣與湖心區(qū)水體與沉積物中AOA與AOB豐度的分布特征及驅(qū)動因子分析

梅梁灣湖區(qū)水體表層、中層和底層水樣中AOAamoA基因的豐度分別為6.70×102、4.24×102和1.60×103copies/ml,AOBamoA基因的豐度分別為1.96×103、5.25×102和7.64×102copies/ml；湖心區(qū)表層、中層和底層水樣中AOAamoA基因的豐度分別為1.54×103、5.48×103和7.69×103copies/ml,AOBamoA基因的豐度分別為2.18×102、4.27×102和5.81×102copies/ml(圖2). 可見,梅梁灣湖區(qū)水體表層、中層和底層水樣中AOAamoA基因豐度分別低于湖心區(qū),而AOBamoA基因豐度分別高于湖心區(qū). 梅梁灣湖區(qū)的AOA/AOB豐度比顯著低于湖心區(qū),且湖區(qū)表層、中層和底層水樣中AOA/AOB豐度比逐步升高.

梅梁灣湖區(qū)和湖心區(qū)表層沉積物AOAamoA基因豐度分別為4.63×107和8.61×108copies/g干土,AOBamoA基因豐度分別為6.53×107和1.32×107copies/g干土. 梅梁灣湖區(qū)的AOA/AOB豐度比低于湖心區(qū).

圖2 太湖水體和沉積物AOA和AOB amoA基因豐度(方框中數(shù)字表示AOA與AOB豐度比)Fig.2 Abundance of AOA and AOB expressed as amoA copy numbers per milliliter in water and per gram in sediment of Lake Taihu(The ratio of AOA to AOB were shown in the box above the chart)

表3 逐步回歸分析篩選的與水體和沉積物中AOA、AOB amoA基因豐度相關(guān)的環(huán)境因子

*表示P<0.05, **表示P<0.01, ***表示P<0.001.

2.3 梅梁灣與湖心區(qū)水體與沉積物中AOA群落組成

梅梁灣湖區(qū)和湖心區(qū)水樣與表層沉積物樣品中AOAamoA基因共有7條DGGE條帶(OTU,圖3). 其中,TH AOA-1、2、3、4是水樣和沉積物樣品共有條帶,TH AOA-5、6是水樣中的特有條帶,TH AOA-7是沉積物中的特有條帶. 聚類分析顯示,梅梁灣湖區(qū)和湖心區(qū)水樣AOA的DGGE帶譜聚在一簇,兩湖區(qū)沉積物AOA的DGGE帶譜也聚在一簇. 梅梁灣湖區(qū)和湖心區(qū)水樣、表層沉積物樣品中AOA群落組成基本相似(圖4).

圖3 太湖水體和表層沉積物氨氧化古菌amoA基因DGGE圖譜Fig.3 DGGE fingerprint of archaeal amoA gene within water and surface sediments in Lake Taihu

圖4 太湖水體和表層沉積物AOA amoA基因DGGE指紋圖譜聚類分析Fig.4 Cluster analysis of DGGE fingerprint of archaeal amoA gene of water and surface sediments in Lake Taihu

克隆測序及系統(tǒng)發(fā)育分析表明(圖5),AOAamoA基因序列可分為3類,TH AOA-1、2和7屬于Nitrosopumilis(1.1a group),與海洋水體和沉積物的AOA親緣關(guān)系較近；TH AOA-4屬于Nitrosophaera(1.1b group),與土壤中的AOA親緣關(guān)系較近. 水樣中特有序列TH AOA-5和6屬于Nitrosotalea(1.1a-associated group),多見于酸性水稻土壤中,且與酸性土壤硝化古菌Nitrosotaleadevanaterra具有最近的親緣關(guān)系.

圖5 太湖水體和表層沉積物氨氧化古菌amoA基因系統(tǒng)發(fā)育分析Fig.5 Phylogenetic tree showing the relationship between the archeal amoA gene sequences obtained from DGGE bands

2.4 梅梁灣與湖心區(qū)沉積物中AOB和NOB群落組成與豐度

基于16S rRNA基因Miseq高通量測序結(jié)果表明,太湖表層沉積物樣品中AOB群落主要包括亞硝化螺旋菌(Nitrosospira)和亞硝化單胞菌(Nitrosomonas)兩大屬,其中梅梁灣湖區(qū)表層沉積物中AOB群落以亞硝化螺旋菌為主,其相對豐度為97.54%；湖心區(qū)表層沉積物中亞硝化螺旋菌與亞硝化單胞菌相對豐度分別為48.57%和51.43%(圖6). 富營養(yǎng)化水平不同的梅梁灣湖區(qū)和湖心區(qū)表層沉積物中AOB群落豐度有明顯差異.

圖6 采樣點HT和ET沉積物中氨氧化細菌及亞硝酸鹽氧化菌16S rRNA基因相對豐度Fig.6 Relative abundance of ammonia-oxidizing and nitrite-oxidizing bacteria 16S rRNA gene sequences of the sediments in HT and ET sites

太湖表層沉積物樣品中NOB群落主要包括硝化螺菌(Nitrospira)和硝化刺菌(Nitrospina)兩大屬,梅梁灣湖區(qū)與湖心區(qū)表層沉積物中均以硝化螺菌為主,2個湖區(qū)硝化螺菌的相對豐度分別為80.0%和90.0%(圖6). 富營養(yǎng)化水平不同的梅梁灣湖區(qū)和湖心區(qū)表層沉積物中NOB群落豐度有明顯差異.

2.5 AOA、AOB與NOB群落豐度與環(huán)境因子相關(guān)性分析

表4 AOA、AOB與NOB群落豐度與環(huán)境因子的逐步回歸分析

**表示P<0.01.

3 討論

3.1 梅梁灣與湖心區(qū)水體和沉積物中硝化微生物群落特征及群落豐度的環(huán)境影響因子

本研究多角度地研究中度富營養(yǎng)化的太湖梅梁灣湖區(qū)和輕度富營養(yǎng)化的湖心區(qū)水體與表層沉積物中氨氧化微生物、亞硝酸氧化微生物的分布、群落組成和豐度等. 梅梁灣湖區(qū)與湖心區(qū)水樣中AOA群落組成基本相似,兩湖區(qū)表層沉積物樣品中AOA群落組成亦基本相似,水體中AOA群落組成與表層沉積物中AOA群落組成有差異；兩湖區(qū)表層沉積物中AOB與NOB群落豐度均有明顯差異.

太湖梅梁灣湖區(qū)和湖心區(qū)表層沉積物AOB群落主要包括亞硝化螺菌和亞硝化單胞菌兩大屬. 亞硝化螺菌和亞硝化單胞菌是淡水湖泊生態(tài)系統(tǒng)最常見的2種AOB[12,26-28]. 中度富營養(yǎng)化的梅梁灣湖區(qū)表層沉積物AOB優(yōu)勢菌為亞硝化螺菌,Xing等[29]也有相似研究結(jié)果. 梅梁灣湖區(qū)表層沉積物中亞硝化螺菌的相對豐度遠高于湖心區(qū),隨著營養(yǎng)化水平的降低,亞硝化螺菌相對豐度下降; 在輕度營養(yǎng)化的湖心區(qū)表層沉積物中,亞硝化螺菌和亞硝化單胞菌豐度基本接近,亞硝化螺菌可能比亞硝化單胞菌更適應富營養(yǎng)水平較高的環(huán)境. 逐步回歸分析結(jié)果表明,太湖表層沉積物中TN含量顯著影響AOB群落豐度. 沉積物中微生物可以促進有機氮的礦化,礦化產(chǎn)生的氨是氨氧化過程的底物和氨氧化微生物的主要能量來源,因此,TN濃度可以提高氨氧化微生物的活性[14],從而影響AOA或AOB的群落組成與豐度[30].

3.2 梅梁灣與湖心區(qū)水體和沉積物中AOA與AOB豐度分布的環(huán)境影響因子

梅梁灣湖區(qū)和湖心區(qū)表層沉積物的AOA/AOB的比率分別為0.71和65.26,梅梁灣湖區(qū)表層沉積物中AOB更具優(yōu)勢,湖心區(qū)則相反,與水體中AOA與AOB的分布趨勢基本一致. 此結(jié)果與Hou等[6]的相似,但與Wu等[12]和Zeng等[14]的結(jié)果有差異,可能是由于采樣時間與季節(jié)不同引起的各湖區(qū)富營養(yǎng)程度的差異而造成的. 研究同時表明,影響表層沉積物AOAamoA基因豐度的主要因素有TN、TP和DO,影響表層沉積物AOBamoA基因豐度的主要因素有TN和DO,影響表層沉積物AOA/AOB豐度比的主要因素有TN,說明氮、磷和溶解氧的含量是影響沉積物中氨氧化微生物豐度的主要因素,此結(jié)果與文獻[8,11,15]一致.

4 結(jié)論

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DistributioncharacteristicsandcontrollingfactorsofnitrifyingmicroorganismsinfreshwaterandsedimentofeutrophiczonesinLakeTaihuinautumn

WU Ling1,2,4, QIN Hongyi1,4, ZHU Mengyuan3, HAN Cheng1,4, ZHU Guangwei3**& ZHONG Wenhui1,4**

(1:JiangsuProvincialKeyLaboratoryofMaterialsCyclingandPollutionControl,SchoolofGeographySciences,NanjingNormalUniversity,Nanjing210023,P.R.China)(2:InstituteofPharmaceuticalandEnvironmentalEngineering,ChangzhouVocationalInstituteofEngineering,Changzhou213164,P.R.China)(3:StateKeyLaboratoryofLakeScienceandEnvironment,NanjingInstituteofGeographyandLimnology,ChineseAcademyofSciences,Nanjing210008,P.R.China)(4:JiangsuCenterforCollaborativeInnovationinGeographicalInformationResourceDevelopmentandApplication,Nanjing210023,P.R.China)

Nitrogen cycling associated with microorganisms is the most important biogeochemical process in eutrophic lakes. In this study, molecular biological techniques including quantitative PCR, PCR-DGGE, and high-throughput sequencing based onamoAand 16S rRNA genes were used to assess the composition and community abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB) in freshwater and sediment samples of Lake Taihu collected during autumn. The key environmental factors affecting the distribution of nitrifying microorganisms were further analyzed. Results showed that theamoAgene abundance of AOA from the upper, middle, and bottom freshwater and surface sediment samples from the mesotrophic Meiliang Bay was lower than those from the less eutrophic central lake and that theamoAgene abundance of AOB from the mesotrophic Meiliang Bay was greater than those from the central lake. The AOA community composition in freshwater was similar in both Meiliang Bay and the central lake, it was the same for AOA in the surface sediment, and the key factors affecting AOA community abundance were nitrate, pH and dissolved oxygen contents, while the community abundance of AOB mainly affected by total nitrogen content in sediments were proven to differ considerably between the two sites. The case was the same for NOB with the key factor nitrite content. AOA of freshwater and surface sediments in Meiliang Bay and central lake included two genera,NitrosopumilisandNitrosotalea. AOB of the surface sediment includedNitrosomonasandNitrosospira. NOB of the surface sediment includedNitrospinaandNitrospira, andNitrospinawas scarce in freshwater. The main environmental factors affecting the abundance of AOA and AOB in Lake Taihu were TN, total phosphorus (TP), and ammonium content. This study proved that the eutrophication indexes (such as total nitrogen, total phosphorus, ammonium, nitrate and nitrite) have significant influences on the abundance of AOA or AOB and community abundance of nitrifying microorganisms in both freshwater and sediments in Lake Taihu, which may help the understanding of the nitrogen cycling in the Lake Taihu ecosystem.

Ammonia-oxidizing bacteria; ammonia-oxidizing archaea; nitrite-oxidizing bacteria; eutrophication; sediment;water; Lake Taihu; Meiliang Bay

*國家自然科學基金項目(41271255)、國家重點基礎(chǔ)研究發(fā)展計劃(2016YFD0200302)和江蘇省高校優(yōu)秀創(chuàng)新團隊項目聯(lián)合資助. 2016-10-12收稿；2017-01-21收修改稿. 吳玲(1981～)，女，博士研究生，講師; E-mail: lwu@czie.edu.cn.

**通信作者；E-mail: zhongwenhui@njnu.edu.cn; gwzhu@niglas.ac.cn.