異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中微生物種群演變特征——典型城市景觀水系

周石磊,張藝冉,孫 悅,楊文麗,黃廷林,李再興,羅 曉,崔建升,周子振,李 揚(yáng)

異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中微生物種群演變特征——典型城市景觀水系

周石磊1*,張藝冉1,孫 悅1,楊文麗1,黃廷林2,李再興1,羅 曉1,崔建升1,周子振3,李 揚(yáng)3

(1.河北科技大學(xué)環(huán)境科學(xué)與工程學(xué)院,河北省污染防治生物技術(shù)實(shí)驗(yàn)室,河北 石家莊 050018；2.西安建筑科技大學(xué)環(huán)境與市政工程學(xué)院,西北水資源與環(huán)境生態(tài)教育部重點(diǎn)實(shí)驗(yàn)室,陜西 西安 710055；3.中原工學(xué)院能源與環(huán)境學(xué)院,河南 鄭州 450007)

為了研究不同異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中水體微生物群落的演變,利用Miseq 高通量測(cè)序法對(duì)景觀水系沉積物富集馴化樣本的微生物信息進(jìn)行統(tǒng)計(jì),對(duì)其微生物群落的多樣性以及多樣性進(jìn)行分析,同時(shí)基于微生物屬的信息進(jìn)行了微生物網(wǎng)絡(luò)分析.結(jié)果顯示,兩種類型培養(yǎng)基在富集馴化完成后氮素得到有效去除,脫氮效果明顯;富集馴化過(guò)程中的OUT主要屬于7個(gè),分別是變形菌門(mén)(Protebacterice)、擬桿菌門(mén)(Bacteroidetes)、綠彎菌門(mén)(Chloroflexi)、厚壁菌門(mén)(Firmicutes)、放線菌門(mén)(Actinobacteria)、藍(lán)藻門(mén)(Cyanobacteria)、酸桿菌門(mén)(Acidobacteria),與此同時(shí),富集馴化過(guò)程中有關(guān)氮循環(huán)的細(xì)菌有上升的變化過(guò)程;主成分分析(PCA),非度量多維尺度分析(NMDS)以及主坐標(biāo)分析(PCoA)表明異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中不同溫度壓力下的細(xì)菌群落組成存在明顯差異,而培養(yǎng)基的類別帶來(lái)的影響相對(duì)較小;網(wǎng)絡(luò)分析顯示模塊核心和網(wǎng)絡(luò)核心均為低豐度的稀有物種;膨脹因子分析(VIF)和冗余分析(RDA)得出溫度、氨氮和硝酸鹽氮是影響群落結(jié)構(gòu)演變的關(guān)鍵環(huán)境因子.綜上可知,Miseq高通量測(cè)序研究異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中微生物種群演變可行,為實(shí)現(xiàn)微生物菌劑“定向-精準(zhǔn)-高效”的篩選提供技術(shù)支撐.

異養(yǎng)硝化-好氧反硝化；景觀水系；Miseq測(cè)序；生物信息分析；微生物群落

好氧反硝化菌[1]是一類在有氧條件下,利用周質(zhì)硝酸鹽還原酶進(jìn)行反硝化作用的脫氮菌,并且大多數(shù)菌同時(shí)具有異養(yǎng)硝化的能力.它的發(fā)現(xiàn)打破了反硝化只能在厭氧缺氧條件下進(jìn)行的傳統(tǒng)反硝化的觀念,為新型生物脫氮提供了新思路.

近年來(lái),相關(guān)研究通過(guò)從土壤[2]、活性污泥[3]、污水[4-5]、河流[6]、湖泊[7-8]、水庫(kù)[9-10]等系統(tǒng),分離了大量高效的好氧反硝化菌.前期文獻(xiàn)報(bào)道主要集中于通過(guò)施加一定選擇壓(間歇曝氣、鹽度或者溫度)或采用特定的選擇性培養(yǎng)基馴化富集.比如,趙驚鴻等[11]通過(guò)采取在好氧反硝化培養(yǎng)基連續(xù)培養(yǎng)中逐步升溫的方式,分離得到耐高溫好氧反硝化菌JH8;成鈺等[12]將通過(guò)在好氧反硝化培養(yǎng)液中添加氯化鈉的方式,分離得到耐鹽的異養(yǎng)硝化-好氧反硝化芽孢桿菌SLWX2;周培等[13]通過(guò)特定方法完成耐受重金屬好氧反硝化菌株的篩選;Carter等[14]利用周質(zhì)硝酸鹽還原酶特定的生化特性和活性位點(diǎn),篩選出多種好氧反硝化菌;Kong等[15]利用氰化鉀作為抑制劑,快速篩選出好氧反硝化菌.然而,關(guān)于在好氧反硝化菌富集馴化過(guò)程中微生物群落的演變過(guò)程鮮有報(bào)道.不同富集馴化方法下微生物群落演變規(guī)律直接影響高效好氧反硝化菌的定向篩選和特性研究,特別是對(duì)高效菌(混合菌群)的潛在功能預(yù)測(cè)以及菌劑的固定化提供必要依據(jù).

石家莊中心城區(qū)水系作為省會(huì)的核心水系,保持健康的生態(tài)功能為城市的經(jīng)濟(jì)發(fā)展提供了重要的生態(tài)保障.由于水系氮素超標(biāo)嚴(yán)重,且主要為氨氮和硝酸鹽氮.完成黑臭水體治理,首先要恢復(fù)水體的自然復(fù)氧功能,使水體呈現(xiàn)好氧狀態(tài).因此,如何在好氧環(huán)境下實(shí)現(xiàn)“定向-精準(zhǔn)-高效”的氮素削減是水體自我修復(fù)功能恢復(fù)的關(guān)鍵,同時(shí)也是一個(gè)亟待解決的科學(xué)問(wèn)題.本文選取石家莊中心城區(qū)典型區(qū)域的沉積物樣品,采用不同的異養(yǎng)硝化-好氧反硝化培養(yǎng)基進(jìn)行常溫和低溫條件下異養(yǎng)硝化-好氧反硝化菌的富集馴化;與此同時(shí)研究富集馴化過(guò)程中的微生物群落演變特征,考察關(guān)鍵異養(yǎng)硝化-好氧反硝化物種,為進(jìn)一步的高效菌劑定向篩選提供必要的技術(shù)支持.

1 材料與方法

1.1 實(shí)驗(yàn)裝置

實(shí)驗(yàn)裝置為2L的燒杯,取石家莊市中心城區(qū)世紀(jì)公園(SJ)(38°11′40″N;114°32′16″E)和民心河裕翔街(YX)(37°58′41″N;114°31′45″E)的沉積物進(jìn)行好氧反硝化菌的富集馴化.通過(guò)恒溫培養(yǎng)箱和充氧泵的間歇曝氣來(lái)控制系統(tǒng)的溫度和溶解氧,實(shí)驗(yàn)的溫度設(shè)為室溫20℃和低溫10℃.每個(gè)2L燒杯中裝有200mL的沉積物和800mL的培養(yǎng)基,其中沉積物樣品用超純水進(jìn)行了洗脫處理.通過(guò)選擇2種類型異養(yǎng)硝化-好氧反硝化培養(yǎng)基來(lái)富集馴化好氧反硝化菌.具體富集馴化過(guò)程為,首次為100%培養(yǎng)基,進(jìn)行培養(yǎng);3d后更換培養(yǎng)基為100%的培養(yǎng)基繼續(xù)培養(yǎng);3d后更換培養(yǎng)基為80%的培養(yǎng)基培養(yǎng);3d后更換為70%的培養(yǎng)基培養(yǎng);3d后更換60%的培養(yǎng)基培養(yǎng);3d后更換50%的滅菌富集培養(yǎng)基培養(yǎng).每次更換期間測(cè)定各個(gè)系統(tǒng)水樣中硝酸鹽氮、亞硝酸鹽氮、氨氮和總氮來(lái)反應(yīng)異養(yǎng)硝化-好氧反硝化菌的富集馴化效果.

1.2 異養(yǎng)硝化-好氧反硝化培養(yǎng)基

異養(yǎng)硝化-好氧反硝化(類型Ⅰ)富集培養(yǎng)基[16](g/L):CH3COONa 0.1;NaNO30.01;NH4Cl 0.0063;K2HPO4·3H2O 0.02;CaCl20.01;MgCl2·6H2O 0.01;蒸餾水1L;pH 7.0 ~7.5.

異養(yǎng)硝化-常規(guī)反硝化(類型Ⅱ)富集培養(yǎng)基[17](g/L):Na2HPO4·7H2O 7.9;KH2PO41.5;MgSO4·7H2O 0.1;丁二酸鈉(琥珀酸鈉)4.7;KNO31.0;微量元素2mL/L;pH值 7.0~7.5.微量元素(g/L):EDTA 50; ZnSO42.2;CaCl25.5;MnCl2·4H2O 5.1;FeSO4·7H2O 5.0;鉬酸鹽1.1;CuSO4·5H2O 1.6;CoCl2·6H2O 1.6;蒸餾水1L; pH 7.0~7.5.

本研究調(diào)整類型Ⅱ的濃度(稀釋30倍),使其與類型Ⅰ的氮素水平相一致,以便城市景觀水系的氮素污染控制.

1.3 微生物多樣性分析

1.3.1 DNA提取與PCR擴(kuò)增 選取初始的世紀(jì)公園和民心河的新鮮沉積物樣品以及富集馴化結(jié)束后的沉積物樣品,通過(guò)土壤DNA試劑盒(美國(guó)MP公司FastDNA SPINTMkit)并參照說(shuō)明書(shū)提取沉積物的總DNA,采用瓊脂糖凝膠電泳法和分光光度計(jì)完成DNA完整性、純度與濃度檢測(cè).沉積物在-80℃下保存,以備高通量測(cè)序.

1.3.2 微生物16S測(cè)序 將提取的沉積物總DNA樣本利用引物338F(ACTCCTACGGGAGGCAGC-AG)和806R(GGACTACHVGGGTWTCTAAT)對(duì)沉積物的16S rRNA基因V4-V5區(qū)進(jìn)行PCR擴(kuò)增,進(jìn)而分析異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中微生物群落的演變過(guò)程.20μL PCR反應(yīng)體系為:5×FastPfu Buffer 4μL, 2.5mmol/L dNTPs 2μL, Forward Primer (5μmol/L) 0.8μL, Reverse Primer(5μmol/L) 0.8μL, FastPfu Polymerase 0.4μL, BSA 0.2μL, Template DNA 10ng, ddH2O 11.8mL.反應(yīng)程序?yàn)?初始變性溫度95℃ 3.0min; 95℃ 30s, 55℃ 30s, 72℃ 45s,循環(huán)30次; 72℃ 10min讀板.將PCR擴(kuò)增的產(chǎn)物委托上海美吉生物科技有限公司利用Miseq進(jìn)行高通量測(cè)序.

1.3.3 生物信息學(xué)分析 對(duì)測(cè)序結(jié)果進(jìn)行數(shù)據(jù)質(zhì)控,依據(jù)相似度97%水平劃分OUT,并按照最小樣本序列進(jìn)行抽平處理.利用R語(yǔ)言分析微生物群落的多樣性以及多樣性分析(http://www.r-project.org/).基于R語(yǔ)言的vegan包完成主成分分析(PCA),非度量多維尺度分析(NMDS)以及主坐標(biāo)分析(PCoA),進(jìn)而分析不同培養(yǎng)基下異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中的微生物群落組成差異.基于方差膨脹因子(VIF)分析以及蒙特卡羅檢驗(yàn)篩選關(guān)鍵影響因子[18],并通過(guò)冗余分析(RDA)得到環(huán)境因子與微生物群落之間的相關(guān)性[19]. 利用Cytoscape軟件進(jìn)行微生物群落的網(wǎng)絡(luò)分析[20],篩選出關(guān)鍵物種[21],進(jìn)而分析不同選擇壓下富集馴化得到的異養(yǎng)硝化-好氧反硝化菌的物種信息.

1.4 水質(zhì)分析方法

水質(zhì)指標(biāo)硝酸鹽氮采用紫外分光光度法,亞硝酸鹽氮為N-(1-奈基)-乙二胺光度法,氨氮為納氏試劑比色法,總氮為過(guò)硫酸鉀氧化-紫外分光光度法.硝酸鹽氮、亞硝酸鹽氮和氨氮的水樣經(jīng)0.45μm醋酸纖維濾膜過(guò)濾.

1.5 數(shù)據(jù)分析方法

繪圖和數(shù)據(jù)統(tǒng)計(jì)分析軟件為R和Origin.其中值<0.05表示存在顯著差異,0.001<值<0.01表示存在極顯著差異,值<0.001表示存在極其顯著差異.參照文獻(xiàn)[22],網(wǎng)絡(luò)分析中對(duì)網(wǎng)絡(luò)中節(jié)點(diǎn)進(jìn)行如下分類:模塊核心(Z32.5,P￡0.62),網(wǎng)絡(luò)核心(Z30.5,P>0.62),外圍節(jié)點(diǎn)(Z<2.5,P￡0.62),連接點(diǎn)(Z<2.5,P>0.62).

2 結(jié)果與分析

2.1 源水脫氮效果分析

如圖1所示,兩種類型培養(yǎng)基在從100%培養(yǎng)基到50%培養(yǎng)基的富集馴化過(guò)程中,都表現(xiàn)出很強(qiáng)的好氧反硝化脫氮能力.類型Ⅰ培養(yǎng)基下,YX采樣點(diǎn)在低溫的反硝化能力要強(qiáng)于常溫,比如在50%的培養(yǎng)基條件下,低溫環(huán)境3d的YX的硝酸鹽氮從5.44mg/L下降到0.30mg/L,而常溫條件下硝酸鹽氮從5.44mg/L下降到1.14mg/L;低溫條件下SJ的硝酸鹽氮從1.30mg/L下降到0.17mg/L,而常溫條件下硝酸鹽氮從1.30mg/L下降到0.34mg/L;YX和SJ低溫與常溫反硝化能力的不同,可能與本底微生物群落組成有關(guān),具體原因還需進(jìn)一步分析.類型Ⅱ的培養(yǎng)基低溫和常溫的好氧反硝化脫氮能力差異不大,YX的硝酸鹽氮去除率82.65%~84.16%;SJ的硝酸鹽氮去除率89.48%~90.77%.然而,在異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中,兩種培養(yǎng)基的氨氮并沒(méi)有表現(xiàn)出明顯的去除效果.其中,在結(jié)束富集馴化時(shí)常溫下的氨氮表現(xiàn)出更好的去除效果.比如,類型Ⅰ的培養(yǎng)基YX的氨氮從3.55mg/L下降到0.52mg/L,SJ的氨氮從0.75mg/L下降到0.69mg/L;類型Ⅱ的培養(yǎng)基YX的氨氮從2.53mg/L下降到1.39mg/L,SJ的氨氮從0.05mg/L上升到1.47mg/L.氨氮去除不明顯的原因可能與沉積物的釋放有關(guān).

圖1 異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中氮素變化特征

Fig.1 The changes of nitrogen concentrations during the enrichment and domestication process of heterotrophic nitrification-aerobic denitrification bacteria

圖2 異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中間隙水氮素變化特征

關(guān)于富集馴化過(guò)程中各個(gè)系統(tǒng)的沉積物間隙水中氮素的變化情況如圖2所示.YX采樣點(diǎn)的氨氮呈現(xiàn)明顯的降低,類型Ⅰ的培養(yǎng)基從初始的32.39mg/L下降到12.14(低溫)和2.07mg/L(常溫);類型Ⅱ的培養(yǎng)基從初始的32.39mg/L下降到6.90(低溫)和3.93mg/L(常溫).SJ采樣點(diǎn)的硝酸鹽氮呈現(xiàn)明顯的降低,類型Ⅰ的培養(yǎng)基從初始的8.67mg/L下降到0.05 (低溫)和0.15mg/L(常溫);類型Ⅱ的培養(yǎng)基從初始的8.67mg/L下降到1.21(低溫)和1.95mg/L(常溫). YX采樣點(diǎn)表現(xiàn)出更高的氨氮去除效果,SJ采樣點(diǎn)表現(xiàn)出更高的硝酸鹽氮去除效果.

2.2 微生物α多樣性分析

通過(guò)對(duì)兩種培養(yǎng)基異養(yǎng)硝化-好氧反硝化菌富集馴化樣本16S rRNA基因進(jìn)行測(cè)序,總共獲得了34881條有效序列,序列平均長(zhǎng)度440bp.通過(guò)計(jì)算微生物多樣性指數(shù)考察不同類型培養(yǎng)基富集馴化異養(yǎng)硝化-好氧反硝化菌實(shí)驗(yàn)過(guò)程中細(xì)菌微生物群落的物種豐度和物種多樣性.

如表1所示,Ace和Chao指數(shù)反映微生物群落的豐富度[23],兩種類型培養(yǎng)基下的微生物豐富度都有明顯的增加,Ace指數(shù)從2120.78增加到2319.69, Chao指數(shù)從2146.76增加到2344.23; Coverage反映微生物群落的覆蓋度[24],在富集馴化過(guò)程中維持在0.9887~0.9925之間,表明測(cè)序可以很好的覆蓋物種信息;Shannon和Simpson指數(shù)反映微生物群落的多樣性[25],Shannon指數(shù)大多數(shù)呈現(xiàn)增加,然而Simpson指數(shù)大多數(shù)為降低,微生物多樣性變化的復(fù)雜原因還需進(jìn)一步分析.

表1 異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中生物多樣性指數(shù)

2.3 細(xì)菌群落組成及β多樣性分析

將異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中的樣本進(jìn)行數(shù)據(jù)庫(kù)比對(duì),同時(shí)分析在各個(gè)水平上的菌群結(jié)構(gòu).結(jié)果顯示屬于52個(gè)門(mén)類866個(gè)屬3081個(gè)OTU,具體結(jié)果如圖3所示.

兩種類型培養(yǎng)基在低溫和常溫條件下富集馴化的OUT主要屬于7個(gè)門(mén)類如圖3(a),分別是變形菌門(mén) (Protebacterice)、擬桿菌門(mén)(Bacteroidetes)、綠彎菌門(mén)(Chloroflexi)、厚壁菌門(mén)(Firmicutes)、放線菌門(mén) (Actinobacteria)、藍(lán)藻門(mén)(Cyanobacteria)、酸桿菌門(mén)(Acidobacteria)和其他少數(shù)細(xì)菌門(mén)類,其中主要的細(xì)菌門(mén)類為變形菌門(mén).特別是,變形菌門(mén)在碳源和氮素代謝過(guò)程中扮演重要的角色[26];擬桿菌門(mén)主要參與硝化過(guò)程[27];綠彎菌門(mén)能夠促進(jìn)水中植物殘留物的降解過(guò)程[28].其中變形菌門(mén)占比34.89%~ 68.06%,擬桿菌門(mén)占比7.29%~15.67%,綠彎菌門(mén)占比6.59%~19.39%.

在變形菌門(mén)中(圖3(b)),各個(gè)異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中共包含Alphaproteobacteria, Betaproteobacteria,Gammaproteobacteria,Deltaproteobacteria和Epsilonproteobacteria 5種.其中, Betaproteobacteria是最大的綱,而且Betaproteobacteria因其具有氮素去除的降解特征[29],廣泛分布污廢水的處理系統(tǒng)[30].本實(shí)驗(yàn)中,YX從初始的13.83%上升到23.22%;SJ從初始的14.62%上升到44.87%,表明在異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中,脫氮微生物得到明顯增加.

與此同時(shí),兩種培養(yǎng)基在富集馴化過(guò)程中微生物種群存在共有的屬種,同時(shí)也存在差異.現(xiàn)將富集馴化過(guò)程中豐度前50的屬如圖3(c)所示,其中很多涉及氮素循環(huán).比如,在培養(yǎng)基Ⅰ中,YX采樣點(diǎn)中的,,,以及得到增加,分別從6.31%, 1.39%, 0.63%, 0.14%, 0.81% 上升到10.56%, 3.56%, 1.93%, 10.46%, 1.81%; SJ采樣點(diǎn)中的,,,以及也得到增加,分別從2.95%, 1.98%, 1.79%, 0.24%, 0.16%上升到4.64%, 3.34%, 2.34%, 1.06%, 1.21%.在培養(yǎng)基Ⅱ中,YX采樣點(diǎn)中的,,和,得到增加,分別從1.39%, 0.14%, 0.63%, 1.11%上升到3.17%, 3.75%, 1.85%, 1.56%;SJ采樣點(diǎn)中的,,,和得到增加,分別從0.24%, 0.74%, 0.01%, 0.16%上升到1.47%, 1.34%, 24.60%, 1.06%.其中,作為綠彎菌門(mén)的代表類群,屬于有機(jī)物降解的一類微生物[31];和促進(jìn)氮素和有機(jī)物的去除[32-33],并且作為異養(yǎng)硝化-好氧反硝化菌主要屬種[34];和是主要的反硝化菌[35-36],反硝化菌在廢水硝酸鹽氮去除過(guò)程中得到增加[37];作為代表性異養(yǎng)硝化-好氧反硝化菌分離于廢水[38]、沉積物[39]和污泥[40]中;與[41]在系統(tǒng)發(fā)育上接近,也是重要的反硝化菌[42].

圖3 異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中種群變化特征

與此同時(shí),基于OTU (97%相似性)水平,對(duì)兩種培養(yǎng)基類型的異養(yǎng)硝化-好氧反硝化菌富集過(guò)程的種群演變進(jìn)行了β-多樣性的分析,具體包括:主成分分析(PCA),非度量多維尺度分析(NMDS)以及主坐標(biāo)分析(PCoA),來(lái)反映不同培養(yǎng)基以及不同溫度下富集馴化的菌種差異性(圖4).PCA分析得出PC1+PC2達(dá)到58.35%,圖中樣本間的組成越相似,反映在PCA圖中的距離越近(圖4(a)).PCA圖中兩類培養(yǎng)基分布在PCA1軸的正負(fù)兩側(cè),同一溫度條件下的樣本聚集相對(duì)緊密,低溫和常溫樣本間分布較分散,顯示2個(gè)采樣點(diǎn)在富集馴化過(guò)程中種群呈現(xiàn)差異性.NMDS中當(dāng)stress<0.05時(shí),則具有很好的代表性[43],本文分析顯示不同采樣點(diǎn)在2種培養(yǎng)基中,在富集馴化過(guò)程中物種呈現(xiàn)顯著差異,同一溫度的差異較小(圖4(b)).通過(guò)基于 Bray–Curtis距離的PCoA分析研究不同培養(yǎng)基類型富集馴化異養(yǎng)硝化-好氧反硝化菌的群落組成(圖4(c)).結(jié)果表明,不同溫度壓力下的細(xì)菌群落組成存在明顯差異,而培養(yǎng)基的類別帶來(lái)的影響相對(duì)較小,跟PCA、NMDS分析以及物種群落組成的結(jié)果相一致.

圖4 異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中種群差異分析

2.4 微生物網(wǎng)絡(luò)分析

基于異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中的微生物群落的屬,利用Cytoscape軟件構(gòu)建微生物的互作網(wǎng)絡(luò).

圖5 異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中微生物網(wǎng)絡(luò)及節(jié)點(diǎn)特征

圖5(a)中展示為斯皮爾曼相關(guān)系數(shù)>0.99的節(jié)點(diǎn),節(jié)點(diǎn)大小為中介中心性,線的顏色綠色表示正相關(guān),紅色表示負(fù)相關(guān).網(wǎng)絡(luò)分析共得到412個(gè)節(jié)點(diǎn),1327條邊;劃分成11個(gè)模塊,模塊1~11的占比分別為15.05%, 18.20%, 6.80%, 9.71%, 7.52%, 5.10%, 9.71%, 7.77%, 3.16%, 16.50%, 0.48%.網(wǎng)絡(luò)中節(jié)點(diǎn)正相關(guān)占比74.00%,負(fù)相關(guān)占比26.00%,表明物種間的關(guān)系呈現(xiàn)共生為主.Roger等[44]定義參數(shù)Z來(lái)衡量一個(gè)點(diǎn)在所在模塊中的作用,值越高說(shuō)明在模塊中的作用越大;定義P來(lái)衡量一個(gè)點(diǎn)參與其他模塊的程度,值越高說(shuō)明與其他模塊的聯(lián)系越密切.有關(guān)節(jié)點(diǎn)的分類如圖5(b)所示,網(wǎng)絡(luò)分析依據(jù)各節(jié)點(diǎn)Z值和P值將所有節(jié)點(diǎn)劃分為模塊核心,網(wǎng)絡(luò)核心,外圍節(jié)點(diǎn)和連接點(diǎn)四類.本網(wǎng)絡(luò)中模塊核心包括14個(gè)物種,網(wǎng)絡(luò)核心包括2個(gè)物種.模塊核心和網(wǎng)絡(luò)核心大多屬于低豐度的稀有物種,其中,能利用各種蛋白質(zhì)進(jìn)行生長(zhǎng)[45],是一種降解有機(jī)物的關(guān)鍵物種[46-47];是污水處理廠的一個(gè)活性反硝化菌[48],說(shuō)明稀有種在異養(yǎng)硝化-好氧反硝化菌富集馴化過(guò)程中對(duì)群落構(gòu)建發(fā)揮著不可替代的作用.

2.5 環(huán)境因子與微生物群落的關(guān)系

基于VIF分析和蒙特卡羅檢驗(yàn),得到關(guān)鍵環(huán)境因子溫度(VIF=1.63<10),氨氮(VIF=1.34<10)和硝酸鹽氮(VIF=1.55<10).RDA分析顯示RDA1和RDA2共解釋了總體變化的48.7%,其中RDA1占主體解釋了42.63%(圖4(d)).其中,溫度與RDA1相關(guān)性達(dá)到-0.14(=0.001<0.05),與RDA2相關(guān)性達(dá)到-0.99(= 0.001<0.05);氨氮與RDA1相關(guān)性達(dá)到-0.89(= 0.03<0.05),與RDA2相關(guān)性達(dá)到-0.45(=0.003< 0.05);硝酸鹽氮與RDA1相關(guān)性達(dá)到0.66(=0.03< 0.05),與RDA2相關(guān)性達(dá)到-0.75(=0.03<0.05).綜上,溫度決定了兩種培養(yǎng)基不同采樣點(diǎn)的不同溫度物種分布,常溫的富集馴化過(guò)程菌群分布在RDA2的正向,低溫的富集馴化過(guò)程菌群分布在RDA2的正向;氨氮和硝酸鹽氮分別與YX和SJ采樣點(diǎn)的富集馴化過(guò)程中菌群成正相關(guān).

3 結(jié)論

3.1 類型Ⅰ培養(yǎng)基下,YX采樣點(diǎn)在低溫的反硝化能力要強(qiáng)于常溫;類型Ⅱ的培養(yǎng)基下2采樣點(diǎn)在低溫和常溫的反硝化脫氮能力差異不大.

3.2 兩種類型培養(yǎng)基在低溫和常溫條件下富集馴化的OUT主要屬于變形菌門(mén)、擬桿菌門(mén)、綠彎菌門(mén)、厚壁菌門(mén)、放線菌門(mén)、藍(lán)藻門(mén)、酸桿菌門(mén).

3.3 不同溫度壓力下的細(xì)菌群落組成存在明顯差異, 而培養(yǎng)基的類別影響相對(duì)較小;溫度、氨氮以及硝酸鹽氮是影響群落結(jié)構(gòu)的關(guān)鍵環(huán)境因子.微生物網(wǎng)絡(luò)中物種大多呈現(xiàn)共生關(guān)系, 關(guān)鍵節(jié)點(diǎn)顯示物種大多為稀有物種.

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Characteristics of bacterial community structure during the enrichment and domestication of heterotrophic nitrification-aerobic denitrification bacteria based on the typical city landscape water.

ZHOU Shi-lei1*, ZHANG Yi-ran1, SUN Yue1, YANG Wen-li1, HUANG Ting-lin2, LI Zai-xing1, LUO Xiao1, CUI Jian-sheng1, ZHOU Zi-zhen3, LI Yang3

(1.Pollution Prevention Biotechnology Laboratory of Hebei Province, School of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China；2.Key Laboratory of Northwest Water Resource, Environment and Ecology, Ministry of Education, School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China；3.School of Energy and Environment, Zhongyuan University of Technology, Zhengzhou 450007, China) ., 2019,39(11)：4831~4839

To explore the effects of different selective pressures on bacterial community structure during enrichment and domestication of heterotrophic nitrification-aerobic denitrification bacteria, bioinformatics analysis of samples taken from enrichment and domestication systems were carried out, using Miseq high-throughput sequencing. In detail,- and-diversity were examined, and network analysis was conducted. Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Actinobacteria, Cyanobacteria, and Acidobacteria were the main phyla identified. Meanwhile, the N-functional bacteria had an increased process. PCA (principal component analysis), NMDS (non-metric multidimensional scaling analysis) and PCoA (principal co-ordinates analysis) showed that microbial community structure was significantly altered with change in temperature, while the influence of different media was small. Network analysis indicated that module hubs and network hubs of bacterial communities were both rare taxa. VIF (variance inflation factor) and RDA (redundancy analysis) showed temperature, ammonia and nitrate were the most important factors affecting bacterial community function and composition. All results together indicate that Miseq high-throughput sequencing was an effective tool to explore changes in bacterial community structure during enrichment and domestication of heterotrophic nitrification-aerobic denitrification bacteria, which could in the future supply a reference to isolate “directional-accurate- efficient” microbial agents.

heterotrophic nitrification-aerobic denitrification；landscape water；Miseq sequencing；bioinformatics analysis；bacterial community structure

X172

A

1000-6923(2019)11-4831-09

周石磊(1987-),男,河北石家莊人,講師,博士,主要從事好氧反硝化菌脫氮機(jī)理與調(diào)控機(jī)制的相關(guān)研究.發(fā)表論文27篇.

2019-04-28

國(guó)家自然科學(xué)基金資助項(xiàng)目(51909056);河北科技大學(xué)引進(jìn)人才科研啟動(dòng)基金(1181278);河北省研究生創(chuàng)新資助項(xiàng)目(CXZZSS2018084)

*責(zé)任作者, 講師, ZSLZhouShilei@126.com