異養硝化-好氧反硝化菌富集馴化過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

異養硝化-好氧反硝化菌富集馴化過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3

(1.河北科技大學環境科學與工程學院,河北省污染防治生物技術實驗室,河北 石家莊 050018；2.西安建筑科技大學環境與市政工程學院,西北水資源與環境生態教育部重點實驗室,陜西 西安 710055；3.中原工學院能源與環境學院,河南 鄭州 450007)

為了研究不同異養硝化-好氧反硝化菌富集馴化過程中水體微生物群落的演變,利用Miseq 高通量測序法對景觀水系沉積物富集馴化樣本的微生物信息進行統計,對其微生物群落的多樣性以及多樣性進行分析,同時基于微生物屬的信息進行了微生物網絡分析.結果顯示,兩種類型培養基在富集馴化完成后氮素得到有效去除,脫氮效果明顯;富集馴化過程中的OUT主要屬于7個,分別是變形菌門(Protebacterice)、擬桿菌門(Bacteroidetes)、綠彎菌門(Chloroflexi)、厚壁菌門(Firmicutes)、放線菌門(Actinobacteria)、藍藻門(Cyanobacteria)、酸桿菌門(Acidobacteria),與此同時,富集馴化過程中有關氮循環的細菌有上升的變化過程;主成分分析(PCA),非度量多維尺度分析(NMDS)以及主坐標分析(PCoA)表明異養硝化-好氧反硝化菌富集馴化過程中不同溫度壓力下的細菌群落組成存在明顯差異,而培養基的類別帶來的影響相對較小;網絡分析顯示模塊核心和網絡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構演變的關鍵環境因子.綜上可知,Miseq高通量測序研究異養硝化-好氧反硝化菌富集馴化過程中微生物種群演變可行,為實現微生物菌劑“定向-精準-高效”的篩選提供技術支撐.

異養硝化-好氧反硝化；景觀水系；Miseq測序；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,利用周質硝酸鹽還原酶進行反硝化作用的脫氮菌,并且大多數菌同時具有異養硝化的能力.它的發現打破了反硝化只能在厭氧缺氧條件下進行的傳統反硝化的觀念,為新型生物脫氮提供了新思路.

近年來,相關研究通過從土壤[2]、活性污泥[3]、污水[4-5]、河流[6]、湖泊[7-8]、水庫[9-10]等系統,分離了大量高效的好氧反硝化菌.前期文獻報道主要集中于通過施加一定選擇壓(間歇曝氣、鹽度或者溫度)或采用特定的選擇性培養基馴化富集.比如,趙驚鴻等[11]通過采取在好氧反硝化培養基連續培養中逐步升溫的方式,分離得到耐高溫好氧反硝化菌JH8;成鈺等[12]將通過在好氧反硝化培養液中添加氯化鈉的方式,分離得到耐鹽的異養硝化-好氧反硝化芽孢桿菌SLWX2;周培等[13]通過特定方法完成耐受重金屬好氧反硝化菌株的篩選;Carter等[14]利用周質硝酸鹽還原酶特定的生化特性和活性位點,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,關于在好氧反硝化菌富集馴化過程中微生物群落的演變過程鮮有報道.不同富集馴化方法下微生物群落演變規律直接影響高效好氧反硝化菌的定向篩選和特性研究,特別是對高效菌(混合菌群)的潛在功能預測以及菌劑的固定化提供必要依據.

石家莊中心城區水系作為省會的核心水系,保持健康的生態功能為城市的經濟發展提供了重要的生態保障.由于水系氮素超標嚴重,且主要為氨氮和硝酸鹽氮.完成黑臭水體治理,首先要恢復水體的自然復氧功能,使水體呈現好氧狀態.因此,如何在好氧環境下實現“定向-精準-高效”的氮素削減是水體自我修復功能恢復的關鍵,同時也是一個亟待解決的科學問題.本文選取石家莊中心城區典型區域的沉積物樣品,采用不同的異養硝化-好氧反硝化培養基進行常溫和低溫條件下異養硝化-好氧反硝化菌的富集馴化;與此同時研究富集馴化過程中的微生物群落演變特征,考察關鍵異養硝化-好氧反硝化物種,為進一步的高效菌劑定向篩選提供必要的技術支持.

1 材料與方法

1.1 實驗裝置

實驗裝置為2L的燒杯,取石家莊市中心城區世紀公園(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)的沉積物進行好氧反硝化菌的富集馴化.通過恒溫培養箱和充氧泵的間歇曝氣來控制系統的溫度和溶解氧,實驗的溫度設為室溫20℃和低溫10℃.每個2L燒杯中裝有200mL的沉積物和800mL的培養基,其中沉積物樣品用超純水進行了洗脫處理.通過選擇2種類型異養硝化-好氧反硝化培養基來富集馴化好氧反硝化菌.具體富集馴化過程為,首次為100%培養基,進行培養;3d后更換培養基為100%的培養基繼續培養;3d后更換培養基為80%的培養基培養;3d后更換為70%的培養基培養;3d后更換60%的培養基培養;3d后更換50%的滅菌富集培養基培養.每次更換期間測定各個系統水樣中硝酸鹽氮、亞硝酸鹽氮、氨氮和總氮來反應異養硝化-好氧反硝化菌的富集馴化效果.

1.2 異養硝化-好氧反硝化培養基

異養硝化-好氧反硝化(類型Ⅰ)富集培養基[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.

異養硝化-常規反硝化(類型Ⅱ)富集培養基[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擴增 選取初始的世紀公園和民心河的新鮮沉積物樣品以及富集馴化結束后的沉積物樣品,通過土壤DNA試劑盒(美國MP公司FastDNA SPINTMkit)并參照說明書提取沉積物的總DNA,采用瓊脂糖凝膠電泳法和分光光度計完成DNA完整性、純度與濃度檢測.沉積物在-80℃下保存,以備高通量測序.

1.3.2 微生物16S測序 將提取的沉積物總DNA樣本利用引物338F(ACTCCTACGGGAGGCAGC-AG)和806R(GGACTACHVGGGTWTCTAAT)對沉積物的16S rRNA基因V4-V5區進行PCR擴增,進而分析異養硝化-好氧反硝化菌富集馴化過程中微生物群落的演變過程.20μL PCR反應體系為: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.反應程序為:初始變性溫度95℃ 3.0min; 95℃ 30s, 55℃ 30s, 72℃ 45s,循環30次; 72℃ 10min讀板.將PCR擴增的產物委托上海美吉生物科技有限公司利用Miseq進行高通量測序.

1.3.3 生物信息學分析 對測序結果進行數據質控,依據相似度97%水平劃分OUT,并按照最小樣本序列進行抽平處理.利用R語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)以及主坐標分析(PCoA),進而分析不同培養基下異養硝化-好氧反硝化菌富集馴化過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篩選關鍵影響因子[18],并通過冗余分析(RDA)得到環境因子與微生物群落之間的相關性[19]. 利用Cytoscape軟件進行微生物群落的網絡分析[20],篩選出關鍵物種[21],進而分析不同選擇壓下富集馴化得到的異養硝化-好氧反硝化菌的物種信息.

1.4 水質分析方法

水質指標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0.45μm醋酸纖維濾膜過濾.

1.5 數據分析方法

繪圖和數據統計分析軟件為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[22],網絡分析中對網絡中節點進行如下分類:模塊核心(Z32.5,P￡0.62),網絡核心(Z30.5,P>0.62),外圍節點(Z<2.5,P￡0.62),連接點(Z<2.5,P>0.62).

2 結果與分析

2.1 源水脫氮效果分析

如圖1所示,兩種類型培養基在從100%培養基到50%培養基的富集馴化過程中,都表現出很強的好氧反硝化脫氮能力.類型Ⅰ培養基下,YX采樣點在低溫的反硝化能力要強于常溫,比如在50%的培養基條件下,低溫環境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,具體原因還需進一步分析.類型Ⅱ的培養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,在異養硝化-好氧反硝化菌富集馴化過程中,兩種培養基的氨氮并沒有表現出明顯的去除效果.其中,在結束富集馴化時常溫下的氨氮表現出更好的去除效果.比如,類型Ⅰ的培養基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.

圖1 異養硝化-好氧反硝化菌富集馴化過程中氮素變化特征

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

圖2 異養硝化-好氧反硝化菌富集馴化過程中間隙水氮素變化特征

關于富集馴化過程中各個系統的沉積物間隙水中氮素的變化情況如圖2所示.YX采樣點的氨氮呈現明顯的降低,類型Ⅰ的培養基從初始的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采樣點的硝酸鹽氮呈現明顯的降低,類型Ⅰ的培養基從初始的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采樣點表現出更高的氨氮去除效果,SJ采樣點表現出更高的硝酸鹽氮去除效果.

2.2 微生物α多樣性分析

通過對兩種培養基異養硝化-好氧反硝化菌富集馴化樣本16S rRNA基因進行測序,總共獲得了34881條有效序列,序列平均長度440bp.通過計算微生物多樣性指數考察不同類型培養基富集馴化異養硝化-好氧反硝化菌實驗過程中細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指數反映微生物群落的豐富度[23],兩種類型培養基下的微生物豐富度都有明顯的增加,Ace指數從2120.78增加到2319.69, Chao指數從2146.76增加到2344.23; Coverage反映微生物群落的覆蓋度[24],在富集馴化過程中維持在0.9887~0.9925之間,表明測序可以很好的覆蓋物種信息;Shannon和Simpson指數反映微生物群落的多樣性[25],Shannon指數大多數呈現增加,然而Simpson指數大多數為降低,微生物多樣性變化的復雜原因還需進一步分析.

表1 異養硝化-好氧反硝化菌富集馴化過程中生物多樣性指數

2.3 細菌群落組成及β多樣性分析

將異養硝化-好氧反硝化菌富集馴化過程中的樣本進行數據庫比對,同時分析在各個水平上的菌群結構.結果顯示屬于52個門類866個屬3081個OTU,具體結果如圖3所示.

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

在變形菌門中(圖3(b)),各個異養硝化-好氧反硝化菌富集馴化過程中共包含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[30].本實驗中,YX從初始的13.83%上升到23.22%;SJ從初始的14.62%上升到44.87%,表明在異養硝化-好氧反硝化菌富集馴化過程中,脫氮微生物得到明顯增加.

與此同時,兩種培養基在富集馴化過程中微生物種群存在共有的屬種,同時也存在差異.現將富集馴化過程中豐度前50的屬如圖3(c)所示,其中很多涉及氮素循環.比如,在培養基Ⅰ中,YX采樣點中的,,,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%.在培養基Ⅱ中,YX采樣點中的,,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,作為綠彎菌門的代表類群,屬于有機物降解的一類微生物[31];和促進氮素和有機物的去除[32-33],并且作為異養硝化-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程中得到增加[37];作為代表性異養硝化-好氧反硝化菌分離于廢水[38]、沉積物[39]和污泥[40]中;與[41]在系統發育上接近,也是重要的反硝化菌[42].

圖3 異養硝化-好氧反硝化菌富集馴化過程中種群變化特征

與此同時,基于OTU (97%相似性)水平,對兩種培養基類型的異養硝化-好氧反硝化菌富集過程的種群演變進行了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)以及主坐標分析(PCoA),來反映不同培養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圖中兩類培養基分布在PCA1軸的正負兩側,同一溫度條件下的樣本聚集相對緊密,低溫和常溫樣本間分布較分散,顯示2個采樣點在富集馴化過程中種群呈現差異性.NMDS中當stress<0.05時,則具有很好的代表性[43],本文分析顯示不同采樣點在2種培養基中,在富集馴化過程中物種呈現顯著差異,同一溫度的差異較小(圖4(b)).通過基于 Bray–Curtis距離的PCoA分析研究不同培養基類型富集馴化異養硝化-好氧反硝化菌的群落組成(圖4(c)).結果表明,不同溫度壓力下的細菌群落組成存在明顯差異,而培養基的類別帶來的影響相對較小,跟PCA、NMDS分析以及物種群落組成的結果相一致.

圖4 異養硝化-好氧反硝化菌富集馴化過程中種群差異分析

2.4 微生物網絡分析

基于異養硝化-好氧反硝化菌富集馴化過程中的微生物群落的屬,利用Cytoscape軟件構建微生物的互作網絡.

圖5 異養硝化-好氧反硝化菌富集馴化過程中微生物網絡及節點特征

圖5(a)中展示為斯皮爾曼相關系數>0.99的節點,節點大小為中介中心性,線的顏色綠色表示正相關,紅色表示負相關.網絡分析共得到412個節點,1327條邊;劃分成11個模塊,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%.網絡中節點正相關占比74.00%,負相關占比26.00%,表明物種間的關系呈現共生為主.Roger等[44]定義參數Z來衡量一個點在所在模塊中的作用,值越高說明在模塊中的作用越大;定義P來衡量一個點參與其他模塊的程度,值越高說明與其他模塊的聯系越密切.有關節點的分類如圖5(b)所示,網絡分析依據各節點Z值和P值將所有節點劃分為模塊核心,網絡核心,外圍節點和連接點四類.本網絡中模塊核心包括14個物種,網絡核心包括2個物種.模塊核心和網絡核心大多屬于低豐度的稀有物種,其中,能利用各種蛋白質進行生長[45],是一種降解有機物的關鍵物種[46-47];是污水處理廠的一個活性反硝化菌[48],說明稀有種在異養硝化-好氧反硝化菌富集馴化過程中對群落構建發揮著不可替代的作用.

2.5 環境因子與微生物群落的關系

基于VIF分析和蒙特卡羅檢驗,得到關鍵環境因子溫度(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相關性達到-0.14(=0.001<0.05),與RDA2相關性達到-0.99(= 0.001<0.05);氨氮與RDA1相關性達到-0.89(= 0.03<0.05),與RDA2相關性達到-0.45(=0.003< 0.05);硝酸鹽氮與RDA1相關性達到0.66(=0.03< 0.05),與RDA2相關性達到-0.75(=0.03<0.05).綜上,溫度決定了兩種培養基不同采樣點的不同溫度物種分布,常溫的富集馴化過程菌群分布在RDA2的正向,低溫的富集馴化過程菌群分布在RDA2的正向;氨氮和硝酸鹽氮分別與YX和SJ采樣點的富集馴化過程中菌群成正相關.

3 結論

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

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

3.3 不同溫度壓力下的細菌群落組成存在明顯差異, 而培養基的類別影響相對較小;溫度、氨氮以及硝酸鹽氮是影響群落結構的關鍵環境因子.微生物網絡中物種大多呈現共生關系, 關鍵節點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理與調控機制的相關研究.發表論文27篇.

2019-04-28

國家自然科學基金資助項目(51909056);河北科技大學引進人才科研啟動基金(1181278);河北省研究生創新資助項目(CXZZSS2018084)

*責任作者, 講師, ZSLZhouShilei@126.com