遼河口蘆葦濕地細(xì)菌和古菌群落周期日變化特征

李明月,米鐵柱*,甄 毓,王勛功

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遼河口蘆葦濕地細(xì)菌和古菌群落周期日變化特征

李明月1,2,3,米鐵柱1,2,3*,甄 毓1,2,3,王勛功2,3

(1.青島海洋科學(xué)技術(shù)國家實(shí)驗(yàn)室海洋生態(tài)與環(huán)境科學(xué)功能實(shí)驗(yàn)室,山東 青島 266071；2.海洋環(huán)境與生態(tài)教育部重點(diǎn)實(shí)驗(yàn)室,山東 青島 266100；3.中國海洋大學(xué)環(huán)境科學(xué)與工程學(xué)院,山東 青島 266100)

采用實(shí)時(shí)熒光定量PCR和Illumina高通量測序技術(shù)對秋季遼河口蘆葦濕地水體中一晝夜細(xì)菌和古菌群落特征進(jìn)行了研究.測序共獲得52802條細(xì)菌和106091條古菌高質(zhì)量序列,以97%的相似性聚類并抽平后共劃分了530和979個(gè)OTU.結(jié)果表明,濕地水體中浮游細(xì)菌和古菌群落豐度均在早上達(dá)到最高值,夜晚達(dá)到最低值.細(xì)菌和古菌群落多樣性指數(shù)均存在周期日變化,不同時(shí)間細(xì)菌和古菌群落結(jié)構(gòu)和組成具有顯著差異.此外,古菌群落組成特征表明遼河口蘆葦濕地具有較高的潛在甲烷生成能力.本研究為進(jìn)一步認(rèn)識(shí)河口濕地細(xì)菌和古菌群落特征和功能奠定了基礎(chǔ).

濕地；16S rRNA；細(xì)菌；古菌；群落結(jié)構(gòu)

濕地是位于陸地和水體的過渡地帶,包括多種類型[1],其中河口濕地位于河流與海洋的交匯處,長期受到?jīng)_刷和斷流等影響,處于不斷的動(dòng)態(tài)變化之中.河口濕地兼具淡水與海水生態(tài)系統(tǒng)的特點(diǎn),各種生物地球化學(xué)過程交織,為大量生物提供了充足的棲息地[2].

微生物是河口濕地的重要組成部分,對河口濕地生態(tài)系統(tǒng)具有重要作用.研究表明,微生物群落及其多樣性影響著濕地生態(tài)系統(tǒng)的特性和功能,是實(shí)現(xiàn)濕地生態(tài)功能、表征濕地環(huán)境變化的關(guān)鍵因素[3].細(xì)菌和古菌群落多樣性、組成和結(jié)構(gòu)與各種環(huán)境因子(例如溫度、鹽度、pH值、氮、硫、總有機(jī)碳、Fe、碳和氮的礦化、水體有機(jī)污染和富營養(yǎng)化等)[4-8]的交互作用,是其影響河口濕地生態(tài)系統(tǒng)的重要途徑之一.對于河口濕地細(xì)菌和古菌群落的研究大多集中于濕地土壤和沉積物中[4-8],而對濕地積水中微生物的研究則相對較少.濕地積水中的微生物群落特征及其與環(huán)境因子關(guān)系的研究,有助于全面理解河口濕地生態(tài)系統(tǒng)的作用機(jī)制,可為河口濕地環(huán)境的原位修復(fù)和污染物微生物處理提供研究基礎(chǔ),這對河口濕地生態(tài)系統(tǒng)的可持續(xù)利用與綜合管理,實(shí)現(xiàn)生態(tài)、經(jīng)濟(jì)、社會(huì)的協(xié)調(diào)發(fā)展具有重要意義.

遼河口濕地位于我國東北部,遼河入?？谔?屬于半濕潤大陸季風(fēng)性氣候,其蘆葦覆蓋密度較大.同時(shí),該區(qū)域具有豐富的天然氣和石油資源[9].這里生長著許多物種,包括珍稀瀕危物種,其生態(tài)系統(tǒng)的穩(wěn)定具有重要意義.

目前,對遼河口濕地微生物群落的相關(guān)研究多集中于濕地土壤[10-12]或蘆葦根際[13],且多采用傳統(tǒng)分子生態(tài)學(xué)方法[10-11,13],如PCR-DGGE技術(shù)[10-11].本研究擬采用實(shí)時(shí)熒光定量PCR和Illumina Miseq高通量測序技術(shù),結(jié)合環(huán)境因子的測定,分析遼河口蘆葦濕地水體中細(xì)菌和古菌群落豐度、多樣性、組成和結(jié)構(gòu)的周期日變化規(guī)律.本研究將為遼河口濕地區(qū)域內(nèi)生態(tài)系統(tǒng)保護(hù)與可持續(xù)發(fā)展提供基礎(chǔ)資料和理論依據(jù).

1 材料與方法

1.1 研究位點(diǎn)及樣品采集

研究區(qū)域位于遼寧省盤錦市雙臺(tái)子河口濕地羊圈子葦場(圖1).于2015年9月13日8:00、12:00和23:00采集300mL水樣(S08、S12和S23)并依次用1μm和0.2μm聚碳酸酯濾膜(47mm, Whatman, UK)過濾,所有樣品儲(chǔ)存于-80℃,以備后續(xù)DNA提取實(shí)驗(yàn).現(xiàn)場采集水樣并測定溫度(T)、pH值、鹽度(Sal)和溶解氧(DO).用于測定溶解有機(jī)碳(DOC)、總氮(TN)、氨氮(NH4-N)、亞硝酸鹽氮(NO2-N)、硝酸鹽氮(NO3-N)、磷酸鹽(PO4-P)和葉綠素a(Chl a)的水樣,經(jīng)過濾后帶回實(shí)驗(yàn)室-20℃保存至分析.

圖1 遼河口蘆葦濕地積水樣品采樣點(diǎn)

1.2 樣品分析

1.2.1 環(huán)境參數(shù)的測定 積水樣品的T、pH值、DO和Sal采用HQ40d (HACH, USA)現(xiàn)場采樣測定.DOC、Chl a和營養(yǎng)鹽的測定詳見已有研究[14].

1.2.3 定量PCR 使用細(xì)菌16S rRNA基因引物338F和806R[15]和古菌16S rRNA基因引物U519F/806R[16],擴(kuò)增16S rRNA基因.退火溫度為58 ℃,擴(kuò)增產(chǎn)物長度約為470bp和300bp.1%瓊脂糖凝膠電泳分離PCR產(chǎn)物,純化目的片段.pMD18-T載體連接純化產(chǎn)物進(jìn)行轉(zhuǎn)化反應(yīng)后,選取陽性克隆提取質(zhì)粒.以梯度稀釋質(zhì)粒標(biāo)準(zhǔn)品作為模板,采用Applied Biosystems 7500 Real Time PCR System (Life Technologies, Forster City, CA, USA)進(jìn)行qPCR.反應(yīng)體系均為:10μL FastStart Universal SYBR Green Master (ROX)(Roche, 德國),0.6μL 10μM正向引物,0.6μL 10μM反向引物(表1),2μL模板DNA,6.6μL去離子水和0.2μL mg/mL牛血清蛋白.每個(gè)樣品設(shè)置3個(gè)平行樣,每組實(shí)驗(yàn)設(shè)置陰性對照.擴(kuò)增條件均為:50℃ 2min;40個(gè)循環(huán),95℃ 10min, 95 ℃ 15s,58℃ 2min.擴(kuò)增完畢后分析熔解曲線,確保模板DNA的特異性擴(kuò)增.以質(zhì)?？截悢?shù)對數(shù)值為橫坐標(biāo),平均Ct值為縱坐標(biāo),繪制細(xì)菌和古菌16S rRNA基因質(zhì)粒標(biāo)準(zhǔn)曲線.

樣品中細(xì)菌和古菌16S rRNA基因qPCR步驟與上述步驟相同,每個(gè)樣品設(shè)置3個(gè)平行樣,并添加陽性對照和陰性對照.

表1 細(xì)菌和古菌16S rRNA基因引物

1.2.4 高通量測序 以樣品DNA為PCR反應(yīng)模板,擴(kuò)增細(xì)菌16S rRNA基因V3-V4區(qū)和古菌16S rRNA基因V4-V5區(qū).細(xì)菌和古菌PCR反應(yīng)引物分別為343F/798R[17]和Arch344F/Arch915R[18](表1).純化后質(zhì)量合格的PCR產(chǎn)物構(gòu)建DNA文庫,合格后使用Illumina Sequencer Miseq平臺(tái)進(jìn)行高通量測序.測序所得原始數(shù)據(jù)已上傳NCBI數(shù)據(jù)庫,序列號(hào)為SRP161823.

1.3 數(shù)據(jù)分析

利用Trimmomatic(v 0.35)[19]軟件對高通量測序得到的雙端序列(raw reads)去雜后,使用FLASH (v 1.2.11)[20]軟件進(jìn)行拼接.拼接后的序列使用QIIME (v 8.0)[21]軟件進(jìn)行降噪和過濾,將含有模糊堿基、單堿基高重復(fù)區(qū)或長度過短的序列去除(clean reads),保留75%以上的堿基質(zhì)量高于Q20的序列.之后對clean reads進(jìn)行去嵌合體序列處理,得到優(yōu)質(zhì)序列(valid reads)用于下游分析.使用VSERCH (v 2.4.2)[22]軟件對所有優(yōu)質(zhì)序列以97%相似度進(jìn)行OTU聚類劃分,并用RDP classifier (v 2.2)[23]將代表序列與Silva (v 123)[24]數(shù)據(jù)庫進(jìn)行比對,得到系統(tǒng)發(fā)育樹和OTU分類表格.保證足夠測序深度的同時(shí),對所有樣品進(jìn)行均一化處理,以避免不同數(shù)據(jù)量大小造成的分析偏差.細(xì)菌和古菌群落16S rRNA基因拷貝數(shù)根據(jù)qPCR結(jié)果和質(zhì)粒標(biāo)準(zhǔn)曲線計(jì)算.利用QIIME軟件計(jì)算Alpha多樣性指數(shù)及稀釋曲線相關(guān)點(diǎn)的抽取.

2 結(jié)果與分析

2.1 理化參數(shù)

濕地積水樣品理化參數(shù)統(tǒng)計(jì)結(jié)果見表2.中午水樣溫度最高,夜晚水樣溫度最低,且DO、TN和NH4-N與溫度具有同樣的變化趨勢,而NO2-N具有與溫度相反的變化趨勢.pH值和DOC在樣品中的濃度在早上、夜晚和中午依次遞減,Chl a則依次遞增,Chl a在中午的濃度為46.16μg/L,遠(yuǎn)高于早晨和夜晚.

表2 遼河口蘆葦濕地積水樣品理化參數(shù)

2.2 細(xì)菌和古菌16S rRNA基因豐度

圖2 遼河口蘆葦濕地不同時(shí)間積水中細(xì)菌和古菌16S rRNA基因豐度

定量PCR結(jié)果表明,遼河口蘆葦濕地積水中細(xì)菌和古菌16S rRNA基因豐度分別為3.30×104~ 7.62×107copy/L和8.40×106~6.56×107copy/L(圖2).從8:00~23:00,細(xì)菌和古菌16S rRNA 基因豐度均呈現(xiàn)出降低趨勢,且早上和中午細(xì)菌和古菌16S rRNA基因豐度遠(yuǎn)高于夜晚.總體來看,古菌16S rRNA 基因豐度略高于細(xì)菌,占細(xì)菌和古菌總16S rRNA 基因豐度的52.54%.

2.3 細(xì)菌和古菌群落多樣性

遼河口蘆葦濕地積水中不同時(shí)間樣品通過高通量測序并質(zhì)控后,共得到52802條高質(zhì)量細(xì)菌序列.樣品序列條數(shù)范圍為15999~18502條,根據(jù)稀釋曲線隨機(jī)抽取15998條序列對所有樣品做均一化處理(圖3).以97%的相似性水平聚類,共得到530個(gè)OTU,OTU數(shù)目最多的時(shí)間點(diǎn)是早上8:00的水樣,最低的是夜晚23:00的水樣.早上、中午和夜晚共有203個(gè)OTU(圖4),占總OTU數(shù)的38.30%.早上獨(dú)有74個(gè)OTU,中午和夜晚則分別獨(dú)有61和44個(gè)OTU,表明從早上到夜晚的過程中細(xì)菌種類隨環(huán)境條件的改變而減少.早上與中午共有79個(gè)OTU,與夜晚共有34個(gè)OTU,而中午與夜晚共有35個(gè)OTU,可見早上與中午細(xì)菌種類差別較小,而夜晚與早上、中午細(xì)菌種類差別較大.

圖4 遼河口蘆葦濕地不同時(shí)間積水中細(xì)菌和古菌OTU韋恩

所有水樣中細(xì)菌測序覆蓋度均在99.36%以上,測序深度能夠基本覆蓋全部物種(表3).Shannon指數(shù)表明早上細(xì)菌群落多樣性最高,夜晚最低,與NO3-N具有相反的變化趨勢.而Chao 1指數(shù)表明中午細(xì)菌群落豐富度略高于早上,夜晚則遠(yuǎn)低于早上和中午,其變化趨勢與T、DO、TN和NH4-N一致,與NO2-N相反.此外,早上水樣中的細(xì)菌群落均勻度最高(Pielou指數(shù)).

表3 遼河口蘆葦濕地不同時(shí)間積水中細(xì)菌和古菌的α-多樣性指數(shù)

注:a序列條數(shù)抽平為15998,b序列條數(shù)抽平為29352.

不同時(shí)間積水中古菌測序并質(zhì)控后,共得到106091條高質(zhì)量序列,平均序列條數(shù)為35363±5708.對所有樣品抽平后序列數(shù)量足夠,測序深度選取比較合理(29352條序列,圖3),共得到979個(gè)OTU(97%的相似性聚類).早上8:00OTU數(shù)量最高,中午12:00OTU數(shù)量最低,分別為839和692個(gè),表明古菌群落對環(huán)境的適應(yīng)性與細(xì)菌不同.3個(gè)時(shí)間點(diǎn)共有530個(gè)OTU(圖4),占所有OTU數(shù)的54.14%.早上獨(dú)有91個(gè)OTU,中午獨(dú)有46個(gè)OTU,夜晚獨(dú)有58個(gè)OTU,表明從早上到夜晚的過程中古菌種類隨環(huán)境條件的改變先減少后升高.早上與中午共有80個(gè)OTU,與夜晚共有138個(gè)OTU,而中午與夜晚共有36個(gè)OTU,可見早上與夜晚古菌種類差別較小,而中午與兩者古菌種類差別較大.

古菌測序覆蓋度在所有樣品中均在99.80%以上,測序深度能夠基本覆蓋全部物種(表3).早上、中午和夜晚古菌群落多樣性和均勻度逐漸降低,與NO3-N具有相反的變化趨勢,而豐富度最高值出現(xiàn)在早上,最低值出現(xiàn)在中午,變化趨勢與DOC相同,與Chl a相反.

2.4 細(xì)菌和古菌群落的組成

通過與Silva (v123)數(shù)據(jù)庫進(jìn)行比對,對OTU進(jìn)行物種注釋和分類.結(jié)果表明,99.92%的細(xì)菌序列被注釋到門分類水平,31.34%的細(xì)菌序列被注釋到屬水平(圖5),河口濕地還蘊(yùn)含著大量的未知細(xì)菌資源.分別在門、綱、目、科、屬水平上對相對豐度較高的細(xì)菌類群做相對豐度圖(圖6).在門分類水平上(圖6),相對豐度大于1%的優(yōu)勢門[25]包括變形菌門(Proteobacteria)、擬桿菌門(Bacteroidetes)、放線菌門(Actinobacteria)和厚壁菌門(Firmicutes).其中,變形菌門(Proteobacteria)、擬桿菌門(Bacteroidetes)和放線菌門(Actinobacteria)的相對豐度總和為96.60%~97.65%.從早上~夜晚,變形菌門(Proteobacteria)、放線菌門(Actinobacteria)和Saccharibacteria相對豐度逐漸增大,而擬桿菌門(Bacteroidetes)和疣微菌門(Verrucomicrobia)相對豐度逐漸減少,且早上變形菌門(Proteobacteria)和擬桿菌門(Bacteroidetes)相對豐度與中午和夜晚差別較大.在綱、目和科水平上(圖6),優(yōu)勢細(xì)菌菌群具有和門水平相似的時(shí)間變化規(guī)律,例如α變形菌綱(Alphaproteobacteria)相對豐度從早上到夜晚逐漸增大,Flavobacteriia相對豐度逐漸減少,且兩者在早上的相對豐度與中午和夜晚差別較大.在屬分類水平上,早上、中午和夜晚積水樣品中細(xì)菌群落組成差別較大(圖6).早上水樣中相對豐度大于1%的優(yōu)勢菌屬[25]為(相對豐度8.65%)、黃桿菌屬(,3.84%)、(1.81%)和(1.66%),中午為嗜甲基菌屬(,2.18%)、(1.34%)、(1.27%)和(1.18%),夜晚為萊茵海默氏菌(, 5.66%)、(2.31%)、交替赤桿菌屬(,2.16%)、交替單胞菌(,1.69%)、(1.51%)、(1.36%)和(1.14%).

圖5 遼河口蘆葦濕地不同時(shí)間積水中細(xì)菌和古菌注釋序列條數(shù)占總條數(shù)的百分比

Fig.5 The proportion of annotated sequences in all sequences of bacterial and archaeal communities in the reed wetland waters of Liaohe estuary

所有樣品共有95.80%古菌序列注釋到門水平,23.95%注釋到屬水平,河口濕地蘊(yùn)含的未知古菌資源更為豐富(圖5).在門分類水平上,所有序列一共注釋了9個(gè)古菌門,其中Woesearchaeota_ (DHVEG_6)、廣古菌門(Euryarchaeota)和Miscellaneous_Euryarchaeotic_Group(MEG)占所有菌群的93.80~94.72%.早上和中午Woesearchaeota_ (DHVEG_6)相對豐度高于夜晚,廣古菌門(Euryarchaeota)則低于夜晚.熱源體綱(Thermoplasmata)、甲烷桿菌綱(Methanobacteria)和甲烷微菌綱(Methanomicrobia)為主要古菌綱類群(圖7),20a_9、熱原體目(Thermoplasmatales)、甲烷桿菌目(Methanobacteriales)、Methanocellales和球菌目(Methanosarcinales)為主要古菌目類群(圖7),甲烷桿菌科(Methanobacteriaceae)、Methanocellaceae和球菌科(Methanosarcinaceae)為主要古菌科類群(圖7),甲烷細(xì)菌屬()、和甲烷八疊球菌屬()為主要古菌屬類群(圖7d).遼河口濕地積水中檢出了7個(gè)產(chǎn)甲烷古菌目中的5個(gè),即熱原體目、甲烷桿菌目、甲烷胞菌目、甲烷八疊球菌目和甲烷微菌目,且具有較高的相對豐度.樣品中古菌群落組成特征表明了遼河口蘆葦濕地活躍的甲烷相關(guān)生物地球化學(xué)過程.

圖6 遼河口蘆葦濕地不同時(shí)間積水中細(xì)菌門、綱、目、科和屬水平相對豐度

圖7 遼河口蘆葦濕地不同時(shí)間積水中古菌綱、目、科、屬水平相對豐度

2.5 細(xì)菌和古菌群落結(jié)構(gòu)

挑選細(xì)菌和古菌相對豐度最高的前30個(gè)OTU做OTU豐度熱圖(圖8).不同時(shí)間積水樣品中細(xì)菌和古菌群落結(jié)構(gòu)明顯不同,且中午和夜晚細(xì)菌群落結(jié)構(gòu)較為相似,聚為一類,早上和夜晚古菌群落結(jié)構(gòu)較為相似,聚為一類.此外,早上、中午和夜晚水樣中細(xì)菌優(yōu)勢OTU明顯各不相同,古菌優(yōu)勢OTU也有一定的差異,表明不同時(shí)間點(diǎn)遼河口蘆葦濕地細(xì)菌群落結(jié)構(gòu)有一定的差異.從屬分類水平挑選豐度最高的前15個(gè)物種做物種豐度熱圖(圖8),細(xì)菌和古菌群落均是早上和中午聚為一類.不同時(shí)間點(diǎn)細(xì)菌和古菌優(yōu)勢種群具有明顯差異,例如,早上古菌優(yōu)勢屬為、甲烷粒菌屬()和鹽球菌屬()等,中午為甲烷桿菌屬()等,而夜晚為甲烷囊菌屬()等.

圖8 遼河口蘆葦濕地不同時(shí)間積水中細(xì)菌OTU、古菌OTU、細(xì)菌屬和古菌屬水平豐度熱圖

3 討論

采用qPCR技術(shù)和Illumina高通量測序技術(shù),結(jié)合環(huán)境因子,對遼河口蘆葦濕地羊圈子葦場內(nèi)積水中細(xì)菌和古菌群落的豐度、多樣性、組成和結(jié)構(gòu)進(jìn)行了分析研究.結(jié)果表明,研究區(qū)域內(nèi)營養(yǎng)鹽濃度整體低于遼河口蘆葦濕地河蟹養(yǎng)殖區(qū)[26]和其他區(qū)域[27],且遠(yuǎn)低于其他濕地水體[28].DOC含量為13m/L左右,與其他濕地相比略低[29]. Chl a表現(xiàn)出顯著的周期日變化特征,與已有報(bào)道一致,最高值出現(xiàn)在正午前后[30].

遼河口蘆葦濕地中細(xì)菌豐度與湖泊濕地浮游細(xì)菌豐度相當(dāng),但低于其他水體環(huán)境[31-32](細(xì)胞個(gè)數(shù)與16S rRNA基因拷貝數(shù)進(jìn)行3.45倍換算[33]).而古菌豐度與其他水體環(huán)境相當(dāng)[34],且古菌豐度高于細(xì)菌,這與其他研究結(jié)果不同[35].本研究位點(diǎn)中細(xì)菌和古菌豐度均存在周期日變化特征(圖2),在溫度最低的夜晚,豐度最低,且顯著低于其他時(shí)間點(diǎn).有研究表明濕地浮游細(xì)菌豐度與溫度和Chl a等具有相關(guān)性[36],異養(yǎng)細(xì)菌豐度周期日變化與微微型真核浮游植物之間具有相關(guān)性,且受到濁度的影響[37].因此,白天水溫升高刺激微生物生長繁殖加速,同時(shí)光合作用增強(qiáng)引起的濕地植物生長繁殖加快間接促進(jìn)微生物的生長,都是細(xì)菌和古菌豐度在早上出現(xiàn)最高值的原因[38].但是,中午細(xì)菌和古菌豐度略低于早上,這則與中午的強(qiáng)光照和紫外線輻射有密切的聯(lián)系[30].本研究中細(xì)菌和古菌豐度的周期日變化趨勢與Chl a變化趨勢不一致,其豐度可能受到溫度、Chl a和早晚透光度等共同作用的影響.

細(xì)菌和古菌群落Alpha多樣性分析包括Shannon指數(shù)、Chao 1指數(shù)和Pielou指數(shù),分別代表多樣性、豐富度和均勻度.遼河口蘆葦濕地積水中細(xì)菌和古菌群落多樣性和豐富度都很高(表3),且古菌群落多樣性、豐富度和均勻度均高于細(xì)菌,這與16S rRNA基因豐度定量結(jié)果一致.此外,遼河口濕地積水中細(xì)菌和古菌群落生物多樣性均具有周期日變化特征,整體表現(xiàn)為早上生物多樣性較高,夜晚較低. 由于對濕地水體中微生物群落的研究較少,具體原因目前還無法判讀,但有報(bào)道發(fā)現(xiàn)海水中細(xì)菌在白天的多樣性高于夜晚,具有明顯的晝夜之分[30]. Zhang等[39]發(fā)現(xiàn)近岸海域中浮游細(xì)菌的豐度、生長和多樣性受到病毒和鞭毛蟲的共同作用影響,而Christian等[40]指出病毒性的細(xì)菌裂解通常發(fā)生在中午~下午,且大都在夜間發(fā)生感染,導(dǎo)致夜間細(xì)菌數(shù)量減少,多樣性也隨之降低.

細(xì)菌主要優(yōu)勢菌群為變形菌門(Proteobacteria)、擬桿菌門(Bacteroidetes)、放線菌門(Actinobacteria),此結(jié)論與其他濕地浮游細(xì)菌優(yōu)勢菌群一致[41-45].遼河口蘆葦濕地積水中檢測到了相對豐度較高的海洋菌屬,說明濕地系統(tǒng)可能受到海水的影響.然而,氮代謝相關(guān)的菌屬相對豐度較小,可能是因?yàn)樵摑竦叵到y(tǒng)氮代謝不活躍.研究表明,遼河口濕地典型的寒冷氣候會(huì)大幅度影響參與氨轉(zhuǎn)化的酶的活性,從而進(jìn)一步影響硝化效率[46],且低溫和高鹽度是遼河口硝化作用的主要限制因子[47].

關(guān)于濕地水體中古菌群落組成的研究較少,濕地土壤或沉積物中古菌優(yōu)勢種群為Euyarchaeota和Thaumarchaeota等[48-49],與本研究濕地水體中主要種群存在一定的差異.濕地是大氣甲烷最大的自然源,遼河口濕地積水中古菌群落組成以產(chǎn)甲烷菌為主,其中極端厭氧甲烷桿菌屬和甲烷八疊菌屬等厭氧菌有較高的相對豐度.但是遼河口濕地積水中DO較為豐富,且有研究發(fā)現(xiàn)在蘆葦濕地土壤中產(chǎn)甲烷菌僅存在于地表下10cm深處[12],這說明濕地積水與沉積物之間可能具有活躍的交換作用.

序列分析表明,不同時(shí)間點(diǎn)積水樣品中細(xì)菌和古菌群落組成和結(jié)構(gòu)表現(xiàn)出了明顯的時(shí)間分布差異.變形菌門和放線菌門晝夜變化表現(xiàn)出相反的趨勢,在海水中也有同樣的發(fā)現(xiàn)[30].-變形菌綱相對豐度在夜間明顯高于白天.有研究發(fā)現(xiàn),由于該類群的光捕獲色素基因在光照下激發(fā)細(xì)胞分裂,其數(shù)量在白天明顯高于夜間[50].甲烷八疊球菌屬在中午的相對豐度最高、夜晚最低,表明濕地中甲基營養(yǎng)型產(chǎn)甲烷途徑在中午最為活躍[51].

微生物作用是濕地系統(tǒng)生態(tài)功能的重要組成部分,生態(tài)環(huán)境的改變與微生物群落的變化息息相關(guān)[52].研究區(qū)域位于羊圈子葦場蘆葦覆蓋密集區(qū),根據(jù)蘆葦不同生長期的需求,積水每年秋季排入遼河.積水微生物群落周期日特征主要受晝夜變化的影響,無潮汐等其他因素的影響.微生物群落豐度、多樣性等均在夜晚較低,若能在夜間排水,蘆葦濕地微生物資源損失較小,蘆葦濕地功能可維持相對穩(wěn)定的狀態(tài).而且,積水中微生物群落對遼河微生物群落也會(huì)有較小的沖擊.遼河口蘆葦濕地位于重要的工業(yè)區(qū)和農(nóng)業(yè)區(qū),受人類活動(dòng)影響較大,微生物群落組成及其多樣性等特性隨之受到影響.對遼河口蘆葦濕地微生物群落的研究不僅有利于了解微生物群落整體特征,而且有助于發(fā)現(xiàn)功能微生物,為遼河口污染物降解和濕地管理等提供理論基礎(chǔ).因此,進(jìn)一步考察遼河口蘆葦濕地中微生物群落的時(shí)空分布特征具有重要意義.由于受各種條件的限制,我們只進(jìn)行了一個(gè)采樣點(diǎn)不同時(shí)段的一次采樣,對濕地積水中微生物群落的周期日變化進(jìn)行了初步探討.在后期研究中,需要進(jìn)一步在不同采樣站位進(jìn)行采樣,以獲得更為全面的微生物群落信息.

4 結(jié)論

4.1 對遼河口蘆葦濕地一晝夜細(xì)菌和古菌群落的實(shí)時(shí)熒光定量PCR和Illumina高通量測序分析結(jié)果表明,細(xì)菌和古菌群落豐度、多樣性、組成和結(jié)構(gòu)具有顯著的周期日變化.早上和中午積水中細(xì)菌和古菌群落豐度差異較小,且遠(yuǎn)高于夜間.早上細(xì)菌和古菌多樣性最高,夜晚最低.

4.2 古菌群落組成特征表明遼河口蘆葦濕地具有較高的潛在甲烷生成能力.

[1] 楊朝飛.中國濕地現(xiàn)狀及其保護(hù)對策 [J]. 中國環(huán)境科學(xué), 1995, 15(6):407-412.Yang C F. Current status and conservation strategy of China’s wetland [J]. China Environmental Science, 1995,15(6):407-412.

[2] 黃桂林,何 平,侯 盟.中國河口濕地研究現(xiàn)狀及展望 [J]. 應(yīng)用生態(tài)學(xué)報(bào), 2006,17(9):1751-1756. Huang G L, He P, Hou M. Present status and prospects of estuarine wetland research in China [J]. Chinese Journal of Applied Ecology, 2006,17(9):1751-1756.

[3] 車玉伶,王 慧,胡洪營,等.微生物群落結(jié)構(gòu)和多樣性解析技術(shù)研究進(jìn)展 [J]. 生態(tài)環(huán)境學(xué)報(bào), 2005,14(1):127-133. Che Y L, Wang H, Hu H Y, et al. Research progresses on analytical technologies used in microbial community structure and diversity [J]. Ecology and Environment, 2005,14(1):127-133.

[4] Franklin R B, Morrissey E Mand Morina J C. Changes in abundance and community structure of nitrate-reducing bacteria along a salinity gradient in tidal wetlands [J]. Pedobiologia, 2017,60:21-26.

[5] Zhang H, Zheng S, Ding J, et al. Spatial variation in bacterial community in natural wetland-river-sea ecosystems [J]. Journal of Basic Microbiology, 2017,57(6):536-546.

[6] Li X, Hou L, Liu M, et al. Primary effects of extracellular enzyme activity and microbial community on carbon and nitrogen mineralization in estuarine and tidal wetlands [J]. Applied Microbiology & Biotechnology, 2015,99(6):2895-2909.

[7] Fei X X, Wang L, Jun H J, et al. Salinity influence on soil microbial respiration rate of wetland in the Yangtze River estuary through changing microbial community [J]. Journal of Environmental Science, 2014,26(12):2562-2570.

[8] Zhang Y, Wang L, Hu Y, et al. Water Organic Pollution and Eutrophication Influence Soil Microbial Processes, Increasing Soil Respiration of Estuarine Wetlands: Site Study in Jiuduansha Wetland [J]. Plos One, 2015,10(5):e0126951.

[9] Lin T, Ye S, Ma C, et al. Sources and preservation of organic matter in soils of the wetlands in the Liaohe (Liao River) Delta, North China [J]. Marine Pollution Bulletin, 2013,71(1/2):276-285.

[10] 關(guān)曉燕,韓家波,王 擺,等.遼東灣大凌河口濕地土壤微生物群落分析 [J]. 生態(tài)環(huán)境學(xué)報(bào), 2012,21(6):1063-1070. Guan X Y, Han J P, Wang B, et al. Analysis of bacterial communities in Liaodong Bay Dalinghe estuarine wetland [J]. Ecology and Environmental Sciences, 2012,21(6):1063-1070.

[11] 趙先麗,周廣勝,周 莉,等.盤錦蘆葦濕地土壤微生物數(shù)量研究 [J]. 土壤通報(bào), 2008,39(6):1376-1379.Zhao X L, Zhou G S, Zhou L, et al. Characteristics of soil microbial community in bulrush wetlands of Panjin, Northeast China [J]. Chinese Journal of Soil Science, 2008,39(6):1376-1379.

[12] 魯青原.遼河三角洲濱海濕地微生物群落組成及其環(huán)境意義 [D]. 北京:中國地質(zhì)大學(xué), 2016. Lu Q Y. The microbial community structure in Liaohe Delta wetlands and its environmental significances [D]. Beijing: China University of Geosciences, 2016.

[13] 田偉君,王勇梅,孫會(huì)梅,等.石油輸入對河口蘆葦濕地根際微生物的影響 [J]. 中國環(huán)境科學(xué), 2014,34(10):2676-2683. Tian W J, Wang Y M, Sun H M, et al. Effect of oil pollution on community structure of plants rhizospheric microorganisms in estuarine wetland [J]. China Environmental Science, 2014,34(10): 2676-2683.

[14] 李 璐,鄒 立,孟泰舟,等.遼河口蘆葦濕地有色溶解有機(jī)物的光譜特征研究 [J]. 中國海洋大學(xué)學(xué)報(bào):自然科學(xué)版, 2017,(12):27-36. Li L, Zou L, Meng T Z, et al. Absorbance and fluorescence spectrums of chromophoric dissolved organic matter in the reed wetland of Liaohe estuary [J]. Periodical of Ocean University of China, 2017, (12):27-36.

[15] Peiffer J A, Spor A, Koren O, et al. Diversity and heritability of the maize rhizosphere microbiome under field conditions [J]. Proceedings of the National Academy of Sciences, 2013,110(16):6548-6553.

[16] Porat I, Vishnivetskaya T A, Mosher J J, et al. Characterization of archaeal community in contaminated and uncontaminated surface stream sediments [J]. Microbial ecology, 2010,60(4):784-795.

[17] Carlos, Nossa, William, et al. Design of 16S rRNA gene primers for 454pyrosequencing of the human foregut microbiome [J]. World Journal of Gastroenterology, 2010,16(33):4135-4144.

[18] Ohene-Adjei S, Teather R M, Ivan M, et al. Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen [J]. Applied and Environmental Microbiology, 2007, 73(14):4609-4618.

[19] Bolger A M, Lohse Mand Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data [J]. Bioinformatics, 2014,30(15):2114- 2120.

[20] Reyon D, Tsai S Q, Khayter C, et al. FLASH assembly of TALENs for high-throughput genome editing [J]. Nature Biotechnology, 2012, 30(5):460-465.

[21] Caporaso J G, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data [J]. Nature Methods, 2010,7(5):335-336.

[22] Rognes T, Flouri T, Nichols B, et al. VSEARCH: a versatile open source tool for metagenomics [J]. Peerj, 2016,4(10):1-22.

[23] Wang Q, Garrity G M, Tiedje J M, et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy [J]. Applied & Environmental Microbiology, 2007,73(16):5261-5267.

[24] Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools [J]. Nucleic Acids Research, 2013,41(Database issue):590-596.

[25] Galand P E, Casamayor E O, Kirchman D L, et al. Ecology of the rare microbial biosphere of the Arctic Ocean [J]. Proceedings of the National Academy of Sciences, 2009,106(52):22427-22432.

[26] 羅西玲,邢 磊,徐賓鐸,等.遼河口蘆葦濕地河蟹養(yǎng)殖區(qū)水體N、P營養(yǎng)鹽和COD的變化 [J]. 海洋湖沼通報(bào), 2016,(3):20-27. Luo X L, Xing L, Xu B Y, et al. Variations in nitrogen and phosphorous nutrients and COD in the river crab aquaculture areas in reed wetland of Liaohe estuary [J]. Transactions of Oceanology and Limnology, 2016,(3):20-27.

[27] 徐微雪,段 亮,宋永會(huì),等.遼河保護(hù)區(qū)七星濕地表層水與間隙水中氮的時(shí)空分布 [J]. 環(huán)境工程技術(shù)學(xué)報(bào), 2014,4(1):40-45. Xu W X, Duan L, Song Y H, et al. Spatio-temporal distributions of nitrogen in surface water and pore water in Qixing Wetland of Liaohe conservation area [J]. Journal of Environmental Engineering Technology, 2014,4(1):40-45.

[28] 吳 明,邵學(xué)新,蔣科毅.西溪國家濕地公園水體和底泥N、P營養(yǎng)鹽分布特征及評價(jià) [J]. 林業(yè)科學(xué)研究, 2008,21(4):587-591. Wu M, Shao X X, Jiang K Y. Characteristics and assessment on nutrient distribution in water and sediments of Xixi National Wetland Park in Hangzhou [J]. Forest Research, 2008,21(4):587-591.

[29] 唐玉姝,王 磊,席雪飛,等.九段沙濕地潮間帶水域有機(jī)碳與無機(jī)碳含量的空間分布特征 [J]. 農(nóng)業(yè)環(huán)境科學(xué)學(xué)報(bào), 2012,31(12):2459- 2465. Tang Y S, Wang L, Xi X F, et al. The spatial distribution characteristics of organic and inorganic carbon content of intertidal water in Jiuduansha Wetland [J]. Journal of Agro-Environment Science, 2012, 31(12):2459-2465.

[30] 劉曉輝.長江口及鄰近海域海水細(xì)菌多樣性及時(shí)空分布規(guī)律研究 [D]. 舟山:浙江海洋大學(xué), 2017. Liu X H. Temporal and spatial distribution of bacterial diversity in seawater of Changjiang estuary and adjacent areas [D]. Zhoushan: Zhejiang Ocean University, 2017.

[31] 宋玉芝,趙淑穎,黃 瑾,等.太湖水體附著細(xì)菌和浮游細(xì)菌的豐度與分布特征 [J]. 環(huán)境工程學(xué)報(bào), 2013,7(8):2825-2831. Song Y Z, Zhao S Y, Huang J, et al. Abundance and distribution of particle-attached bacteria and free-living bacteria in Lake Taihu [J]. Chinese Journal of Environmental Engineering, 2013,7(8):2825-2831.

[32] Wang L, Zhang J, Li H, et al. Shift in the microbial community composition of surface water and sediment along an urban river [J]. Science of the Total Environment, 2018,627:600-612.

[33] Li J, Zhou H, Fang J, et al. Microbial distribution in different spatial positions within the walls of a black sulfide hydrothermal chimney [J]. Marine Ecology Progress Series, 2014,508:67-85.

[34] Yang Y, Dai Y, Wu Z, et al. Temporal and spatial dynamics of archaeal communities in two freshwater lakes at different trophic status [J]. Frontiers in Microbiology, 2016,7:451.

[35] Hu A, Hou Land Yu C-P. Biogeography of planktonic and benthic archaeal communities in a subtropical eutrophic estuary of China [J]. Microbial Ecology, 2015,70(2):322-335.

[36] 崔尹贍,李 珊,魏云林,等.納帕海濕地浮游細(xì)菌的豐度及可培養(yǎng)低溫細(xì)菌多樣性研究 [J]. 上海環(huán)境科學(xué), 2017,36(3):93-138.Cui Y S, Li S, Wei Y L, et al. Studies on the abundance of bacterioplankton and diversity of culturable low-temperature bacteria in Napahai Wetland [J]. Shanghai Environmental Science, 2017,36(3): 93-138.

[37] 李 云,李道季,張利華,等.長江口極微型和微微型浮游生物的垂向變化與周日波動(dòng) [J]. 海洋科學(xué), 2011,35(9):24-30. Li Y, Li D J, Zhang L H, et al. Vertical characteristics and diel variations of femtoplankton and picoplankton at the Changjiang estuary [J]. Marine Science, 2011,35(9):24-30.

[38] Barbosa A B, Galva?o H M, Mendes P A, et al. Short-term variability of heterotrophic bacterioplankton during upwelling off the NW Iberian margin [J]. Progress in Oceanography, 2001,51:339-359.

[39] Zhang R, Weinbauer M G, Qian P Y. Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters [J]. Environmental Microbiology, 2007,9(12):3008-3018.

[40] Winter C, Herndl G J, Weinbauer M G. Diel cycles in viral infection of bacterioplankton in the North Sea [J]. Aquatic Microbial Ecology, 2004,35:207-216.

[41] Zhang J, Zhang X, Liu Y, et al. Bacterioplankton communities in a high-altitude freshwater wetland [J]. Annals of Microbiology, 2014, 64(3):1405-1411.

[42] Elsayed O, Maillard E, Vuilleumier S, et al. Bacterial communities in batch and continuous-flow wetlands treating the herbicide S-metolachlor [J]. Science of the Total Environment, 2014,499:327- 335.

[43] Zhang L, Gao G, Tang X, et al. Bacterial community changes along a salinity gradient in a Chinese wetland [J]. Canadian Journal of Microbiology, 2013,59(9):611-619.

[44] Baik K S, Park S C, Kim E M, et al. Diversity of bacterial community in freshwater of Woopo wetland [J]. The Journal of Microbiology, 2008,46(6):647-655.

[45] Buesing N, Filippini M, Bürgmann H, et al. Microbial communities in contrasting freshwater marsh microhabitats [J]. FEMS Microbiology Ecology, 2009,69(1):84-97.

[46] Wan T, Zhang G, Du F, et al. Combined biologic aerated filter and sulfur/ceramisite autotrophic denitrification for advanced wastewater nitrogen removal at low temperatures [J]. Frontiers of Environmental Science & Engineering, 2014,8(6):967-972.

[47] Huang X, Bai J, Li K R, et al. Characteristics of two novel cold- and salt-tolerant ammonia-oxidizing bacteria from Liaohe Estuarine Wetland [J]. Marine Pollution Bulletin, 2017,114(1):192-200.

[48] Li B, Chen H, Li N, et al. Spatio-temporal shifts in the archaeal community of a constructed wetland treating river water [J]. Science of the Total Environment, 2017,605:269-275.

[49] Long Y, Yi H, Chen S, et al. Influences of plant type on bacterial and archaeal communities in constructed wetland treating polluted river water [J]. Environmental Science and Pollution Research, 2016,23(19): 19570-19579.

[50] Giovannoni S J, Stinql U. Molecular diversity and ecology of microbial plankton [J]. Nature, 2005,437(7057):343-348.

[51] Liu Y, Whitman W B. Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea [J]. Annals of the New York Academy of Sciences, 2008,1125(1):171-189.

[52] 張 健,趙陽國,李海艷,等.黃海西北近岸沉積物中細(xì)菌群落空間分布特征 [J]. 海洋學(xué)報(bào), 2010,32(2):118-127.Zhang J, Zhao Y G, Li H Y, et al. Temporal and spatial distribution characterization of bacterial community in sediments from the inshore of the northwest Huanghai Sea [J]. Acta Oceanologica Sinica, 2010, 32(2):118-127.

致謝：感謝中國海洋大學(xué)環(huán)境科學(xué)與工程學(xué)院鄒立副教授、李璐同學(xué)在樣品采集和環(huán)境因子分析中提供的大力支持與辛勤工作.

Diurnal variations of bacterial and archaeal communities in the reed wetland of Liaohe estuary.

LI Ming-yue1,2,3, MI Tie-zhu1,2,3*, ZHEN Yu1,2,3, WANG Xun-gong2,3

(1.Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China；2.Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao 266100, China；3.College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China)., 2019,39(2)：849~858

The present study investigated the diurnal variations of bacterial and archaeal communities in Liaohe estuarine wetland in autumn, using the real-time fluorescence quantitative PCR method and Illumina high-throughput sequencing. The results showed that 52802 bacterial and 106091 archaeal sequences were obtained, which could be classified into 530 and 979 OTUs in 97% similarity level, respectively. The results showed that bacterial and archaeal 16S rRNA gene abundances both had the highest values in the morning, and the lowest ones at midnight. There were diurnal variations in the bacterial and archaeal community α-diversity indices, and obvious temporal changes were also observed in the bacterial and archaeal community compositions and structures. In addition, Liaohe estuarine wetland had high potential ability of methanogenesis which could be reflected by the composition of archaeal community. This study provides the basic information and knowledge to further understand the characteristics and functions of bacterial and archaeal communities in estuarine wetlands.

wetland；16S rRNA；bacteria；archaea；community structure

X172

A

1000-6923(2019)02-0849-10

李明月(1990-),女,河南周口人,博士,主要從事海洋生態(tài)學(xué)研究.發(fā)表論文4篇.

2018-06-21

國家重點(diǎn)研發(fā)計(jì)劃課題(2017YFC1404402);鰲山科技創(chuàng)新計(jì)劃項(xiàng)目(2016ASKJ02);國家自然科學(xué)基金資助項(xiàng)目(41620104001)

* 責(zé)任作者, 副教授, mitiezhu@ouc.edu.cn