氯代烴污染深層土壤細(xì)菌群落結(jié)構(gòu)及組裝機(jī)制

樊艷玲,茍雅玲,王紅旗,劉增俊,許賀峰,楊 碩,梁 競

氯代烴污染深層土壤細(xì)菌群落結(jié)構(gòu)及組裝機(jī)制

樊艷玲1,2,茍雅玲1,3,王紅旗1*,劉增俊2**,許賀峰4,楊 碩2,梁 競2

(1.北京師范大學(xué)水科學(xué)研究院,北京 100875；2.北京市生態(tài)環(huán)境保護(hù)科學(xué)研究院,北京 100037；3.北京市科學(xué)技術(shù)研究院資源環(huán)境研究所,北京 100095；4.中國科學(xué)院生態(tài)環(huán)境研究中心,北京 100085)

為研究氯代烴污染土壤中細(xì)菌群落結(jié)構(gòu)特征和組裝機(jī)制,在某氯代烴污染場地不同污染區(qū)域采集2～10m范圍內(nèi)非飽和帶土壤,基于高通量測序技術(shù)分析了細(xì)菌群落結(jié)構(gòu),揭示了群落結(jié)構(gòu)變化的主要驅(qū)動因子、環(huán)境影響因子及組裝機(jī)制.結(jié)果表明:輕污染區(qū)細(xì)菌群落結(jié)構(gòu)變化的主要驅(qū)動因子是土壤類型,β多樣性主要受物種替換影響,貢獻(xiàn)度為53.9%,群落組成與水溶性硫酸鹽(=0.61,=0.0002)和總有機(jī)碳含量(=0.42,=0.0005)顯著相關(guān);重污染區(qū)細(xì)菌群落結(jié)構(gòu)變化的主要驅(qū)動因子是污染程度,β多樣性主要受豐富度差異影響,貢獻(xiàn)度為52.9%,群落組成與三氯乙烯(=0.17,=0.0425)、三氯甲烷(=0.22,=0.0375)、水溶性硫酸鹽(=0.36,=0.0074)、總有機(jī)碳(=0.29,=0.0168)、全氮(=0.20,=0.0130)含量顯著相關(guān).氯代烴脅迫降低了大多數(shù)物種的生態(tài)位寬度和生態(tài)位重疊指數(shù),但適應(yīng)性物種除外,最終導(dǎo)致變形菌門(Proteobacteria)的豐度增加,放線菌門(Actinobacteriota)、厚壁菌門(Firmicutes)和綠彎菌門(Chloroflexi)豐度出現(xiàn)降低.在污染物濃度較低時,菌群組裝以隨機(jī)性過程為主,貢獻(xiàn)度為65.6%,污染物較高時,隨機(jī)性過程下降為27.7%,組裝由確定性過程主導(dǎo).

深層土壤；細(xì)菌群落；結(jié)構(gòu)特征；組裝機(jī)制

氯代烴(CAHs)作為一種重要的有機(jī)溶劑和產(chǎn)品中間體,被廣泛應(yīng)用于工業(yè)領(lǐng)域,在其生產(chǎn)、儲存和使用過程中,容易通過泄露、排放或跑冒滴漏等對土壤和地下水造成污染.我國學(xué)者對有機(jī)場地的資料統(tǒng)計顯示,地下水和土壤中出現(xiàn)鹵代有機(jī)物的場地分別占84%和57%,這些鹵代有機(jī)物主要為氯代石油烴和氯代苯類[1].美國超級基金修復(fù)報告中統(tǒng)計顯示,地下水中需開展三氯乙烯、四氯乙烯、氯乙烯、順-1,2-二氯乙烯污染修復(fù)的分別占到55%、41%、37%和34%,土壤中需開展三氯乙烯和四氯乙烯污染修復(fù)的占21%和20%[2].

與物理、化學(xué)修復(fù)方式相比,生物修復(fù)方式具有擾動小、經(jīng)濟(jì)節(jié)約、不產(chǎn)生二次污染物、能最大限度降低污染物濃度等優(yōu)勢.氯乙烯在地下的生物降解途徑主要包含厭氧還原脫氯、好氧共代謝[3]和直接氧化[4-5],研究中發(fā)現(xiàn)的還原脫氯菌主要有地桿菌屬()、梭菌屬(. DC-1)[6],梭菌屬(spKYT-1)[7],脫鹵桿菌屬(),脫鹵擬球菌屬()[8],脫硫單胞菌屬(),脫硫桿菌屬(),sp. Y51[9],梭狀芽胞桿菌()[10],丙酸桿菌(spHK-1),硫螺旋菌屬()[11]等,氧化共代謝菌群主要有亞硝化單胞菌(),假單胞菌屬(), 紅球菌屬(),黃桿菌屬(sp)等[12].不同氯代烴場地中的優(yōu)勢微生物差異較大[13],研究表明污染土壤和清潔土壤中微生物群落結(jié)構(gòu)存在顯著差異[14-15],但不同土壤環(huán)境下,觸發(fā)群落結(jié)構(gòu)發(fā)生變化的污染程度不同,有室內(nèi)實驗研究表明三氯乙烯在1′10-6mg/kg水平下即可對土壤微生物群落結(jié)構(gòu)造成影響[16],但現(xiàn)場數(shù)據(jù)的研究結(jié)果表明多環(huán)芳烴[17]和多氯聯(lián)苯[18]在高濃度水平下才會引發(fā)群落結(jié)構(gòu)的變化.為有效指導(dǎo)污染場地的微生物修復(fù),應(yīng)充分了解場地中的土著菌群分布特征.

微生物的多樣性和群落結(jié)構(gòu)是影響環(huán)境介質(zhì)功能的主要因素[19],因此研究氯代烴實際場地中的微生物群落結(jié)構(gòu)及其組裝機(jī)制對氯代烴污染土壤的微生物修復(fù)具有重要的指導(dǎo)意義.由于氯代烴的遷移性較強(qiáng),實際場地中污染深度往往貫穿整個非飽和帶,為了解場地深層土壤中微生物群落結(jié)構(gòu)特征及其組裝機(jī)制,本研究基于華北某氯代烴污染場地,分析比較了2～10m深度范圍內(nèi)不同污染程度和土壤質(zhì)地條件下土壤微生物群落結(jié)構(gòu),探討了群落組成的影響因素和組裝機(jī)制,以期為氯代烴污染場地的微生物修復(fù)提供參考.

1 材料與方法

1.1 土壤樣品采集

土壤樣品采集于華北某氯代烴污染場地,該場地包氣帶巖性以填土、粉質(zhì)粘土和砂質(zhì)粉土為主,污染物類型為氯代烴.2022年10月,選擇2個不同污染程度的區(qū)域進(jìn)行鉆孔取樣,分別為輕污染區(qū)(經(jīng)緯度為39.43463883,116.2539744,定義為L區(qū))和重污染區(qū)(經(jīng)緯度為39.43455549,116.2537315,定義為H區(qū)).樣品采集深度為2,4,6,8,10m.每個區(qū)域在1m′1m范圍內(nèi)均勻布設(shè)3個鉆孔,在同一深度進(jìn)行取樣,作為3個重復(fù).

現(xiàn)場采用30鉆機(jī)進(jìn)行鉆孔取樣.參照《巖土工程勘察規(guī)范》[GB50021-2001][20]判斷土壤性質(zhì),用無菌鏟和VOCs土壤采樣器采集土壤樣品,每個采樣點采集3份土壤樣品,用于微生物測試的土壤裝入無菌離心管裝入裝有干冰的保溫箱中運輸至實驗室,用于污染物含量和土壤理化性質(zhì)測試的土壤分別裝入40mL棕色瓶和棕色廣口瓶,低溫送至實驗室.

1.2 土壤理化性質(zhì)測定

參照《土壤干物質(zhì)和水分的測定重量法》[HJ 613-2011][21]測定土壤含水率,檢出限為0.1%;參照《土壤 pH值的測定電位法》[HJ 962-2018][22]測定土壤pH值,檢出限為0.01;參照《沉積巖中總有機(jī)碳的測定》[GB/T 19145-2003][23]測定土壤總有機(jī)碳(TOC)含量,檢出限為0.10%;參照《土壤質(zhì)量全氮的測定凱式法》[HJ 717-2014][24]測定土壤總?cè)?TN)含量,檢出限為48mg/kg;參照《土壤總磷的測定堿熔-鉬銻抗分光光度法》[HJ 632-2011][25]測定土壤總磷(TP)含量,檢出限為20mg/kg;參照《土壤水溶性和酸溶性硫酸鹽的測定》[HJ 635-2012][26]測定土壤硫酸鹽(S)含量,檢出限為20mg/kg;參照《土壤和沉積物揮發(fā)性有機(jī)物的測定吹掃捕集/氣相色譜-質(zhì)譜法》[HJ 605-2011][27]測定土壤VOCs含量,檢出限為0.05～0.1mg/kg.

1.3 土壤DNA抽提和PCR擴(kuò)增

根據(jù)E.Z.N.A.? soil DNA kit(Omega Bio-tek, Norcross, GA, U.S.)說明書進(jìn)行微生物群落總DNA抽提,使用1%的瓊脂糖凝膠電泳檢測DNA的提取質(zhì)量,使用NanoDrop2000測定DNA濃度和純度;使用338F(5’-ACTCCTACGGGAGGCAGCAG-3’)和806R(5’-GGACTACHVGGGTWTCTAAT-3’)對16S rRNA基因V3-V4可變區(qū)進(jìn)行PCR擴(kuò)增,擴(kuò)增程序如下:95℃預(yù)變性3min,27個循環(huán)(95℃變性30s,55℃退火30s,72℃延伸30s),然后72℃穩(wěn)定延伸10min,最后在4℃進(jìn)行保存(PCR儀:ABI GeneAmp? 9700型).PCR反應(yīng)體系為:5×TransStart FastPfu緩沖液4μL,2.5mM dNTPs 2μL,上游引物(5umol/L)0.8μL,下游引物(5umol/L)0.8μL,TransStart FastPfuDNA聚合酶0.4μL,模板DNA 10ng,ddH2O補足至20μL.每個樣本3個重復(fù).

1.4 Illumina Miseq測序

1.5 微生物群落組成分析

使用fastp[28](https://github.com/OpenGene/fastp, version 0.20.0)軟件對原始測序序列進(jìn)行質(zhì)控,使用FLASH[29](http://www.cbcb.umd.edu/software/flash,version 1.2.7)軟件進(jìn)行拼接.使用UPARSE[30]軟件(http://drive5.com/uparse/,version 7.1),根據(jù)97%[30-31]的相似度對序列進(jìn)行OTU聚類并剔除嵌合體.利用RDP classifier[32](http://rdp.cme.msu.edu/,version 2.2)對每條序列進(jìn)行物種分類注釋,比對Silva 16S rRNA數(shù)據(jù)庫(version 138),設(shè)置比對閾值為70%.

1.6 統(tǒng)計分析

數(shù)據(jù)處理采用Microsoft Excel 2016軟件完成.多樣性是指某個群落或生境內(nèi)部的物種多樣性[33],采用R語言Vegan包計算如下指數(shù)對其進(jìn)行表征:sobs指數(shù)和chao指數(shù)表征物種豐富度,既一個群落或生境中種的數(shù)目的多寡,數(shù)值越大,豐富度越大.shannon指數(shù)和simpson指數(shù)表征總多樣性,是對豐富度和均勻度的綜合反映,shannon指數(shù)越大,多樣性越高,simpson指數(shù)越大,多樣性越低.利用Kruskal-Wallis秩和檢驗進(jìn)行多樣性指數(shù)的組間差異性檢驗.多樣性是指在一個梯度上從一個生境到另一個生境所發(fā)生的物種多樣性變化的速率和范圍[33],是種數(shù)改變一半的測度.采用R語言Vegan包,基于bray-curtis進(jìn)行層級聚類分析和PCoA分析,基于euclidean進(jìn)行置換多元方差分析(PERMANOVA),體現(xiàn)物種多樣性的變化.采用R語言adespatial包進(jìn)行β多樣性分解,spaa包進(jìn)行生態(tài)位寬度和生態(tài)位重疊計算.基于Spearman相關(guān)性,采用R語言linkET包進(jìn)行mantel test檢驗.中性群落模型分析采用R語言minpack.lm包.

2 結(jié)果與討論

2.1 土壤污染特征及理化性質(zhì)分析

土壤樣品采集位置及檢出污染物濃度如表1所示.L區(qū)檢出污染物主要為三氯乙烯、三氯甲烷、順式-1,2-二氯乙烯,最大值分別為1.99,0.1和0.15mg/kg. H區(qū)檢出污染物為三氯乙烯、三氯甲烷和順式-1,2-二氯乙烯,濃度分布范圍分別為1.16～17.20mg/kg, 0.21～10.50mg/kg和0.08～0.42mg/ kg.H區(qū)樣品中三氯乙烯和三氯甲烷濃度普遍高于L區(qū),且H區(qū)深層(6,8和10m)三氯乙烯和三氯甲烷濃度高于淺層(2和4m).

表1 土壤樣品采集位置及污染物濃度

注:低于檢出限的樣品濃度用檢出限數(shù)值的1/2代替,3個平行樣品中當(dāng)未檢出個數(shù)32個時標(biāo)記為NA.

土壤類型及理化性質(zhì)如表2所示.

表2 土壤類型及理化性質(zhì)

2.2 土壤剖面細(xì)菌群落a多樣性

對L區(qū)和H區(qū)樣品進(jìn)行高通量測序,處理后共獲得10910個OTU,分類為55門、187科、456目、729科、1491屬、3139種.從樣本中隨機(jī)抽取一定數(shù)量的序列,統(tǒng)計這些序列對應(yīng)樣本的Shannon指數(shù),結(jié)果如圖1所示,曲線均趨于平緩,表明本次測序數(shù)據(jù)量足夠.

圖1 基于OTU水平香農(nóng)指數(shù)稀釋曲線

圖2為土壤剖面細(xì)菌群落多樣性隨深度的變化.L區(qū)2～10m范圍內(nèi)Sobs指數(shù)和Chao指數(shù)變化不顯著(圖2(a)和(b)),表明深度的變化未顯著影響物種的豐富度.Shannon指數(shù)變化不顯著(圖2(c)),但10m處Simpson指數(shù)顯著高于2和4m處(圖2(d)),表明10m處物種多樣性顯著低于2和4m處.

自閉癥譜系障礙兒童確診前后其家庭有效應(yīng)對模式的構(gòu)建——基于一名中度自閉癥兒童6年康復(fù)歷程的考察……………………………………………………劉英玲（95）

H區(qū)淺層(2和4m)Sobs指數(shù)、Chao指數(shù)、Shannon指數(shù)和Simpson指數(shù)均無顯著差異,表明淺層土壤內(nèi)物種豐富度和多樣性無顯著差異.深層(6～10m)Sobs指數(shù)和Chao指數(shù)無顯著差異,表明深層土壤內(nèi)物種豐富度未發(fā)生顯著變化.6m處Shannon指數(shù)和Simpson指數(shù)分別顯著高于和低于10m處,表明深層土壤內(nèi)物種多樣性發(fā)生了變化.淺層Sobs指數(shù)、Chao指數(shù)和Shannon指數(shù)均顯著高于深層,Simpson指數(shù)顯著低于10m處,均表明淺層物種豐富度和多樣性均顯著高于深層.

2.3 土壤剖面細(xì)菌群落b多樣性

基于bray_curtis距離,分別對L區(qū)和H區(qū)樣本進(jìn)行層級聚類分析和PCoA分析,結(jié)果如圖3所示.樣本層級聚類結(jié)果(圖3(a))將L區(qū)全部樣本劃分為回填土(2m)和原土(4,6,8和10m)兩大類,其中,原土中又進(jìn)一步分為兩個小類,分別為粉質(zhì)黏土(4和6m)和砂質(zhì)粉土(6和8m).以土壤類型作為分組依據(jù)對L區(qū)所有樣本進(jìn)行PCoA分析(圖3(b)),結(jié)果顯示,回填土、粉質(zhì)黏土和砂質(zhì)粉土之間顯著區(qū)分,主成分PC1和PC2的累計解釋方差為49.65%,置換多元方差分析(PERMANOVA)2值為0.4683,P值為0.001,回填、粉質(zhì)黏土和砂質(zhì)粉土組間差異顯著大于組內(nèi)差異,表明氯代烴低污染脅迫下土壤類型是細(xì)菌群落結(jié)構(gòu)差異的主要驅(qū)動因子.

樣本層級聚類結(jié)果(圖3(c))將H區(qū)全部樣本劃分為淺層(2和4m)和深層(6,8和10m)兩大類.根據(jù)氯代烴分布規(guī)律,H區(qū)淺層樣品中污染物濃度較低,深層樣品中污染物濃度較高,因此,本文依據(jù)污染程度將H區(qū)鉆孔分為低濃度組(D)和高濃度組(G)進(jìn)行PCoA分析,結(jié)果如圖3(d)所示,D組和G組之間顯著區(qū)分,主成分PC1和PC2的累計解釋方差為73.12%,置換多元方差分析(PERMANOVA)2值為0.4891,P值為0.002,表明D組和G組組間差異顯著大于組內(nèi)差異,表明氯代烴高污染脅迫下污染物濃度驅(qū)動微生物群落結(jié)構(gòu)發(fā)生了變化.

圖2 土壤剖面細(xì)菌群落α多樣性

a, b, c, d分別為L區(qū)土壤剖面sobs, chao, Shannon和simpson指數(shù); e, f, g和h分別為H區(qū)土壤剖面sobs, chao, shannon和simpson指數(shù)

圖3 基于OTU水平的聚類分析與PCoA分析

a,c分別為L區(qū)和H區(qū)樣品聚類分析;b,d分別為L區(qū)和H區(qū)樣品PCoA分析

群落間物種組成差異起源于兩種不同的過程:物種周轉(zhuǎn)或豐富度差異,其中,物種周轉(zhuǎn)表示不同群落間的物種替換,而物種喪失會導(dǎo)致群落間物種豐富度產(chǎn)生差異[34-35].例如,由于物種對環(huán)境變化的敏感性不同,受脅迫物種容易因為環(huán)境壓力而在生境中消失,而對環(huán)境變化具有較高容忍度的物種則能存活下來[36].Beta多樣性分解結(jié)果顯示,L區(qū)土壤細(xì)菌群落的差異主要受物種替換過程影響,貢獻(xiàn)度為53.9%,豐富度差異貢獻(xiàn)為23.7%,相似性為22.4%(圖4(a)),H區(qū)土壤細(xì)菌群落的差異主要受豐富度差異影響,貢獻(xiàn)度為52.9%,物種替換貢獻(xiàn)度為27%,相似性為20.1%(圖4(b)),表明H區(qū)污染物的積累造成了不耐受物種的喪失.

圖4 L區(qū)和H區(qū)的b多樣性分解三元圖

2.4 細(xì)菌群落結(jié)構(gòu)

土壤剖面門水平細(xì)菌群落相對豐度如圖5所示.L區(qū)2和4m處優(yōu)勢菌分別為放線菌門(Actinobacteriota)(26.02%)和綠彎菌門(Chloroflexi) (24.39%),6～8m間優(yōu)勢菌為變形菌門(Proteobacteria) (24.44%、28.89%、37.55%).變形菌門相對豐度隨深度逐漸增加,從2m至10m相對豐度依次從9.05%增加至37.55%(圖5(a)).結(jié)合L區(qū)PCoA分組結(jié)果(圖3(b)),放線菌門(Actinobacteriota)在不同土質(zhì)中相對豐度不同,在回填土中的相對豐度較大(26.02%),其次是砂質(zhì)粉土(14.13%～18.34%),粉質(zhì)黏土中相對豐度最小(7.23%～10.62%).

H區(qū)2和4m處優(yōu)勢菌為放線菌門(Actinobacteriota) (26.42%、22.85%),6～10m間優(yōu)勢菌為變形菌門(Proteobacteria)(32.74%、51.74%、84.12%)(圖5(b)).結(jié)合H區(qū)PCoA分組結(jié)果(圖3(d)),氯代烴低濃度脅迫下(D組)放線菌門(Actinobacteriota)是優(yōu)勢菌,氯代烴高濃度脅迫下(G組)放線菌門(Actinobacteriota)、厚壁菌門(Firmicutes)和綠彎菌門(Chloroflexi)相對豐度均出現(xiàn)了下降,變形菌門(Proteobacteria)出現(xiàn)富集,成為優(yōu)勢菌.

生態(tài)位寬度反映了種群對生境資源利用程度和對環(huán)境適應(yīng)能力,物種的生態(tài)位越寬越具有競爭力,生態(tài)位越窄在競爭中越處于劣勢[37-38].D組樣品中各菌群生態(tài)位寬度顯著高于G組,Wilcox檢驗p值為0.00295(圖6(b)),氯代烴脅迫造成了大部分菌群生態(tài)位的減小,只有變形菌門(Proteobacteria)、放線菌門(Actinobacteriota)、Methylomirabilota、疣微菌門(Verrucomicrobiota)等出現(xiàn)了生態(tài)位寬度增加(圖6(a)).

當(dāng)兩個物種利用同一資源或共同占有某一資源時,就會出現(xiàn)生態(tài)位重疊現(xiàn)象, D組樣品中各菌群生態(tài)位寬度顯著高于G組,Wilcox檢驗p值為0.0000(圖6(c)).當(dāng)生態(tài)位重疊值大于0.6時,物種對之間的生態(tài)位重疊顯著[39].G組中優(yōu)勢菌群物種關(guān)聯(lián)圖顯示(圖6(d)),變形菌門(Proteobacteria)與放線菌門(Actinobacteriota)重疊指數(shù)為0.63,與剩余8個優(yōu)勢菌群的生態(tài)位重疊指數(shù)均小于0.6,變形菌門(Proteobacteria)與其他菌群的生態(tài)位重疊現(xiàn)象不明顯.放線菌門(Actinobacteriota)與剩余8個優(yōu)勢菌群的生態(tài)位重疊指數(shù)均大于0.8,生態(tài)位重疊現(xiàn)象顯著.厚壁菌門(Firmicutes)、綠彎菌門(Chloroflexi)、放線菌門(Actinobacteriota)、擬桿菌門(Acidobacteriota)、芽單胞菌門(Gemmatimonadota)之間有明顯的重疊.

圖5 L區(qū)和H區(qū)土壤剖面細(xì)菌門水平群落組成

菌群生態(tài)位寬度和生態(tài)位重疊指數(shù)分析結(jié)果顯示,氯代烴脅迫下,變形菌門(Proteobacteria)和放線菌門(Actinobacteriota)生態(tài)位寬度變大,但由于前者生態(tài)位重疊指數(shù)較低,相對豐度出現(xiàn)了增大,后者生態(tài)位重疊指數(shù)較高,相對豐度出現(xiàn)了降低.厚壁菌門(Firmicutes)和綠彎菌門(Chloroflexi)在氯代烴脅迫下生態(tài)位寬度減小,且生態(tài)位重疊指數(shù)較高,因此兩者的相對豐度均出現(xiàn)了降低.

圖6 D組和G組樣本生態(tài)位特征

Fig.6 Niche characteristics of samples from Group D and Group G

a:物種生態(tài)位寬度;b:生態(tài)位寬度wilcox檢驗;c:生態(tài)位重疊指數(shù)wilcox檢驗;d:G組物種關(guān)聯(lián)

2.5 細(xì)菌群落與環(huán)境因子的相關(guān)性

Mantel檢驗結(jié)果顯示(圖7),L區(qū)細(xì)菌群落組成與土壤中水溶性硫酸鹽含量(S)(=0.61,=0.0002)和TOC含量(=0.42,=0.0005)顯著相關(guān),雖然土壤中也含有較低濃度的三氯乙烯(TCE),但細(xì)菌群落結(jié)構(gòu)與其無顯著相關(guān)性,表明氯代烴入侵的初始階段或污染程度較低時,細(xì)菌群落結(jié)構(gòu)具有一定的穩(wěn)定性. H區(qū)細(xì)菌群落組成與水溶性硫酸鹽含量(S)(=0.36,= 0.0074)、TOC含量(=0.29,=0.0168)、三氯乙烯(TCE) (=0.17,=0.0425)、三氯甲烷(chloroform)(= 0.22,= 0.0375)和全氮(TN)(=0.20,=0.0130)相關(guān),表明氯代烴高濃度脅迫下,污染物影響了細(xì)菌群落組成.

圖7 L區(qū)和H區(qū)細(xì)菌群落物種組成與環(huán)境因子的相關(guān)性分析

顏色梯度和方塊大小代表Spearman相關(guān)系數(shù),紅色代表正相關(guān),紫色代表負(fù)相關(guān).對細(xì)菌群落結(jié)構(gòu)與環(huán)境因子做Mantel分析,線條寬度對應(yīng)Mantel檢驗r值,線條顏色表示9999次置換檢驗的統(tǒng)計顯著性

2.6 群落組裝機(jī)制

傳統(tǒng)的生態(tài)位理論認(rèn)為群落結(jié)構(gòu)的形成是物種特征、種間相互作用和環(huán)境條件控制的確定性過程[40-41].中性理論認(rèn)為群落結(jié)構(gòu)是由出生、死亡、遷移、物種形成和擴(kuò)散限制等隨機(jī)過程塑造的[42].微生物組裝過程中,生態(tài)位理論和中心理論同時發(fā)生[41,43-44].Sloan等[45]提出了中性群落模型(NCM),量化了中性過程的重要性,模型中2表示模擬的擬合程度,2越高擬合效果越好,即越接近中性模型,m為物種的遷移率,越高表明物種受到擴(kuò)散限制越低.

圖8 基于中性群落模型(NCM)評估群落組裝過程

藍(lán)色實線表示NCM的預(yù)測曲線,藍(lán)色虛線表示NCM預(yù)測的95%置信區(qū)間

對H區(qū)D組和G組樣本進(jìn)行中性群落模型評估,結(jié)果如圖8所示,D組擬合度2為0.656,遷移率m為1.08(圖8(a)),G組擬合度2為0.277,遷移率m為0.087(圖8(b)).隨機(jī)過程的相對貢獻(xiàn)度在G組中有了明顯的降低,擴(kuò)散受到了限制,這可能是因為較高的環(huán)境異質(zhì)性會限制細(xì)菌群落的擴(kuò)散,推動環(huán)境選擇的確定性過程[46].Mo等[47]研究了在不同鹽度環(huán)境中真菌組裝過程,發(fā)現(xiàn)隨機(jī)過程的相對貢獻(xiàn)隨著鹽度的增加而逐漸降低.G組物種的生態(tài)位寬度較D組出現(xiàn)了顯著降低(圖6(b)),有研究證明,相較于具有較寬生態(tài)位的物種,較窄生態(tài)位物種更容易受確定性過程的影響[48].

3 討論

3.1 細(xì)菌群落多樣性

在污染物濃度較低的L區(qū),以2m為間隔的樣本之間豐富度和多樣性并未發(fā)生顯著變化,表明深度未引起物種多樣性的顯著差異.樣本層級聚類和PCoA結(jié)果均顯示填土、粉質(zhì)粘土和砂質(zhì)粉土細(xì)菌群落顯著區(qū)分,表明土壤類型是物種差異的主要驅(qū)動力.前期已有研究證明,土壤類型是影響微生物活動和群落結(jié)構(gòu)的重要因素之一[49-50],Bai等[51]基于稻田土柱實驗研究結(jié)果表明,細(xì)菌和古菌群落結(jié)構(gòu)能夠根據(jù)土壤類型進(jìn)行明顯的區(qū)分.但由于土壤類型代表了土壤母質(zhì)以及過去和現(xiàn)在復(fù)雜的氣候條件的綜合影響,很難將土壤類型對微生物群落的影響歸因于單一因素[51].本研究的mantel檢驗結(jié)果顯示L區(qū)群落組成與總有機(jī)碳和水溶性硫酸鹽含量顯著相關(guān).前期已有研究證明,生物多樣性與土壤中碳含量和pH值相關(guān)[52-53],但是當(dāng)pH值范圍較窄時,pH值對群落組成的影響不明顯[53].本研究中全部樣品pH值范圍為8.18～8.96,未對群落組成造成明顯的影響.

H區(qū)的淺層樣本與深層樣本間多樣性存在顯著差異,層級聚類和PCoA結(jié)果表明,氯代烴污染程度是物種差異的主要驅(qū)動力.mantel檢驗結(jié)果也驗證了三氯乙烯和三氯甲烷濃度變化與物種組成變化呈顯著相關(guān)性.H區(qū)低濃度組微生物生態(tài)位重疊指數(shù)顯著高于高濃度組(圖6(c)),Galand[54]認(rèn)為,物種的重疊能夠為生態(tài)系統(tǒng)提供功能冗余,緩沖多樣性的喪失,這與淺層樣本物種多樣性顯著高于深層的現(xiàn)象相吻合.

有研究報道,當(dāng)環(huán)境條件超出了它們的“歷史生活”中的環(huán)境范疇時,微生物群落組成和功能將會發(fā)生變化[55],雖然NEMIR等[16]基于室內(nèi)實驗確定微生物群落結(jié)構(gòu)受到顯著影響的明顯閾值約為1×10-6mg/kgTCE,但本文結(jié)論顯示,L區(qū)TCE濃度范圍為0.05～1.99mg/kg時mental檢驗顯示TCE濃度與群落組成之間不存在顯著相關(guān)性.H區(qū)TCE濃度范圍介于1.16～17.20mg/kg,三氯甲烷濃度范圍介于0.21～10.50mg/kg時才驅(qū)動了群落結(jié)構(gòu)的變化,類似的結(jié)論在石油和多環(huán)芳烴類污染場地中也得到了驗證:鄭一鳴等[56]在石油類污染場地中發(fā)現(xiàn)高濃度石油污染是引起土壤和沉積物中微生物群落組成變化的主要驅(qū)動因子,Liu等[17]在多環(huán)芳烴污染土壤中也發(fā)現(xiàn),多環(huán)芳烴總濃度為3166.52mg/kg樣品中的細(xì)菌群落與34.26～258.20mg/kg樣品中的細(xì)菌群落差異顯著.

3.2 細(xì)菌群落結(jié)構(gòu)

變形菌門在土壤環(huán)境中非常常見,與碳、氮、硫循環(huán)功能有關(guān)[57].L區(qū)土壤中變形菌門相對豐度隨深度的增加而增大,最大為37.55%, H區(qū)高濃度氯代烴脅迫下,變形菌的相對豐度最大豐度達(dá)到84.14%,這表明,變形菌是氯代烴耐受菌,在氯代烴脅迫下,變形菌門的生態(tài)位寬度增加了45.4%,與放線菌的生態(tài)位重疊指數(shù)為0.63,與剩余8個優(yōu)勢菌門的重疊指數(shù)均小于0.54,這也表明變形菌在氯代烴環(huán)境下具有相對較強(qiáng)的競爭優(yōu)勢.孫仲平等[58]研究發(fā)現(xiàn),在TCE污染沉積物中變形菌是優(yōu)勢菌(相對豐度84.9%),但隨著TCE的降解,變形菌門相對豐度分別下降為66.1%和49.8%,與之趨勢相反的是厚壁菌門,污染樣品中相對豐度較低,但隨著污染物濃度的降低,相對豐度分別增大至22.1%和47.9%.Koner等[59]在9m深度處高氯代烴污染濃度土壤也發(fā)現(xiàn)了豐度較高的變形菌門.

同樣在氯代烴脅迫下生態(tài)位寬度增加的還有放線菌門,增幅為17.8%,但由于放線菌門與其他優(yōu)勢菌群的生態(tài)位重疊顯著,因此其相對豐度出現(xiàn)了下降.厚壁菌門和綠彎菌門在高氯代烴濃度環(huán)境下出現(xiàn)了生態(tài)位寬度變窄,同時具有較高的生態(tài)位重疊,因此均出現(xiàn)了相對豐度的降低,孫仲平等[58]的研究中也發(fā)現(xiàn)了厚壁菌門在TCE污染沉積物中的降低.

3.3 細(xì)菌群落組裝機(jī)制

目前群落組裝機(jī)制的研究中應(yīng)用最多的是生態(tài)位理論和中性理論,前者認(rèn)為群落的形成是環(huán)境狀況、生境異質(zhì)性和物種間相互作用等確定性過程影響了物種的有無及豐度的大小[60],后者則把群落的形成歸結(jié)為隨機(jī)性的過程,例如物種的出生、死亡、殖民、移民、物種形成和擴(kuò)散限制[61].確定性和隨機(jī)性過程共同作用于微生物群落的形成[43],但兩者相對重要性的大小取決于環(huán)境的類型、環(huán)境條件和生物特性[62].

當(dāng)環(huán)境條件充足,大部分物種能夠較好的生長時,隨機(jī)性過程起主導(dǎo)作用,物種多樣性較高[63],Lan等[64]發(fā)現(xiàn)森林土壤中細(xì)菌群落組裝主要以隨機(jī)性過程為主.隨著環(huán)境條件變得苛刻,確定性過程起主導(dǎo)作用,優(yōu)勢菌種占據(jù)主導(dǎo)地位,物種多樣性也隨著降低[65].Xun等[66]在研究中發(fā)現(xiàn),群落組裝過程與菌群多樣性有關(guān),隨著土壤細(xì)菌物種豐富度和功能多樣性的降低,細(xì)菌群落組成過程由隨機(jī)過程向確定性過程轉(zhuǎn)變.H區(qū)2～4m深度內(nèi)細(xì)菌群落多樣性水平較高,細(xì)菌群落組裝過程中隨機(jī)性過程貢獻(xiàn)度為65.6%,遷移率為1.8,細(xì)菌群落遷移率較大,組裝以隨機(jī)性過程為主.而在濃度較高的6～10m深度內(nèi),細(xì)菌群落組裝過程中隨機(jī)性過程貢獻(xiàn)度下降為27.7%,確定性過程占主導(dǎo),擴(kuò)散受到限制,遷移率下降為0.087,部分菌群受到氯代烴的抑制作用,耐受菌得以富集.研究中也發(fā)現(xiàn)H區(qū)α多樣性隨著深度的增加逐漸減小,這一現(xiàn)象與組裝機(jī)制分析結(jié)果相吻合.

4 結(jié)論

4.1 輕污染區(qū)土壤中,土壤類型是造成細(xì)菌群落差異的主要驅(qū)動因子,群落組成與水溶性硫酸鹽和總有機(jī)碳含量顯著相關(guān).細(xì)菌群落的差異主要受物種替換影響.

4.2 重污染區(qū)土壤中,污染脅迫是造成細(xì)菌群落差異的主要驅(qū)動因子,除水溶性硫酸鹽和總有機(jī)碳以外,群落組成還受三氯乙烯、三氯甲烷和全氮顯著影響.細(xì)菌群落的差異主要受豐富度差異影響.

4.3 氯代烴脅迫顯著降低了細(xì)菌群落的豐富度和多樣性,除適應(yīng)性物種外,多數(shù)物種在氯代烴脅迫下降低了生態(tài)位寬度和生態(tài)位重疊指數(shù),致使變形菌門的豐度增加,放線菌門、厚壁菌門和綠彎菌門豐度降低.

4.4 氯代烴濃度較低時,組裝中隨機(jī)性過程貢獻(xiàn)度為65.6%,隨著污染物濃度的增大,隨機(jī)性過程下降為27.7%,組裝以確定性過程為主.

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Structure and assembly mechanism of bacterial communities in deep soil contaminated by chlorinated hydrocarbons.

FAN Yan-ling1,2, GOU Ya-ling1,3, WANG Hong-qi1*, LIU Zeng-jun2**, XU He-feng4, YANG Shuo2, LIANG Jing2

(1.College of Water Sciences, Beijing Normal University, Beijing 100875, China；2.Beijing Municipal Research Institute of Eco-Environmental Protection, Beijing 100037, China；3.Institute of Resources and Environment, Beijing 100095, China；4.Research Center for Eco-Environmental Science, Chinese Academy of Sciences, Beijing 100085, China)., 2023,43(10)：5550～5561

To study the structural characteristics and assembly mechanism of bacterial community in chlorinated hydrocarbons contaminated soil, unsaturated-zone soil within 2～10m was collected from different contaminated areas. Based on high-throughput sequencing technology, the bacterial community was analyzed and the main drivers, environmental influencing factors and assembly mechanism of the community structure changes were revealed. The results showed that the main driver of bacterial community structure change in the lightly polluted area was soil type. The β-diversity mainly influenced by species replacement with a contribution of 53.9%, and community composition significantly correlated with water-soluble sulfate(=0.61,=0.0002) and total organic carbon content(=0.42,=0.0005). Furthermore, the main driver of bacterial community structure change in the heavily polluted area was the degree of pollution. The β-diversity mainly influenced by the differences in abundance with a contribution of 52.9%, community composition was significantly correlated with trichloroethylene(=0.17,=0.0425), chloroform (=0.22,=0.0375), water-soluble sulfate (=0.36,=0.0074), total organic carbon (=0.29,=0.0168), and total nitrogen content (=0.20,=0.0130). Chlorinated hydrocarbons stress narrowed the niche width and reduced the niche overlap index of most species except adaptive ones, and led to an increase in the abundance of Proteobacteria, while that of Actinobacteriota, Firmicutes, and Chloroflexi decreased. In the soil with low pollutant concentration, the bacterial community assembly was dominated by random process, with a contribution of 65.6%. In the soil with high pollutant concentration, the random process decreased to 27.7%, and the assembly was dominated by deterministic process.

deep soil；bacterial community；structural characteristics；assembly mechanism

X53

A

1000-6923(2023)10-5550-12

2023-02-24

北京市生態(tài)環(huán)境保護(hù)學(xué)科研究院基金項目(Y2020-003)

* 責(zé)任作者, 教授, ambar@bnu.edu.cn, 副研究員, lzengj@126.com

樊艷玲(1984-),女,山東淄博人,高級工程師,北京師范大學(xué)博士研究生,主要從事污染土壤與地下水修復(fù)治理研究.發(fā)表論文20余篇.flylinger@163.com.

樊艷玲,茍雅玲,王紅旗,等.氯代烴污染深層土壤細(xì)菌群落結(jié)構(gòu)及組裝機(jī)制 [J]. 中國環(huán)境科學(xué), 2023,43(10):5550-5561.

Fan Y L,Gou Y L, Wang H Q, et al. Structure and assembly mechanism of bacterial communities in deep soil contaminated by chlorinated hydrocarbons [J]. China Environmental Science, 2023,43(10):5550-5561.