酸性礦山廢水對(duì)沉積物真核微生物群落的影響

劉 帆,張曉輝,唐 宋,王 茂,劉紅玲*

酸性礦山廢水對(duì)沉積物真核微生物群落的影響

劉 帆1,張曉輝1,唐 宋2,王 茂3,劉紅玲1*

(1.南京大學(xué)環(huán)境學(xué)院,污染控制與資源化研究國(guó)家重點(diǎn)實(shí)驗(yàn)室,江蘇南京 210023；2.中國(guó)疾病預(yù)防控制中心環(huán)境與健康相關(guān)產(chǎn)品安全所,北京 100021；3.中山大學(xué)公共衛(wèi)生學(xué)院,廣東 廣州 510080)

對(duì)受酸性礦山廢水污染的橫石河上游到下游水體和沉積物中理化參數(shù)以及沉積物中真核微生物的多樣性進(jìn)行系統(tǒng)調(diào)查,使用斯皮爾曼相關(guān)性分析和典范對(duì)應(yīng)分析(CCA)甄別真核微生物群落的主要環(huán)境脅迫因子.結(jié)果表明:橫石河中真核微生物優(yōu)勢(shì)種群為真菌門(Fungi)(4.51%~86.69%)、綠藻門(Chlorophyta)(0.61%~77.36%)和纖毛蟲門(Ciliophora)(0.81%~34.91%).沉積物中真核微生物群落Alpha多樣性隨酸性礦山廢水污染梯度降低而逐漸升高.與原核微生物群落不同,酸性礦山廢水環(huán)境沉積物中真核微生物群落結(jié)構(gòu)的變化主要受硫酸根和電導(dǎo)率的影響.

酸性礦山廢水；真核微生物；硫酸根；電導(dǎo)率；下一代測(cè)序

酸性礦山廢水(AMD)是含硫的礦物在氧氣和水存在情況下,經(jīng)風(fēng)化、淋溶和微生物共同作用而形成[1].由于處理工藝缺失,處理費(fèi)用昂貴,大部分酸性礦山廢水未經(jīng)處理便直接排放到自然環(huán)境中[1].酸性礦山廢水具有pH值低,硫酸鹽、電導(dǎo)率和重金屬濃度高的特點(diǎn)[2], 對(duì)大多數(shù)生命來說都是難以生存的極端環(huán)境[3-5].然而,這種有極端環(huán)境脅迫的獨(dú)特生境卻孕育著大量嗜酸、耐酸、耐重金屬的微生物.由于環(huán)境脅迫較為明確,物種豐度相對(duì)較低,生物學(xué)與地球化學(xué)過程關(guān)聯(lián)緊密,AMD環(huán)境被認(rèn)為是基于基因組定量研究微生物生態(tài)和群落功能的理想“龕位”[6-7].另外,由于AMD污染,環(huán)境中微生物豐度也較低,這種復(fù)雜性較低的系統(tǒng)為研究較小尺度內(nèi)相關(guān)微生物基因異質(zhì)性以及為推斷復(fù)雜微生物群落進(jìn)化過程提供了一個(gè)理想的條件[7].

隨著第二代測(cè)序技術(shù)的發(fā)展,對(duì)這種酸性極端環(huán)境中微生物群落多樣性以及群落功能和進(jìn)化的了解越來越清晰.以往的研究往往集中在原核微生物群落,以致對(duì)真核微生物群落的了解較為薄弱[4,8-10].然而,真核微生物群落在酸性礦山廢水的形成、沉積物生態(tài)系統(tǒng)重金屬耐受、物質(zhì)循環(huán)和能量流動(dòng)等方面的作用不容忽視[11].

大寶山礦區(qū)位于廣東省韶關(guān)市曲江區(qū)境內(nèi),自19世紀(jì)70年代以來,采礦和冶煉活動(dòng)產(chǎn)生的大量礦渣和酸性礦山廢水排入到下游的橫石河中,而橫石河是當(dāng)?shù)鼐用褶r(nóng)業(yè)灌溉的主要水源之一.因此,橫石河的嚴(yán)重污染不僅給當(dāng)?shù)厣鷳B(tài)環(huán)境造成了極大的風(fēng)險(xiǎn),還威脅到當(dāng)?shù)鼐用竦慕】礫12].本研究選取廣東省大寶山礦區(qū)污染的橫石河作為研究點(diǎn)位,從上游到下游采樣,覆蓋不同的礦山廢水污染梯度,從極度污染區(qū)域到輕微污染區(qū)域.系統(tǒng)調(diào)查不同污染梯度中真核微生物群落多樣性的差異,并結(jié)合化學(xué)分析方法和生物信息學(xué)方法對(duì)引起真核微生物群落結(jié)構(gòu)和多樣性變化的主要脅迫因子進(jìn)行甄別.本研究拓展了對(duì)酸性礦山廢水污染環(huán)境中真核微生物群落多樣性的了解,為酸性礦山廢水污染生物修復(fù)提供了理論參考.

1 材料和方法

1.1 樣品采集

圖1 廣東省大寶山橫石河和對(duì)照河流采樣點(diǎn)位

選取大寶山礦區(qū)橫石河流域(約25km)作為研究區(qū)域.在2014年10月,沿橫石河從主污染源(大寶山礦區(qū))到下游輕微污染區(qū)域設(shè)置采樣點(diǎn),現(xiàn)場(chǎng)測(cè)定水樣pH值,采集500mL水樣4℃保存待分析.抓斗式采泥器(HYDRO-BIOS Apparatebau GmbH, Kiel- Holtenau, Germany)采集22個(gè)(1~22號(hào))表面沉積物(河流底部0~5cm,約500g)樣品.根據(jù)距主污染源的距離和酸性礦山廢水污染梯度將橫石河分為4個(gè)污染區(qū)域(圖1):區(qū)域1-極端污染區(qū)域(1~3號(hào)樣品);區(qū)域2-重度污染區(qū)域(4~11號(hào)樣品);區(qū)域3-中度污染區(qū)域(12~17號(hào)樣品);區(qū)域4-輕度污染區(qū)域(18~22號(hào)樣品).此外,采集周邊未受采礦活動(dòng)影響的河流中(青云山河)水體和沉積物樣品為無AMD污染對(duì)照(23~27號(hào))樣品.一周內(nèi)完成樣品凍干、磨細(xì)、分裝后保存于-80℃冰箱用于后續(xù)實(shí)驗(yàn).

1.2 理化參數(shù)測(cè)定

取0.5g干燥的沉積物于10mL 1mol/L的偏磷酸鈉((NaPO3)6)中分散24h后,使用Malvern Mastersizer 2000粒度儀(Malvern Instruments GmbH, Rigipsstr. Herrenberg, Germany)測(cè)定沉積物的粒度.剩余沉積物干樣經(jīng)球磨儀磨碎過200目的尼龍篩,分別測(cè)定過氧化氫酶活力、pH值和電導(dǎo)率 (EC)、總有機(jī)碳(TOC)含量[13-15].取適量土壤于純水中震蕩提取,然后用Thermo ICS-5000離子色譜(Thermo Fisher Scientific Inc, Waltham, MA, USA)測(cè)定硫酸根(SO42-)含量.沉積物中重金屬含量篩查,將0.2g底泥樣品加入到12mL反王水(硝酸、鹽酸體積比為3:1)中,使用MILESTONE ETHOS UP (Milestone Office, Shelton, CT, USA)微波消解儀消解,趕酸稀釋后使用電感耦合等離子體發(fā)射光譜(ICP-OES) (PerkinElmer Office, Akron, OH, United States)篩查金屬含量(As、Ca、Cd、Co、Cr、Cu、K、Mg、Mn、Mo、Ni、Pb、Sr、V和Zn).然后對(duì)橫石河沉積物中顯著高于對(duì)照河流中的金屬(As、Cd、Cu、Pb和Zn)使用BCR連續(xù)提取法[16]進(jìn)行重金屬4種形態(tài)分析,并測(cè)定水樣中這5種重金屬含量(ICP-MS (PerkinElmer Office, Akron, OH, United States)).

1.3 沉積物eDNA提取

使用土壤基因組DNA提取試劑盒,根據(jù)試劑盒建議的方法(基于MO BIO Powersoil DNA Isolation Kit)提取沉積物中的總DNA.使用Nanodrop (Nanodrop 2000, ThermoFisher, USA)檢測(cè)所提取DNA的濃度和純度,如果濃度小于20ng/μL,使用乙醇進(jìn)行沉淀濃縮.

1.4 18S rDNA PCR擴(kuò)增

用于18S rRNA基因V9區(qū)域的引物序列為: 1380F(5’-CCCTGCCHTTTGTACACAC-3’)和1510R (5’-CCTTCYGCAGGTTCACCTAC-3’)[17].上游引物5’端連接有Golay Barcode sequence用于區(qū)別樣品[18].PCR擴(kuò)增條件為:退火溫度98℃反應(yīng)30s,以下擴(kuò)增步驟進(jìn)行20個(gè)循環(huán):變性(98℃,5s),退火(62℃, 30s),延伸(72℃,15s);循環(huán)結(jié)束后末次延伸條件為72℃反應(yīng)7min.每次進(jìn)行PCR反應(yīng)時(shí)需設(shè)置對(duì)照組:陽性對(duì)照,陰性對(duì)照,無模板陰性對(duì)照.PCR反應(yīng)結(jié)束后,1.5%瓊脂糖凝膠電泳檢驗(yàn)PCR產(chǎn)物的大小,PCR產(chǎn)物經(jīng)純化后以等物質(zhì)的量混合組成測(cè)序文庫(kù)立即用于下游測(cè)序.

1.5 Ion Torrent測(cè)序和測(cè)序數(shù)據(jù)分析

測(cè)序工作以Ion Torrent Proton測(cè)序儀器相關(guān)試劑用戶手冊(cè)為依據(jù).測(cè)序數(shù)據(jù)已上傳到NCBI Sequence Read Archive (SRA)數(shù)據(jù)庫(kù)(Accession SRP094407).生物信息學(xué)分析系統(tǒng)環(huán)境為Bio-Linux 8[19],測(cè)序數(shù)據(jù)處理的主要程序?yàn)镼IIME[20]和USEARCH V9.0[21].通過RDP classifier[22]搜尋Protist Ribosomal Reference database (PR2)[23]進(jìn)行比對(duì)注釋.所得高質(zhì)量結(jié)果用于后續(xù)分析.

1.6 統(tǒng)計(jì)分析

所有統(tǒng)計(jì)分析主要是基于R語言(V3.6.1),對(duì)理化參數(shù)在橫石河不同區(qū)域以及橫石河和對(duì)照河流之間的差異都進(jìn)行Kruskal-Wallis秩和檢驗(yàn),<0.05視為有顯著差異.使用斯皮爾曼相關(guān)性分析來研究Alpha多樣性與理化變量的相關(guān)性.使用典范對(duì)應(yīng)分析(CCA)來確定重要的理化參數(shù)對(duì)微生物群落的影響,理化參數(shù)選擇依據(jù)CCA計(jì)算過程中VIF (variance inflation factors)值來確定.

2 結(jié)果

2.1 理化參數(shù)

表1 橫石河主要理化參數(shù)

續(xù)表1

橫石河理化參數(shù)如表1所示,分析結(jié)果顯示,上下游水體和沉積物理化參數(shù)差異巨大,大部分參數(shù)的數(shù)值有沿橫石河顯著下降的趨勢(shì).橫石河極端污染區(qū)域酸度極高,水體和沉積物中的pH值分別為2.98和2.64,到下游pH值逐漸回升,達(dá)到5.79和7.60.水體中所有重金屬和沉積物中大部分重金屬濃度有從上游到下游顯著下降的趨勢(shì),但是,沉積物中Cu的濃度在橫石河下游反而上升.沉積物中電導(dǎo)率和硫酸根濃度也有沿橫石河顯著降低的趨勢(shì).

2.2 測(cè)序結(jié)果

(A) 各采樣點(diǎn)真核微生物“門”分類水平相對(duì)豐度(B) 橫石河不同污染區(qū)域獨(dú)有和共有OTU數(shù)目.韋恩圖相對(duì)豐度小于0.5%的門類未展示

在橫石河和對(duì)照河流沉積物樣品中共獲得392596條reads,去除低質(zhì)量樣品(8號(hào)樣品),根據(jù)97%的相似度可以分為3436個(gè)OTU,其中,在橫石河4個(gè)污染區(qū)域和對(duì)照河流都存在的OTU有756個(gè).通過物種注釋結(jié)果,優(yōu)勢(shì)種群為:后鞭毛生物類(Opisthokonta)的真菌門(Fungi)(4.51%~ 86.69%)、原始色素體生物的(Archaeplastida)的綠藻門(Chlorophyta)(0.61%~77.36%)、囊泡蟲類(Alveolata)的纖毛蟲門(Ciliophora)(0.81%~34.91%).其余豐度較高的門類還有古蟲類(Excavata)的Discoba(0.51%~22.67%)、有孔蟲類的(Rhizaria)絲足蟲類(Cercozoa)(0.65%~19.50%)、原始色素體生物的(Archaeplastida)的扭鞘藻門(Streptophyta) (0.56%~ 82.64%)、后鞭毛生物類(Opisthokonta)的Metazoa (1.82%~11.45%)、茸鞭生物界(Stramenopiles)的褐藻門(Ochrophyta)(1.11%~12.23%)和不等鞭毛門(Stramenopiles-X)(0.60%~18.26%)、變形蟲門(Amoebozoa)的錐足亞門(Conosa)(0.56%~11.08%) (圖2).

2.3 Alpha多樣性

Alpha多樣性指數(shù)可以反映特定環(huán)境中微生物群落豐富度和均勻度,是根據(jù)OTU數(shù)目評(píng)估生態(tài)系統(tǒng)多樣性的綜合性指標(biāo).一般來說,環(huán)境越惡劣,生態(tài)系統(tǒng)物種多樣性就越低[24],因此,可以根據(jù)群落的Alpha多樣性大小評(píng)估生態(tài)系統(tǒng)所承受的環(huán)境壓力大小.整體來看,橫石河沉積物種真核微生物群落的Alpha多樣性(Chao1指數(shù)和Shannon指數(shù))(圖3(A)和(B))從上游到下游呈顯著上升的趨勢(shì),并且橫石河內(nèi)4個(gè)污染區(qū)域Alpha多樣性指數(shù)存在顯著差異.所以,橫石河上游到下游,隨著污染梯度下降,酸性礦山廢水污染對(duì)環(huán)境的脅迫水平逐漸降低.使用秩和檢驗(yàn)對(duì)橫石河不同污染區(qū)域Alpha多樣性差異性進(jìn)行檢驗(yàn)(值<0.05*、<0.01**、<0.001***)

(A),(B) Alpha多樣性.;(C) Beta多樣性

2.4 Beta多樣性

根據(jù)各采樣點(diǎn)真核微生物群落Bray-Curtis相異性,使用非度量多元尺度分析(non-metric multidimensional scaling, NMDS)方法評(píng)估橫石河和對(duì)照河流沉積物樣本中微生物群落結(jié)構(gòu)的差異性,并進(jìn)一步使用非參數(shù)多元方差分析(PERMANOVA).結(jié)果顯示,橫石河不同污染區(qū)域(區(qū)域1、區(qū)域2、區(qū)域3和區(qū)域4)和對(duì)照河流沉積物中真核微生物群落結(jié)構(gòu)存在顯著差異(=0.0214*);如果將橫石河當(dāng)做整體來看,其沉積物中真核微生物群落結(jié)構(gòu)和對(duì)照河流相比存在顯著差異(=0.0028**)(圖3(C)).

2.5 物種網(wǎng)絡(luò)分析

圖4 真核微生物“屬”分類水平相對(duì)豐度(相對(duì)豐度大于0.5%)和Alpha多樣性與橫石河理化參數(shù)相關(guān)性關(guān)系矩陣圖

右側(cè)數(shù)值為斯皮爾曼相關(guān)系數(shù)

使用Gephi v0.9.1和FruchtermanReingold放置算法的交互平臺(tái)[25],對(duì)橫石河真核微生物的關(guān)系進(jìn)行網(wǎng)絡(luò)可視化和模塊檢測(cè).在“科(family)”一級(jí)挑選橫石河沉積物中相對(duì)豐度大于0.5%,具有顯著共現(xiàn)關(guān)系(斯皮爾曼相關(guān)系數(shù)||>0.6且<0.01)的真核微生物進(jìn)行共現(xiàn)網(wǎng)絡(luò)關(guān)系分析.結(jié)果發(fā)現(xiàn)橫石河中核心真核微生物有:傘菌綱下屬科(Agaricomycets)、異葉足綱下屬科(Heterolobosea_XX)、毛霉菌亞門下屬科(Mucoromycotina_X)、棘阿米巴科(Acanthamoebidae)、三足蟲科(Trinematidae)、Allapsidae沙棘科(Sandonidae)、Cercomonadidae、Schizoplasmodiids、卵菌門下屬科(Oomycota_XX)、Glissomonadida_X、多枝藻目下屬科(Chaetophorales)、小球藻科(Chlorellales_X)、衣藻(CW-Chlamydomonad)、Vermamoebidae、Neobodonid、鉤端螺旋體科(Leptophryidae)、擬阿米巴科(Paramoebidae)、顎足綱下屬科(Maxillopoda)、環(huán)藻目下屬科(Sphaeropleales)等.

2.6 真核微生物群落與理化參數(shù)的關(guān)系

橫石河中不同污染區(qū)域水體和沉積物中的理化參數(shù),沉積物中真核微生物群落結(jié)構(gòu)都顯著不同.為了了解環(huán)境因子對(duì)真核微生物群落的影響,首先將所測(cè)得的環(huán)境理化參數(shù)與在“屬”分類水平上相對(duì)豐度大于5%的真核微生物以及Alpha多樣性進(jìn)行斯皮爾曼相關(guān)性分析.隨后、通過CCA分析影響微生物群落結(jié)構(gòu)的主要環(huán)境理化因子.相關(guān)性分析結(jié)果顯示,與物種相對(duì)豐度和Alpha多樣性相關(guān)性較強(qiáng)的環(huán)境理化因子主要有:水體pH值(Aq. pH)、水體中銅(Aq. Cu)、鋅(Aq. Zn)、鎘(Aq. Cd)、鉛(Aq. Pb);沉積物pH值(Sd.pH)、沉積物中電導(dǎo)率(Sd.EC)、硫酸根(Sd.Sulfate)、三價(jià)鐵離子(Sd. Ferric)、總鐵(Sd.Total.Fe)、可提取態(tài)銅(Cu.Exchangeable)、可提取態(tài)鋅(Zn.Exchangeable)、可提取態(tài)鎘(Cd.Exchangeable)和還原態(tài)鎘(Cd.Reducible)(圖4).CCA結(jié)果顯示,對(duì)橫石河中真核微生物群落影響最大的環(huán)境理化因子是沉積物中硫酸根離子(Sd.Sulfate)和電導(dǎo)率(Sd.EC);其次則是水體的pH值(Aq.pH),水體中鉛濃度(Aq.Pb)、沉積物中三價(jià)鐵的濃度(Sd.Ferric)和沉積物中鉛(Sd.Pb)的濃度,隨后水體中鋅(Aq.Zn)的濃度、沉積物中二價(jià)鐵(Sd.Ferrous)的濃度、沉積物中總有機(jī)碳的含量(Sd.TOC)等環(huán)境因子也對(duì)真核生物的群落結(jié)構(gòu)變化有著顯著的影響(圖5).

圖5 真核微生物和理化參數(shù)典型相關(guān)分析

每個(gè)點(diǎn)代表相應(yīng)樣品中的真核微生物,半透明的橢圓代表95%的置信區(qū)間,箭頭的方向和長(zhǎng)短代表該理化參數(shù)和真核微生物群落結(jié)構(gòu)的關(guān)系

3 討論

目前真核微生物群落響應(yīng)酸性廢水只報(bào)道過真菌和綠藻,不同酸性礦山廢水環(huán)境中真核微生物占整體生物量比重差異巨大,如西班牙Rio Tinto礦區(qū)的酸性礦山廢水環(huán)境中真核微生物生物量占比很大,其生物量和生物多樣性遠(yuǎn)超原核微生物[26],而其它一些酸性礦山廢水環(huán)境,如美國(guó)加利福尼亞的Richmond Mine,其極端污染區(qū)域真核微生物多樣性比細(xì)菌甚至古菌都要低[27].真核生物在礦山廢水生態(tài)環(huán)境物質(zhì)和能量流動(dòng)方面所起到的作用不容忽視[10],如耐酸的藻類和真菌能夠給異養(yǎng)菌(如硫酸鹽還原菌(SRB)和Fe3+還原菌)提供必需的有機(jī)碳[10],通過吞食影響其它細(xì)菌和古菌的群落結(jié)構(gòu),或通過鐵氧化速率來調(diào)節(jié)酸性礦山廢水生成速率[28],并在酸性礦山廢水生態(tài)系統(tǒng)鐵/硫循環(huán)過程中發(fā)揮著重要作用[10].

結(jié)合對(duì)照河流沉積物中真核微生物分析,橫石河優(yōu)勢(shì)種群有真菌門(Fungi)(4.51%~86.69%)、綠藻門(Chlorophyta)(0.61%~77.36%)和纖毛蟲門(Ciliophora) (0.81%~34.91%).不同污染區(qū)域物生物群落結(jié)構(gòu)和多樣性有顯著差異,在上游污染較為嚴(yán)重的區(qū)域纖毛蟲門(Ciliophora)豐度較低,而隨著污染程度的降低,纖毛蟲門相對(duì)豐度逐漸升高.真核微生物群落的豐富度(Chao1)和均勻度(Shannon)也隨著污染程度的降低而顯著升高.

共現(xiàn)關(guān)系網(wǎng)絡(luò)分析發(fā)現(xiàn)一些真核微生物的存在與群落中其他真核微生物有著密切聯(lián)系,對(duì)這些微生物深入了解將有助于揭開真核微生物對(duì)酸性礦山廢水污染響應(yīng)機(jī)制的神秘面紗.Acanthamoebidae和Sandonidae廣泛存在于淡水、土壤等各類環(huán)境介質(zhì)中[29];Agaricomycetes可以存在于酸性環(huán)境中[30],具有金屬氧化功能,尤其能夠?qū)⑷芙鈶B(tài)的二價(jià)錳氧化為礦物態(tài)的三價(jià)/四價(jià)錳[31],多用于錳污染修復(fù); Chlorellales[32]和Chlamydomona[33]是極端環(huán)境中常見的異養(yǎng)藻類,能夠在碳源稀缺,理化條件惡劣的環(huán)境中生存[34];Heterolobosea是一種在酸性礦山廢水環(huán)境中廣泛存在[35]并且能夠在極端環(huán)境中保持極高的代謝活力[6];Mucoromycotina之前多在植物根際土壤中發(fā)現(xiàn),能夠幫助植物吸收礦物質(zhì)[35]; Strombidiidae可以在高鹽水體中作為優(yōu)勢(shì)種群生存[36-37].這些物種在重金屬降解、固碳、礦物吸收和能量代謝方面發(fā)揮著重要作用.

在酸性礦山廢水環(huán)境中,對(duì)原核微生物群落影響較大的環(huán)境理化因子主要有pH值[38-39]、鐵濃度梯度[40]、氧化梯度[41]、重金屬濃度[42]等.對(duì)大寶山礦區(qū)污染的橫石河區(qū)域的研究表明影響原核微生物Alpha多樣性的前3個(gè)因素分別是沉積物中電導(dǎo)率(Sd.EC)、沉積物pH值(Sd.pH)和水體中(Aq.Pb)[9].在本研究中,斯皮爾曼相關(guān)性分析結(jié)果顯示,與橫石河沉積物中真核微生物群落Alpha多樣性(Chao1和Shannon指數(shù))顯著正相關(guān)的環(huán)境理化因子有:水體pH值(Aq.pH)和沉積物pH值(Sd.pH);與其顯著負(fù)相關(guān)的環(huán)境理化因子有:水體中Cu(Aq.Cu)、Cd(Aq. Cd)、Zn(Aq.Zn)、Pb(Aq.Pb)的濃度.另外,沉積物中三價(jià)鐵(Sd.Ferric)和總鐵(Sd.Total.Fe)、硫酸根(Sd.Sulfate)、可提取態(tài)銅(Cu.Exchangeable)、鋅(Zn.Exchangeable)、鎘(Cd.Exchangeable)和還原態(tài)鎘(Cd.Reducible)也和多種真核微生物相對(duì)豐度有著顯著的相關(guān)性關(guān)系,表明這些環(huán)境理化因子或許是酸性礦山廢水環(huán)境中真核微生物群落面臨的主要環(huán)境壓力.隨后使用CCA分析進(jìn)一步對(duì)影響微生物群落結(jié)構(gòu)的環(huán)境因子進(jìn)行甄別,限制性排序分析結(jié)果顯示對(duì)真核微生物群落結(jié)構(gòu)影響最大的環(huán)境理化因子是沉積物中硫酸根(Sd.Sulfate)和電導(dǎo)率(Sd.EC),其次才是水體pH值(Aq.pH)、水中鉛的含量(Aq.Pb)和沉積物中三價(jià)鐵的含量(Sd.Ferric).由此可見原核微生物和真核微生物對(duì)同樣環(huán)境脅迫響應(yīng)并不相同,原核微生物對(duì)河流環(huán)境中pH值、鐵含量、氧化梯度、重金屬濃度的變化比較敏感,而真核微生物則對(duì)硫酸根和電導(dǎo)率變化響應(yīng)更為強(qiáng)烈.與Alpha多樣性顯著相關(guān)的環(huán)境理化因子中Aq.pH、AqPb和Aq.Zn對(duì)真核微生物群落結(jié)構(gòu)有較為顯著的影響,而從CCA結(jié)果來看,Aq.Cu和Aq.Cd對(duì)真核微生物群落結(jié)構(gòu)影響不大.之前有學(xué)者對(duì)西藏地區(qū)森林生態(tài)系統(tǒng)研究發(fā)現(xiàn),pH值能夠決定土壤真菌群落的Alpha多樣性,但是對(duì)Beta多樣性沒有顯著的影響[43].因此,對(duì)真核微生物群落Alpha多樣性產(chǎn)生顯著影響的環(huán)境理化因子與影響B(tài)eta多樣性的環(huán)境理化因子可能不同,橫石河沉積物中對(duì)真核微生物群落影響最大的環(huán)境因子是硫酸根和電導(dǎo)率.

4 結(jié)論

4.1 沉積物中真核微生物群落Alpha多樣性隨酸性礦山廢水污染梯度降低而逐漸升高.

4.2 原核微生物和真核微生物對(duì)同樣環(huán)境脅迫響應(yīng)并不相同.酸性礦山廢水環(huán)境沉積物中真核微生物群落結(jié)構(gòu)的變化主要受硫酸根和電導(dǎo)率的影響.

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Effects of acid mine drainage on eukaryotic community in river sediments.

LIU Fan1, ZHANG Xiao-hui1, TANG Song2, WANG Mao3, LIU Hong-ling1*

(1.State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China；2.National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing 100021, China；3.School of Public Health, Sun Yat-sen University, Guangzhou 510080, China)., 2019,39(12)：5285~5292

Analytical and molecular biology methods were integrated together to investigate the physicochemical factors in water and sediments of Hengshi River as well as the diversity of eukaryotic microorganism community in the sediments. Spearman correlation and canonical correspondence analysis (CCA) were further applied to identify the major factors affecting eukaryotic microorganism community. Results of 18S rRNA gene sequencing indicated that Eukaryotic community of Hengshi River was dominated by Fungi (4.51%~86.69%), Chlorophyta (61%~77.36%) and Ciliophora (0.81%~34.91%). Both the abundance and evenness of eukaryotic communities in sediments significantly and gradually increased along the Hengshi River with decreasing contamination, which suggested that composition of eukaryotic microorganism community partly reflected changes in physicochemical conditions. Sulfate and electric conductivity of the sediments were the major abiotic factors altering the eukaryotic communities in the sediments of AMD impacted Hengshi River, which was different from the findings of prokaryotic microbial communities.

acid mine drainage；eukaryotic microorganisms；sulfate；electric conductivity；next-generation sequencing

X172

A

1000-6923(2019)12-5285-08

劉 帆(1995-),女,山東濟(jì)南人,南京大學(xué)碩士研究生,研究方向?yàn)榄h(huán)境毒理學(xué)與風(fēng)險(xiǎn)評(píng)價(jià).

2019-05-24

國(guó)家自然科學(xué)基金(21677073);國(guó)家重點(diǎn)研發(fā)項(xiàng)目(2018YFC1801505);國(guó)家科技重大專項(xiàng)(2018ZX07208001,2017ZX07301002)

* 責(zé)任作者, 副教授, hlliu@nju.edu.cn