彌河沉積物的反硝化和厭氧氨氧化過(guò)程

李佳霖,秦 松

彌河沉積物的反硝化和厭氧氨氧化過(guò)程

李佳霖1,2,秦 松1,2*

(1.中國(guó)科學(xué)院煙臺(tái)海岸帶研究所海岸帶生物學(xué)與生物資源保護(hù)實(shí)驗(yàn)室,山東 煙臺(tái) 264003；2.中國(guó)科學(xué)院海洋大科學(xué)研究中心,山東 青島 266071)

本研究選取彌河4個(gè)站點(diǎn)為研究對(duì)象,在不同季節(jié)分別采集沉積物樣品,測(cè)定理化指標(biāo),并采用同位素配對(duì)技術(shù)和分子生物學(xué)方法,研究了沉積物中的反硝化和厭氧氨氧化作用及其影響因素.結(jié)果表明,彌河沉積物中的反硝化速率變化范圍為151.75~2847.86μmol/(m2×h),厭氧氨氧化速率的變化范圍為149.57~2109.17μmol/(m2×h),厭氧氨氧化在氮去除中的貢獻(xiàn)量平均達(dá)到56.1%.沉積物中的反硝化細(xì)菌以K型為主,豐度為0.19×106~5.12×106個(gè)/g,主要是-和-變形菌門(mén);厭氧氨氧化細(xì)菌以A為標(biāo)記基因的豐度范圍是2.58×102~1.14×104個(gè)/g,主要為浮霉菌門(mén)的屬細(xì)菌.反硝化速率與沉積物中的TN含量和間隙水PO43-呈正相關(guān)關(guān)系,厭氧氨氧化作用與沉積物中的TN含量呈正相關(guān),而與沉積物密度呈負(fù)相關(guān)關(guān)系,沉積物的理化指標(biāo)是決定氮去除速率的主要環(huán)境條件.彌河的反硝化和厭氧氨氧化作用明顯,對(duì)減輕氮超標(biāo)具有重要意義,合理改變沉積環(huán)境是有效提高氮去除速率的可參考方式.

反硝化作用；厭氧氨氧化作用；功能微生物；影響因素；彌河

受人類(lèi)活動(dòng)的影響,世界范圍內(nèi)的近海入海河流普遍存在無(wú)機(jī)氮污染的環(huán)境問(wèn)題.根據(jù)美國(guó)環(huán)境保護(hù)署2012年的調(diào)查報(bào)告,全美河流41%的河段氮含量較高;在我國(guó)實(shí)施監(jiān)測(cè)的55條入海河流中,過(guò)半數(shù)河流入海斷面水質(zhì)劣于V類(lèi)地表水水質(zhì)標(biāo)準(zhǔn),主要污染要素中包括氨氮[1].無(wú)機(jī)氮污染不僅會(huì)造成區(qū)域性河流的富營(yíng)養(yǎng)化,而且隨河流入海后將產(chǎn)生近海海域嚴(yán)重的生態(tài)問(wèn)題,如生物多樣性降低、發(fā)生有害藻華甚至出現(xiàn)低氧“死區(qū)”[2-3].無(wú)機(jī)氮污染主要來(lái)源于非點(diǎn)源的農(nóng)業(yè)化肥,由于未能有效利用,全球超過(guò)一半的施用化肥排入河流,而隨著糧食需求及施肥強(qiáng)度的加大,未來(lái)全球范圍內(nèi)排入河流的氮總量仍將增加[4],因此對(duì)河流生態(tài)系統(tǒng)中無(wú)機(jī)氮的生物地化循環(huán)進(jìn)行研究具有重要意義.

微生物介導(dǎo)的無(wú)機(jī)氮生物地化循環(huán)過(guò)程包括固氮、硝化、硝酸鹽異化還原成銨、反硝化和厭氧氨氧化等作用[5].其中,河流的反硝化和厭氧氨氧化過(guò)程將無(wú)機(jī)氮轉(zhuǎn)化為氮?dú)夂脱趸瘉喌?可以實(shí)現(xiàn)流域中無(wú)機(jī)氮的去除,既是計(jì)算氮循環(huán)通量的關(guān)鍵內(nèi)容,也是潛在治理氮富營(yíng)養(yǎng)化、實(shí)現(xiàn)生態(tài)修復(fù)的有效途徑.反硝化和厭氧氨氧化是厭氧反應(yīng)過(guò)程,普遍存在于水體懸浮顆粒物、沉積物環(huán)境中,在河流中去除氮的貢獻(xiàn)量達(dá)到40%~80%[6].利用同位素配對(duì)技術(shù)對(duì)反硝化和厭氧氨氧化進(jìn)行測(cè)定,發(fā)現(xiàn)多數(shù)河流沉積物的反硝化作用占主要貢獻(xiàn)量[7-9],但隨著無(wú)機(jī)氮濃度的升高,提供反硝化作用所需電子的有機(jī)物相對(duì)不足,厭氧氨氧化作用會(huì)成為無(wú)機(jī)氮去除的主要過(guò)程[10].研究發(fā)現(xiàn)河流中的環(huán)境因子,包括鹽度、無(wú)機(jī)氮、有機(jī)質(zhì)、沉積物的理化特征與反硝化和厭氧氨氧化速率均存在相關(guān)性[11-13];同時(shí)由于是微生物介導(dǎo)的過(guò)程,反硝化和厭氧氨氧化細(xì)菌的豐度和多樣性能夠直接影響反應(yīng)速率[14-16].由于河流的環(huán)境條件變化較大,反硝化和厭氧氨氧化速率及其主要的影響因素也存在明顯差異.

彌河是萊州灣南岸的主要入海河流之一.受溫帶季風(fēng)氣候冷暖氣流的影響,彌河具有我國(guó)北方河流的顯著特點(diǎn),即春秋旱易形成斷流現(xiàn)象、夏季降雨較多流量激增、冬季少雪存在冰期.由于常年開(kāi)采地下水用于城市供水及農(nóng)業(yè)生產(chǎn),造成流域內(nèi)大范圍的地下水超采,彌河中下游的咸水入侵較為嚴(yán)重,河水的鹽度尤其是在旱季有顯著升高[17].彌河上游的壽光是北方重要的蔬菜生產(chǎn)基地,中下游流經(jīng)化工產(chǎn)業(yè)園,大量工農(nóng)業(yè)含氮污水及沿岸生活污水造成彌河的氮污染較高[18].彌河入海后匯入萊州灣,萊州灣尤其是西部海區(qū)水體呈現(xiàn)嚴(yán)重的氮富營(yíng)養(yǎng)化狀態(tài),其主要的來(lái)源是沿岸河流的輸入[19].本研究通過(guò)對(duì)彌河沉積物的反硝化和厭氧氨氧化過(guò)程向河水中釋放氮?dú)馔康难芯?探討了其對(duì)流域氮循環(huán)過(guò)程的影響;同時(shí)對(duì)沉積物中反硝化和厭氧氨氧化功能菌群的豐度和多樣性進(jìn)行分析,結(jié)合環(huán)境因子,評(píng)估了影響沉積物反硝化和厭氧氨氧化速率的影響因子.本研究結(jié)果深化了河流中對(duì)氮自去除過(guò)程的認(rèn)識(shí),有助于實(shí)現(xiàn)氮收支的核算,為河流富營(yíng)養(yǎng)化的生態(tài)治理和修復(fù)提供科學(xué)參考.

1 材料與方法

1.1 研究站位與樣品采集

彌河主要的環(huán)境問(wèn)題是咸水入侵和氮富營(yíng)養(yǎng)化,咸水入侵造成沉積物生境的顯著變化.根據(jù)咸水入侵范圍[17]和主要陸源污染輸入地域,沿彌河選取4個(gè)采樣站位,分別位于鹵水區(qū)的彌河橋下(M1)、咸淡水混合區(qū)的南半截河村(M2)、張家北樓村(M3)和入侵范圍外的壽光市內(nèi)(M4),于2012年4月、8月和10月進(jìn)行了春、夏和秋季的調(diào)查和采樣(圖1).由于采樣年份冬季彌河存在冰期,本研究未對(duì)彌河進(jìn)行冬季的研究.

沉積物樣品采集于河邊淺水浸沒(méi)區(qū),采取多點(diǎn)取樣法.用有機(jī)玻璃柱(長(zhǎng)度20cm,直徑3.4cm)采集表層5cm沉積物柱樣,每個(gè)站位采集12個(gè)平行樣,上下部用膠塞塞好;采集采樣點(diǎn)原位水2.0L,用0.22μm濾膜過(guò)濾水樣后裝入滅菌廣口瓶中,于4℃保溫箱保存帶回實(shí)驗(yàn)室,用于反硝化和厭氧氨氧化通量測(cè)定培養(yǎng)實(shí)驗(yàn)[20].用無(wú)菌針筒采樣管采集表層5cm沉積物保存于4℃保溫箱,用于間隙水營(yíng)養(yǎng)鹽、孔隙度、密度、含水量、有機(jī)碳、總氮和粒度測(cè)定,采集表層5cm沉積物保存于液氮中帶回置于-80℃冰箱中,用于功能群落豐度和多樣性分析[21].

圖1 采樣點(diǎn)分布

★為采樣點(diǎn)

1.2 環(huán)境因子測(cè)定

河水的溫度、鹽度和pH值在采樣現(xiàn)場(chǎng)用YSI556MPS采集數(shù)據(jù).密度和含水率采用稱(chēng)量法測(cè)定.沉積物粒度采用激光粒度儀直接測(cè)定(Matersizer 2000F laser particle size analyzer, Malvern, England). 間隙水經(jīng)離心采集后用連續(xù)流動(dòng)分析儀(AAA3, Seal Analytical GmbH, German)的標(biāo)準(zhǔn)方法進(jìn)行測(cè)定[22].元素分析儀 (Elementar, Vario Micro, German)用于測(cè)定沉積物的有機(jī)碳和總氮.重金屬含量采用ICP-MS (PerkinElmer, Elan DRC II, Hong Kong)測(cè)定.所有環(huán)境參數(shù)進(jìn)行3個(gè)重復(fù)測(cè)定,結(jié)果取平均值.

1.3 反硝化與厭氧氨氧化速率測(cè)定

反硝化和厭氧氨氧化速率采用同位素配對(duì)技術(shù)通過(guò)培養(yǎng)實(shí)驗(yàn)的方法進(jìn)行測(cè)定.將采集的沉積物帶回實(shí)驗(yàn)室后插入培養(yǎng)箱中,在有機(jī)玻璃管內(nèi)注滿原位采集的上覆水,密封平衡24h.將培養(yǎng)管分為2組,每組6個(gè),室溫下培養(yǎng),分別加入125和250μL的Na15NO3溶液(0.1mol/L,99.5%15N,Sigma),培養(yǎng)管用帶磁轉(zhuǎn)子的膠塞封住,在磁轉(zhuǎn)子60r/min條件下放置0.5h使15NO3－在培養(yǎng)體系中均勻分布.培養(yǎng)0時(shí)刻,在對(duì)照組6個(gè)培養(yǎng)管中加入0.5mL的ZnCl2(50%,/)溶液,培養(yǎng)3h后,在對(duì)照組6個(gè)培養(yǎng)管中加入0.5mL ZnCl2溶液,混勻后取上層泥漿樣品12.5mL放入容量瓶(Labco, UK),加入100μL ZnCl2溶液密封保存.培養(yǎng)樣品中的28N2、29N2和30N2含量比值利用氦氣頂空平衡法抽取出頂空氣體,采用穩(wěn)定同位素質(zhì)譜儀(Gasbench-MAT253, Thermo Fisher, USA)測(cè)定[20],計(jì)算公式如下[23]:

式中:是不同濃度15NO-添加體系中15NO-濃度的比值;29N2是29N2-N生成量,μmol;30N2是30N2-N生成量,μmol;(1)和(2)分別表示加入125和250μL Na15NO3的培養(yǎng)體系;14是還原體系中14NO-/15NO-的比值;14是N2-N實(shí)際生成量,μmol;28是厭氧氨氧化作用生成的28N2的量,μmol;28是反硝化作用生成的28N2的量,μmol.

1.4 功能微生物豐度與多樣性

稱(chēng)取0.50g沉積物樣品,用土壤DNA提取試劑盒(Mobio, USA)提取DNA,并將平行的3個(gè)樣品混合后進(jìn)行后續(xù)PCR反應(yīng).用反硝化細(xì)菌亞硝酸鹽還原酶的編碼基因S和K及厭氧氨氧化功能基因片段A的特異性引物進(jìn)行片段擴(kuò)增,引物及反應(yīng)條件見(jiàn)表1.用絕對(duì)實(shí)時(shí)定量PCR方法測(cè)定樣品中的功能基因豐度,并以沉積物濕重計(jì)量.用變性梯度聚丙烯酰胺凝膠電泳法對(duì)功能菌群多樣性進(jìn)行聚類(lèi)分析,選取樣品用克隆建庫(kù)方法分析功能基因的群落結(jié)構(gòu)和組成.功能基因序列提交NCBI數(shù)據(jù)庫(kù),S和K的序列號(hào)分別為MT532558 - MT532658和MT532681 - MT532729,A的序列號(hào)為MT532659 - MT532680.測(cè)定的功能基因序列通過(guò)Mothur采用最遠(yuǎn)鄰算法進(jìn)行操作分類(lèi)單元(OTU)分類(lèi),翻譯為相應(yīng)的氨基酸序列,與GenBank數(shù)據(jù)庫(kù)中相關(guān)氨基酸的近似序列進(jìn)行比對(duì),并采用最大似然法用Mega-X構(gòu)建系統(tǒng)發(fā)育樹(shù).

表1 反硝化和硝化作用功能基因片段引物序列信息

1.5 統(tǒng)計(jì)分析方法

單因子方差分析用于檢驗(yàn)環(huán)境因子、功能菌群豐度及氮去除速率是否存在顯著性差異,<0.05表示兩者間存在差異,<0.01表示兩者間存在顯著性差異.采用皮爾遜相關(guān)系數(shù)衡量變量間的線性關(guān)系.通過(guò)多元回歸方法分析上覆水、沉積物和間隙水的理化性質(zhì)及功能細(xì)菌豐度對(duì)反硝化作用和厭氧氨氧化作用變化的影響[28-29].

2 結(jié)果與討論

2.1 彌河主要環(huán)境因子

環(huán)境條件通過(guò)影響微生物的種群豐度和結(jié)構(gòu),制約生化反應(yīng)速率,進(jìn)而影響微生物參與的生物地化循環(huán)作用過(guò)程.彌河樣品環(huán)境因子數(shù)據(jù)見(jiàn)表2,各因子在站位和季節(jié)間存在較大差異.研究站位上覆水的鹽度顯示,彌河研究河段盡管處于咸水入侵區(qū)附近,但鹽度相比于同樣處于咸水入侵區(qū)的小清河(2.73‰)和膠萊河(7.96‰)偏低[30];所有樣品的pH值均偏堿性,平均值為8.57.沉積物間隙水中無(wú)機(jī)氮NH4+-N和NO3--N含量均值為0.534和0.397mg/L, NH4+-N含量隨季節(jié)變化較大,春季明顯較高; PO43-P的含量較低,與無(wú)機(jī)氮的濃度相比差異明顯偏低,是沉積物生態(tài)環(huán)境的主要限制因子;沉積物中重金屬含量較高,按重金屬的物質(zhì)的量濃度比較,Zn的濃度最高,其次是Cu, Pb和Cr的濃度較低.

彌河處于明顯的季風(fēng)氣候區(qū),夏季雨水充沛,徑流量上升,上覆水鹽度夏季偏低,但站位間和季節(jié)間的環(huán)境因子差異并不顯著.

表2 站點(diǎn)上覆水、沉積物和間隙水的物理化學(xué)性質(zhì)

2.2 反硝化和厭氧氨氧化速率

如圖2所示,彌河的反硝化速率范圍為151.75~ 2847.86μmol/(m2×h),均值為680.83μmol/ (m2×h),季節(jié)間差異較為顯著(=0.02,=7.39),春季的反硝化速率明顯較高,平均達(dá)到1512.58μmol/(m2×h),夏秋季則相對(duì)較低.厭氧氨氧化速率范圍為149.57~ 2109.17μmol/(m2×h),均值為898.32μmol/(m2×h),也表現(xiàn)出較為顯著的季節(jié)間差異(=0.04,=6.01),春季最高,夏季較高,秋季則最低.彌河厭氧氨氧化在氮去除中的貢獻(xiàn)量是42.5%~78.0%,平均達(dá)到56.1%,說(shuō)明在彌河的無(wú)機(jī)氮去除過(guò)程中,厭氧氨氧化過(guò)程與反硝化過(guò)程的貢獻(xiàn)量相當(dāng),兩者都是氮去除的重要過(guò)程.

反硝化作用是在厭氧條件下NO3-經(jīng)多步反應(yīng)依次還原為NO2-、NO、N2O,最終生成N2(或不完全反應(yīng)條件下終產(chǎn)物為N2O)的過(guò)程.彌河的反硝化作用速率與三峽庫(kù)區(qū)小江支流泄水期的速率相當(dāng)[647.20μmol/(m2×h)],與小江支流蓄水期的速率[24.04μmol/(m2×h)]和淀山湖區(qū)的速率[14.60μmol/ (m2×h)]相比較高[31-32];對(duì)全球河流沉積物的反硝化作用估算范圍是100.00~685.71μmol/(m2×h)[33],與之相比彌河的反硝化作用速率春季明顯偏高,夏秋兩季處于正常范圍內(nèi).彌河的厭氧氨氧化速率經(jīng)沉積物密度值換算后均值約為11.2nmol/(g×h),與長(zhǎng)江口低鹽度河域[0.23nmol/(g×h)]和美國(guó)的Cape Fear河的速率[0.17~4.77nmol/(g×h)]相比偏高[34-35].而白洋淀河畔的速率是7~20nmol/(g×h),與彌河速率相當(dāng).彌河較高的厭氧氨氧化速率造成其相對(duì)貢獻(xiàn)量也較高,而珠江河口、白洋淀河畔和法國(guó)的Seine灣厭氧氨氧化過(guò)程的貢獻(xiàn)量分別是3.8%~7%[7],11%~ 35%[8]和3%~8%[9],其氮去除過(guò)程均以反硝化過(guò)程為主.厭氧氨氧化過(guò)程的貢獻(xiàn)量較高可能與河水沉積物中NO3-含量較高,而有機(jī)碳含量相對(duì)不足有關(guān)[36].

2.3 反硝化細(xì)菌豐度與功能基因多樣性

亞硝酸鹽還原為NO既是反硝化過(guò)程的重要限速步驟,也是反硝化過(guò)程區(qū)別于其它硝酸鹽代謝的標(biāo)志性反應(yīng),其功能酶即亞硝酸鹽還原酶(Nir)包括K和S,分別是銅型亞硝酸鹽還原酶和細(xì)胞色素cd1型亞硝酸鹽還原酶,是反硝化微生物的重要分子標(biāo)志物[37].彌河的反硝化細(xì)菌豐度如圖3(a)所示,其中K型豐度范圍是0.19× 106~5.12×106個(gè)/g,均值為1.38×106個(gè)/g;S型豐度范圍是7.19×103~2.23×105個(gè)/g,均值為8.41× 104個(gè)/g.反硝化細(xì)菌以K型為主,豐度約是S型的16倍.一般較淺的河流中K型反硝化菌是主要類(lèi)群,而湖泊和海灣中S型反硝化細(xì)菌則是主要類(lèi)群[31],主要原因是S反硝化細(xì)菌對(duì)環(huán)境因子的變化更敏感,河流出現(xiàn)斷流現(xiàn)象造成的表層沉積物條件的改變,以及河流氮富營(yíng)養(yǎng)化造成的相對(duì)磷限制,會(huì)顯著影響S型反硝化細(xì)菌豐度[38].

DGGE的條帶圖譜如圖3(b)所示,根據(jù)條帶位置和豐度計(jì)算香農(nóng)指數(shù)的范圍分別是2.64~3.08和1.32~2.42.K型反硝化細(xì)菌相似度范圍是34.9%~ 69.2%;在相似度40%條件下聚類(lèi)為3類(lèi),季節(jié)差異性高于采樣點(diǎn)間的差異性.而S型反硝化細(xì)菌所有站位的相似度范圍是8.1%~52.1%,在相似度10%條件下聚類(lèi)成2類(lèi),樣品的季節(jié)和采樣點(diǎn)間差異均較大,反映出彌河的反硝化細(xì)菌中K型的多樣性明顯較高,且主要隨季節(jié)變化有所差異,而S型反硝化細(xì)菌樣品間差異更大,說(shuō)明其群落組成波動(dòng)較大.

圖3 反硝化細(xì)菌功能基因nirK和nirS豐度、聚類(lèi)分析及系統(tǒng)進(jìn)化樹(shù)

4,8,10表示采樣時(shí)間為4月、8月、10月,下同

選取M1-4、M3-8和M2-10樣品構(gòu)建彌河K型反硝化細(xì)菌功能基因的克隆文庫(kù)(圖3c).以陽(yáng)性克隆的菌株進(jìn)行測(cè)序,并將測(cè)序結(jié)果的氨基酸序列與Pfam數(shù)據(jù)庫(kù)進(jìn)行比對(duì)確認(rèn),共獲得101條K功能基因序列,不同序列間的相似性為32%以上.在80%相似度下,共獲得23個(gè)OTU,以的亞硝酸鹽還原酶A的氨基酸序列為外群,構(gòu)建了K功能基因氨基酸序列的系統(tǒng)進(jìn)化樹(shù).彌河K型反硝化細(xì)菌的相近序列為-和變形菌門(mén),相近序列多為不可培養(yǎng)的環(huán)境細(xì)菌,來(lái)源包括農(nóng)田土壤、活性污泥、生物膜反應(yīng)器、以及湖泊和近海的沉積物.S型反硝化細(xì)菌在彌河沉積物中的占比較低,以M3-8樣品構(gòu)建了克隆文庫(kù),共獲得49條功能基因序列,不同序列間的相似性達(dá)到45%以上.按照80%相似度共獲得15個(gè)OTU,以IM2的亞硝酸鹽還原酶氨基酸序列為外群,與相近序列編碼的氨基酸序列構(gòu)建了系統(tǒng)進(jìn)化樹(shù).S型反硝化細(xì)菌的相近序列包含-、和-變形菌門(mén),相近序列都來(lái)自于不同河流和河口的沉積物.

2.4 厭氧氨氧化細(xì)菌豐度與功能基因多樣性

聯(lián)氨合成酶Hzs是亞硝酸鹽還原成NO后與NH4+合成聯(lián)氨即肼(NH2NH2)的功能酶,其編碼基因A是厭氧氨氧化細(xì)菌的分子標(biāo)志物,常用于研究厭氧氨氧化細(xì)菌的群落組成和環(huán)境豐度[27].彌河的A基因豐度如圖4(a)所示,范圍為2.58×102~ 1.14×104個(gè)/g,均值為2.04×103個(gè)/g,與東江河[39]、珠江入?？赱40]及法國(guó)Cape Fear河[41]的厭氧氨氧化細(xì)菌豐度相比偏低約一數(shù)量級(jí).本研究中以聯(lián)氨氧化酶功能基因AB為標(biāo)志物對(duì)厭氧氨氧化細(xì)菌進(jìn)行了目標(biāo)基因擴(kuò)增,但未獲得目標(biāo)條帶.

已知的厭氧氨氧化細(xì)菌均屬于浮霉菌目(Planctomycetes),由于其A序列差異較小,DGGE條帶分離不明顯,直接選取M3-8和M2-10的樣品構(gòu)建了克隆文庫(kù),驗(yàn)證了彌河厭氧氨氧化細(xì)菌的存在及優(yōu)勢(shì)種群.測(cè)序共獲得22條A序列,不同序列間的相似性為19%以上,3%相似度下共獲得10個(gè)OTU.通過(guò)與NCBI數(shù)據(jù)庫(kù)A基因氨基酸序列比對(duì),發(fā)現(xiàn)均為“”屬,以另一主要屬的A氨基酸序列為外群,構(gòu)建系統(tǒng)進(jìn)化樹(shù)(圖4b).在珠江口入海斷面沉積物厭氧氨氧化細(xì)菌多樣性的研究也發(fā)現(xiàn),鹽度較低的站位中,屬厭氧氨氧化菌是優(yōu)勢(shì)種群[42].

圖4 厭氧氨氧化細(xì)菌功能基因hzsA豐度及系統(tǒng)進(jìn)化樹(shù)

2.5 影響反硝化和厭氧氨氧化速率的主要因素

彌河的反硝化和厭氧氨氧化作用具有協(xié)同性,兩者顯著正相關(guān),相關(guān)系數(shù)為0.72(=0.008).Zhou等[43]的研究發(fā)現(xiàn),通過(guò)氯酸鹽抑制反硝化NO3-還原為NO2-的步驟,反硝化作用和厭氧氨氧化作用均會(huì)顯著降低,說(shuō)明在NO2-含量較低的沉積環(huán)境中,厭氧氨氧化作用的進(jìn)行依賴(lài)于反硝化作用第一步還原反應(yīng)生成的NO2-,在長(zhǎng)江口河域[44]以及沉水農(nóng)田[37]中均觀測(cè)到厭氧氨氧化作用與反硝化作用的關(guān)聯(lián)性.目前在氮富營(yíng)養(yǎng)化嚴(yán)重的河道,依賴(lài)反硝化的厭氧氨氧化作用作為生物修復(fù)手段被證實(shí)是同時(shí)去除NO3-和NH4+的有效途徑[45].

彌河的反硝化作用與沉積物間隙水中的PO43-呈顯著正相關(guān)(= 0.76,= 0.005),與沉積物的TN呈正相關(guān)(= 0.63,= 0.029);厭氧氨氧化作用與沉積物的TN呈顯著正相關(guān)(= 0.85,= 0.001),與沉積物密度呈負(fù)相關(guān)(= - 0.66,= 0.018).沉積物中的N素是氮去除過(guò)程的主要底物,是影響彌河反硝化和厭氧氨氧化速率的主要環(huán)境因子.受農(nóng)業(yè)施肥非點(diǎn)源污染的影響,河流湖泊和近岸水體表現(xiàn)為磷限制的富營(yíng)養(yǎng)化,氮磷比極高,磷成為重要的限制因子.在磷酸鹽限制條件下,反硝化作用將受到抑制,這一現(xiàn)象在磷限制水體的室內(nèi)培養(yǎng)實(shí)驗(yàn)中也得到證實(shí),在PO43-濃度低于0.15mg/L時(shí)通過(guò)填加磷酸鹽可以顯著提高反硝化作用的速率,可能的原因是磷限制了反硝化細(xì)菌的生長(zhǎng)尤其是反硝化聚磷菌這一類(lèi)對(duì)磷酸鹽有較高依賴(lài)性的菌的生長(zhǎng)[46].密度是沉積物重要的物理指標(biāo),但其對(duì)微生物及其地化循環(huán)的影響認(rèn)知還較少,可能與厭氧氨氧化作用的底物NO2-來(lái)源于反硝化作用中間過(guò)程有關(guān),沉積物的物理性質(zhì)影響了NO2-在功能細(xì)菌間的輸運(yùn).

對(duì)上覆水、沉積物和間隙水的理化性質(zhì)及功能細(xì)菌豐度對(duì)反硝化作用和厭氧氨氧化作用變化的影響分析發(fā)現(xiàn)沉積物的理化性質(zhì)是影響反硝化作用和厭氧氨氧化作用的主要解釋因子,2分別為0.656和0.593,而功能細(xì)菌豐度的影響均較小.自然河流中,由于環(huán)境條件的波動(dòng)性較大,細(xì)菌的生長(zhǎng)和生理活動(dòng)都會(huì)受到環(huán)境因子的限制,因此功能細(xì)菌和相應(yīng)生態(tài)功能速率間的相關(guān)性并不顯著,在長(zhǎng)江流域的河流中也發(fā)現(xiàn)沉積物的反硝化作用與細(xì)菌群落間不存在相關(guān)性[14].彌河的反硝化和厭氧氨氧化作用去除氮的效率主要取決于沉積物的理化性質(zhì),這一結(jié)果可以指導(dǎo)彌河富營(yíng)養(yǎng)化的修復(fù)工作.

3 結(jié)論

3.1 彌河沉積物中存在反硝化作用和厭氧氨氧化作用,反硝化速率范圍為151.75~2847.86μmol/(m2×h),不同季節(jié)的均值為680.83μmol/(m2×h),厭氧氨氧化速率范圍為149.57~2109.17μmol/(m2×h),不同季節(jié)均值為898.32μmol/(m2×h),兩者在氮去除過(guò)程中的貢獻(xiàn)量相當(dāng).

3.2 彌河沉積物反硝化作用的功能細(xì)菌以K型為主,豐度0.19×106~5.12×106個(gè)/g,S型則較少,豐度范圍是7.19×103~2.23×105個(gè)/g,反硝化細(xì)菌主要為-和變形菌門(mén);厭氧氨氧化細(xì)菌以A為基因標(biāo)記的豐度為2.58×102~1.14×104個(gè)/g,主要是浮霉菌門(mén)的類(lèi)群.

3.3 彌河的反硝化和厭氧氨氧化作用具有協(xié)同性,依賴(lài)于反硝化的厭氧氨氧化作用可能是彌河沉積物厭氧氨氧化作用的主要機(jī)制.沉積物的理化性質(zhì)是解釋彌河反硝化和厭氧氨氧化速率差異的主要環(huán)境因子,其中TN作為反硝化作用和厭氧氨氧化作用的底物,與兩個(gè)過(guò)程呈正相關(guān)關(guān)系,此外PO43-濃度較低,作為環(huán)境限制因子與反硝化作用呈顯著正相關(guān)關(guān)系,而沉積物密度與厭氧氨氧化作用呈負(fù)相關(guān)關(guān)系.

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Denitrification and anammox processes in sediment of Mihe River, China.

LI Jia-lin1,2, QIN Song1,2*

(1.Laboratory of Coastal Biology and Bioresource Protection, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China；2.Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China)., 2021,41(4)：1588~1596

Four sites were selected along Mihe River to sample the sediments and characterize physiochemical property to reveal denitrification/anammox processes and their constraints by isotope tracing experiment and molecular biological analysis. The results showed that the rates of denitrification varied from 151.75 to 2847.86μmol/(m2×h), and the anammox rates were in the range of 149.57 to 2109.17μmol/(m2×h). The relative contribution of anammox in the nitrogen removal was in an average of 56.1%. TheK type was the dominant denitrifier with the abundance of 0.19′106~5.12′106copies/g in the sediment, which was in the phylum of- and- Proteobacteria. The anammox bacteria marked byA gene was in the range of 2.58′102~1.14′104copies/g, which belonged to the Ca. Brocadia clades in Planctomycetes. Furthermore, the denitrification rate was positively related with TN content and PO43-concentration, while the anammox rate was positively related with TN content and negatively related with sediment density. The physiochemical property of sediment was the main condition determining the nitrogen removal rates. The results highlight that denitrification and anammox were of great significance to reduce nitrogen eutrophication, and sediment environment treatment was a considerable way to regular nitrogen removal rate.

denitrification；anammox；functional bacteria；impact factor；Mihe River

X524

A

1000-6923(2021)04-1588-09

李佳霖(1980-),女,山東煙臺(tái)人,副研究員,博士,主要從事環(huán)境微生物生態(tài)學(xué)研究.發(fā)表論文12篇.

2020-08-10

國(guó)家自然科學(xué)基金資助項(xiàng)目(41106100)

*責(zé)任作者, 研究員, sqin@yic.ac.cn