賀 惠,甄 毓,米鐵柱*,于志剛(1.中國海洋大學(xué)海洋生命學(xué)院,山東 青島 266003；2.中國海洋大學(xué)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.青島海洋科學(xué)與技術(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；4.中國海洋大學(xué)環(huán)境科學(xué)與工程學(xué)院,山東 青島 266100；5.中國海洋大學(xué)海洋化學(xué)理論與教育部重點(diǎn)實(shí)驗(yàn)室,山東 青島 266100)

乳山灣近海沉積物潛在硝化速率的測定

賀 惠1,2,3,甄 毓2,3,4,米鐵柱2,3,4*,于志剛3,5(1.中國海洋大學(xué)海洋生命學(xué)院,山東 青島 266003；2.中國海洋大學(xué)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.青島海洋科學(xué)與技術(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；4.中國海洋大學(xué)環(huán)境科學(xué)與工程學(xué)院,山東 青島 266100；5.中國海洋大學(xué)海洋化學(xué)理論與教育部重點(diǎn)實(shí)驗(yàn)室,山東 青島 266100)

在測定乳山灣近海沉積物潛在硝化速率(PNR)的過程中發(fā)現(xiàn),培養(yǎng)前后溶解無機(jī)氮(DIN)含量顯著下降,表明體系中存在DIN損失,且損失的溶解無機(jī)氮含量與硝化總量的比值為 2.72%～40.02%.進(jìn)一步通過實(shí)時(shí)熒光定量 PCR技術(shù)測定培養(yǎng)過程中亞硝酸鹽還原酶基因(nitrite reductase gene, nirK)的表達(dá)情況,發(fā)現(xiàn)氨氧化古菌(AOA)和好氧氨氧化細(xì)菌(AOB)均有nirK基因的表達(dá),表明硝化微生物的反硝化過程(ND)是導(dǎo)致無機(jī)氮損失的原因之一.若僅根據(jù)測得的硝酸鹽和亞硝酸鹽濃度估算乳山灣近海沉積物的潛在硝化速率會(huì)低估 PNR(C0和C2 2個(gè)站位,考慮ND過程得到的總PNR分別是未考慮ND過程的15.9倍和22.1倍,而對(duì)于AOA的PNR則是22.3倍和46.1倍),因此在計(jì)算時(shí),必須將體系中損失的無機(jī)氮計(jì)算在內(nèi).

乳山灣；潛在硝化速率；硝化微生物的反硝化過程；亞硝酸鹽還原酶

沉積物潛在硝化速率(potential nitrification rates, PNR)是表征好氧氨氧化微生物硝化作用的重要化學(xué)指標(biāo).潛在硝化速率的測定方法主要有15N同位素法[1]和培養(yǎng)法[2-3],其中培養(yǎng)法由于方法簡單易于施行,是最常用的測定方法[4-5].潛在硝化速率可以比較不同環(huán)境樣品中微生物將銨鹽轉(zhuǎn)化為硝酸鹽的最大硝化能力,反映微生物的豐度和活性[6-7].通過添加氨芐青霉素抑制氨氧化細(xì)菌的活性,可以進(jìn)一步區(qū)分氨氧化古菌(ammonia-oxidizing archaea, AOA)和氨氧化細(xì)菌(aerobic ammonia-oxidizing bacteria, AOB)在硝化作用中的相對(duì)貢獻(xiàn),如 Zheng等[4]通過測定長江口潮間帶沉積物的PNR推測該環(huán)境中的氨氧化過程主要由AOA推動(dòng).

硝化作用是氮素循環(huán)的重要過程,不僅影響銨態(tài)氮的轉(zhuǎn)化,同時(shí)也與過量氮素導(dǎo)致的水體富營養(yǎng)化和溫室氣體N2O釋放等環(huán)境問題直接相關(guān)[8].隨著技術(shù)手段的不斷進(jìn)步,越來越多的研究表明好氧氨氧化微生物在進(jìn)行氨氧化的同時(shí)也存在反硝化過程[9-10],即硝化微生物的反硝化作用(nitrififer denitrification, ND).該過程能將亞硝酸鹽轉(zhuǎn)化為N2O等氣體,導(dǎo)致培養(yǎng)體系中氮元素?fù)p失.然而,關(guān)于硝化微生物的反硝化作用對(duì)測定和計(jì)算潛在硝化速率的影響鮮見報(bào)道.

乳山灣位于山東半島,為一半封閉的淺水海灣,是重要的經(jīng)濟(jì)貝類養(yǎng)殖區(qū).隨著近年來工農(nóng)業(yè)和養(yǎng)殖業(yè)的發(fā)展,大量氮、磷營養(yǎng)鹽的輸入使得乳山灣近海水質(zhì)環(huán)境發(fā)生了一系列變化,富營養(yǎng)化趨勢明顯,2008年該海域暴發(fā)了海洋卡盾藻(Chattonella marina)為主要原因種的赤潮[11].基于以上考慮,本研究選擇受人類活動(dòng)擾動(dòng)大、富營養(yǎng)化現(xiàn)象突出的乳山灣為研究海域,通過實(shí)時(shí)熒光定量PCR技術(shù)和潛在硝化速率模擬培養(yǎng)實(shí)驗(yàn),對(duì)硝化微生物的反硝化過程進(jìn)行研究,為準(zhǔn)確計(jì)算該海域沉積物潛在硝化速率提供依據(jù).

1 材料與方法

1.1 樣品采集

2015年2月采集乳山灣鄰近海域表層沉積物樣品,采樣站位為C0站(121.47°E, 36.74°N)和C2站(121.49°E, 36.66°N) (圖1).沉積物樣品置于4°C低溫保存,立即帶回實(shí)驗(yàn)室進(jìn)行潛在硝化速率測定的培養(yǎng)實(shí)驗(yàn).

1.2 潛在硝化速率測定

按照 Bernhard等[3]的方法測定潛在硝化速率.取1.0g沉積物加入30mL配制的水樣(現(xiàn)場海水經(jīng)0.22μm濾膜過濾后,加入NH4Cl和KH2PO4使其終濃度分別為300,60μmol/L)中,黑暗條件下連續(xù)振蕩培養(yǎng),每個(gè)樣品設(shè)置 3個(gè)平行培養(yǎng)組.0,1,2,3d分別取樣、離心、過濾,水樣于-20°C保存,用于NH4-N、NO3-N和NO2-N的測定.共設(shè)置2個(gè)培養(yǎng)組,一組加入1.0g/L氨芐青霉素[4],抑制AOB活性,計(jì)算AOA的PNR;另一組不添加氨芐青霉素,計(jì)算AOA與AOB的PNR之和,兩組相減即為AOB的PNR.

圖1 乳山灣近海采樣站位示意Fig.1 Sampling sites in surface sediments from adjacent waters of Rushan Bay

1.3 沉積物微生物RNA提取

培養(yǎng)過程中于第0,1,2,3d分別取沉積物樣品2.0g,置于液氮中保存.利用 RNA PowerSoil?Total RNA Isolation kit(MOBIO,USA)提取沉積物中微生物總 RNA,以 Transcriptor First Strand cDNA Synthesis kit(Roche,Germany)將RNA反轉(zhuǎn)錄為cDNA.將得到的cDNA置于-80℃保存.

1.4 nirK基因質(zhì)粒標(biāo)準(zhǔn)曲線的建立及樣品測定

利用引物分別擴(kuò)增AOA和AOB nirK基因(表1).PCR產(chǎn)物經(jīng)電泳分離,檢測目的條帶.將目的條帶純化、連接到pMD18-T載體(寶生物,大連)后轉(zhuǎn)化到Escherichia coli Trans 5α感受態(tài)細(xì)胞中,通過藍(lán)白斑篩選含有目的基因片段的陽性克隆.快速質(zhì)粒小提試劑盒(康為,北京)制備質(zhì)粒后,送測序公司測序(華大基因,北京).用超微量紫外分光光度計(jì)(Picodrop,UK)測定重組質(zhì)粒的濃度,計(jì)算重組質(zhì)粒的拷貝濃度.將得到的質(zhì)粒 10倍梯度稀釋后于-80℃保存,用于構(gòu)建定量 PCR體系的標(biāo)準(zhǔn)曲線.

表1 AOA和AOB nirK基因引物Table 1 Primers for AOA and AOB nirK gene

以梯度稀釋的質(zhì)粒標(biāo)準(zhǔn)品為模板,利用FastStart Universal SYBR Green Master(ROX)試劑盒(Roche,Germany)在ABI 7500實(shí)時(shí)熒光定量PCR儀(Applied Biosystems,USA)上進(jìn)行檢測,反應(yīng)體系中加入 0.20μg/μL牛血清蛋白(Bovine Serum Albumin,BSA)[14].退火溫度如表1.每個(gè)質(zhì)粒濃度3個(gè)平行樣,實(shí)驗(yàn)體系中同時(shí)添加陰性對(duì)照.以質(zhì)?？截悢?shù)對(duì)數(shù)值為橫坐標(biāo),Ct值為縱坐標(biāo),繪制標(biāo)準(zhǔn)曲線.

樣品測定時(shí),定量PCR的體系和條件與標(biāo)準(zhǔn)曲線相同,實(shí)驗(yàn)體系中同時(shí)包括陽性對(duì)照和陰性對(duì)照,每個(gè)樣品測定3個(gè)平行樣.通過標(biāo)準(zhǔn)曲線換算樣品中nirK基因的拷貝數(shù).

2 結(jié)果與分析

2.1 培養(yǎng)過程中溶解無機(jī)氮含量的變化

圖2 培養(yǎng)過程中硝酸鹽與亞硝酸鹽、銨鹽及溶解無機(jī)氮含量的變化Fig.2 The content variations of nitrate and nitrite, ammonium, dissolved inorganic nitrogen during cultivation

計(jì)算無機(jī)氮損失量與硝化總量的比值發(fā)現(xiàn)(圖 3),該比值較高.由此表明,計(jì)算潛在硝化速率時(shí)無機(jī)氮的損失量不可忽略.在潛在硝化模擬培養(yǎng)條件下,導(dǎo)致無機(jī)氮損失的生物過程主要有好氧反硝化過程和硝化微生物的反硝化過程等.

圖3 培養(yǎng)過程中無機(jī)氮損失量與硝化總量的比值Fig.3 The ratio of inorganic nitrogen loss to nitrified nitrogen accumulation during cultivation

2.2 硝化微生物的反硝化過程中nirK基因表達(dá) 量的變化

圖4 培養(yǎng)過程中AOB亞硝化單胞菌屬和AOA nirK基因拷貝數(shù)的變化Fig.4 The variations of Nitrosomonas and AOA nirK gene copy numbers during cultivation

對(duì)含有目的基因的質(zhì)粒標(biāo)準(zhǔn)品進(jìn)行定量PCR反應(yīng)后,得到不同質(zhì)粒濃度對(duì)應(yīng)的 Ct值.以質(zhì)?？截悢?shù)的對(duì)數(shù)值為橫坐標(biāo),Ct值為縱坐標(biāo)計(jì)算回歸方程.AOA nirK基因的回歸方程為y=-3.230x+38.511,回歸系數(shù) r=-0.999;熔解曲線只在78.1℃處有一單峰出現(xiàn),說明qPCR反應(yīng)過程中無引物二聚體或其他非特異性擴(kuò)增.AOB亞硝化單胞菌屬Nitrosomonas nirK基因的回歸方程為y=-3.179x+37.569,回歸系數(shù) r=-0.998;熔解曲線只在81.6℃處有一單峰出現(xiàn),說明qPCR反應(yīng)過程中無引物二聚體或其他非特異性擴(kuò)增.

cDNA定量結(jié)果顯示,培養(yǎng)過程中 AOA和AOB的nirK基因均有表達(dá)(圖4).未添加氨芐青霉素組,C0和C2 2個(gè)站位AOB亞硝化單胞菌屬Nitrosomonas nirK 基因的表達(dá)量分別為(1.1×103)～(2.8×103)copies/g和(9.4×102)～(1.9×103) copies/g,并隨時(shí)間呈現(xiàn)緩慢增加的趨勢;AOA nirK 基因的表達(dá)量分別為(4.5×105)～(4.1× 106)copies/g和(5.1×105)～(3.4×106)copies/g,且分別在第2d和第3d達(dá)到最大值.

添加氨芐青霉素組,C0和 C2 2個(gè)站位中AOA nirK 基因表達(dá)量分別為(5.0×105)～(2.3× 106)copies/g和(1.2×106)～(2.4×106)copies/g,在第2d達(dá)到最大值.

2.3 潛在硝化速率分析

潛在硝化速率的計(jì)算分別采用2種方法.方法 1是通過測得的硝酸鹽和亞硝酸鹽增加量計(jì)算PNR(以N/g沉積物計(jì),式1).

式中:ΔC(NO3+NO2)表示測得的硝酸鹽和亞硝酸鹽含量的變化值,μmol N;t表示培養(yǎng)時(shí)間,d.

方法2則是在方法1的基礎(chǔ)上,考慮無機(jī)氮的損失(損失的溶解無機(jī)氮)計(jì)算PNR(以N/g沉積物計(jì),式2).

式中:ΔCDIN表示培養(yǎng)過程中溶解無機(jī)氮含量變化值的絕對(duì)值,μmol N.若整個(gè)系統(tǒng)沒有無機(jī)氮的損失,則ΔCDIN為零,與式(1)的結(jié)果一致.

表層沉積物的潛在硝化速率如圖5所示.若不考慮反硝化過程的存在,即實(shí)際生成的硝酸鹽和亞硝酸鹽沒有損失時(shí),由式(1)計(jì)算PNR,C0和C2兩個(gè)站位的總潛在硝化速率分別為0.019和0.018μmol N/(d·g沉積物),添加氨芐青霉素組抑制細(xì)菌活性后得到的AOA的PNR分別為0.008和0.006μmol N/(d·g沉積物),差減得到AOB的PNR分別為0.011和0.012μmol N/(d·g沉積物).若將硝化微生物的反硝化過程考慮在內(nèi),根據(jù)式(2)校正DIN損失后,C0和C2兩個(gè)站位的總PNR分別為0.295和0.398μmol N/(d·g沉積物),AOA的PNR分別為0.174和0.290μmol N/(d·g沉積物),AOB的 PNR分別為 0.121和 0.108μmol N/(d·g沉積物).

圖5 乳山灣近海表層沉積物的潛在硝化速率Fig.5 Potential nitrification rates in surface sediments from adjacent waters of Rushan Bay

3 討論

通常在計(jì)算潛在硝化速率時(shí)有以下幾個(gè)方面的假設(shè)條件:(1)培養(yǎng)體系中不存在有機(jī)氮和無機(jī)氮間的相互轉(zhuǎn)化(氨化作用與氨同化作用),(2)培養(yǎng)過程中不存在反硝化過程,(3)硝化過程不受銨鹽及其它營養(yǎng)元素的限制.通過設(shè)置適宜的培養(yǎng)時(shí)間、避光抑制光合作用以及較高的氨氮濃度,可基本滿足第1個(gè)和第3個(gè)條件;而對(duì)于第2個(gè)假設(shè),早期的研究中認(rèn)為通過攪拌充氣等措施,將培養(yǎng)體系保持在富氧狀態(tài),即可避免反硝化過程.近年來有研究表明,好氧反硝化過程和硝化微生物的反硝化過程都能在有氧條件下將亞硝酸鹽轉(zhuǎn)化為N2O或N2等氣體[9,15-16],使得這一問題變得復(fù)雜.AOA和AOB均存在亞硝酸鹽還原酶基因nirK[12,16-19],可以進(jìn)行反硝化作用;采用15N同位素技術(shù)對(duì)土壤中N2O的來源進(jìn)行研究,發(fā)現(xiàn)硝化微生物的反硝化過程對(duì)其平均貢獻(xiàn)率為34%～80%[20-21],說明該過程在N2O釋放過程中發(fā)揮了重要作用.

在PNR測定的培養(yǎng)實(shí)驗(yàn)中,添加的NH4+濃度遠(yuǎn)高于背景值,可認(rèn)為其是硝化作用的主要氮源.根據(jù)氮元素物質(zhì)的量守恒,消耗的銨鹽應(yīng)全部轉(zhuǎn)化為硝酸鹽和亞硝酸鹽,對(duì)于整個(gè)培養(yǎng)系統(tǒng),溶解無機(jī)氮的含量應(yīng)保持穩(wěn)定.而本研究中,消耗的銨鹽量大于生成的硝酸鹽和亞硝酸鹽含量,溶解無機(jī)氮含量降低,說明培養(yǎng)體系中存在無機(jī)氮損失.進(jìn)一步以硝化微生物的反硝化過程關(guān)鍵酶nirK基因?yàn)槟康幕?采用特異引物進(jìn)行定量PCR反應(yīng),在培養(yǎng)過程中檢測到 AOA和 AOB nirK基因的大量表達(dá).微生物基因組 DNA中存在nirK基因說明其具有反硝化能力,但可能由于環(huán)境不適宜而不會(huì)轉(zhuǎn)錄表達(dá),不發(fā)揮相應(yīng)的生理生化功能,而在RNA水平上檢測到nirK基因的表達(dá)則可以明確表明微生物正在進(jìn)行反硝化作用.微生物的轉(zhuǎn)錄和翻譯過程是同時(shí)進(jìn)行的,通常在轉(zhuǎn)錄RNA之后即合成具有相應(yīng)功能的蛋白質(zhì)(酶).本研究在RNA水平上檢測到AOA與AOB nirK基因的表達(dá),說明培養(yǎng)體系中正在進(jìn)行反硝化作用,硝化微生物的反硝化作用是造成無機(jī)氮損失的原因之一.

由于乳山灣近海沉積物中硝化微生物的反硝化過程的存在,培養(yǎng)體系中存在亞硝酸鹽向N2O的轉(zhuǎn)化,導(dǎo)致DIN損失.若只根據(jù)測得的硝酸鹽和亞硝酸鹽的增加量計(jì)算PNR,會(huì)低估該海域?qū)嶋H的潛在硝化速率,因此必須考慮DIN的損失量加以校正,即上文提出的式(2).本研究中C0和C2 2個(gè)站位校正后的總 PNR是未校正數(shù)值的15.9倍和22.1倍,校正后AOA的PNR是未校正數(shù)值的22.3倍和46.1倍,考慮無機(jī)氮損失前后潛在硝化速率差異較大.

分析AOA amoA基因與nirK基因表達(dá)量發(fā)現(xiàn),二者存在顯著的正相關(guān)關(guān)系(未發(fā)表數(shù)據(jù)),并且 nirK基因表達(dá)量約為 amoA基因表達(dá)量的10～40倍,說明培養(yǎng)體系中ND過程強(qiáng)烈,這與較高的DIN損失相一致;Lund等[12]對(duì)Monterey海灣的研究結(jié)果亦是如此,表明AOA中nirK基因和amoA基因關(guān)系密切,相伴而生.乳山灣近海沉積物中能夠檢測到硝化微生物的反硝化過程的存在,并且其強(qiáng)度可能隨氨氧化作用的增強(qiáng)而增高,由此導(dǎo)致的無機(jī)氮損失必須加以關(guān)注.

4 結(jié)論

4.1 乳山灣近海沉積物的潛在硝化速率培養(yǎng)試驗(yàn)中存在無機(jī)氮損失,且損失的溶解無機(jī)氮含量與硝化總量的比值為2.72%～40.02%.

4.2 培養(yǎng)過程中AOA和AOB nirK基因活性較高,表明硝化微生物的反硝化過程是造成無機(jī)氮損失的原因之一.

4.3 對(duì)于C0和C2 2個(gè)站位,考慮硝化微生物的反硝化過程得到的總PNR分別是未考慮硝化微生物的反硝化過程的 15.9倍和 22.1倍,而對(duì)于AOA的PNR則分別是22.3倍和46.1倍.因此,若僅根據(jù)測得的硝酸鹽和亞硝酸鹽的增加量計(jì)算該海域沉積物潛在硝化速率會(huì)導(dǎo)致計(jì)算結(jié)果偏低,計(jì)算時(shí)應(yīng)將培養(yǎng)過程中無機(jī)氮的損失納入其中.

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致謝：感謝中國海洋大學(xué)海洋生命學(xué)院王華龍同學(xué)和國家海洋局第一海洋研究所海洋生態(tài)研究中心劉軍同學(xué)在野外采樣中給予的幫助,感謝中國海洋大學(xué)化學(xué)化工學(xué)院谷文艷同學(xué)在溶解無機(jī)氮測定中給予的幫助.

Measurement of potential nitrification rates in sediments from adjacent waters of Rushan Bay.

HE Hui1,2,3, ZHEN Yu2,3,4, MI Tie-zhu2,3,4*, YU Zhi-gang3,5(1.College of Marine Life Science, Ocean University of China, Qingdao 266003, China；2.Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao 266100, China；3.Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China；4.College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China；5.Key Laboratory of Marine Chemical Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China). China Environmental Science, 2017,37(3)：1082～1088

In this paper, dissolved inorganic nitrogen (DIN) contents declined significantly when potential nitrification rates (PNR) were measured in sediments from adjacent waters of Rushan Bay, which indicated DIN loss occurred during cultivation, and the ratio of dissolved inorganic nitrogen loss to nitrification content ranged from 2.72% to 40.02%. Moreover, the expressions of copper-containing nitrite reductase gene (nirK) were analyzed with real-time quantitative polymerase chain reaction (qPCR), and the results showed that the gene expressed in both ammmonia-oxidizing archaea (AOA) and aerobic ammonia-oxidizing bacteria (AOB), and nitrifier denitrification was one of the reasons that led to DIN loss. PNR would be underestimated if only nitrate and nitrite concentrations were taken into account. For station C0 and C2, total PNR considered ND was 15.9 and 22.1 times of that not considered ND, respectively; and PNR of AOA considered ND was 22.3 and 46.1 times of that not considered ND, respectively. Therefore, DIN loss should be considered when PNR in sediments from adjacent waters of Rushan Bay were calculated.

Rushan Bay；potential nitrification rates；nitrifier denitrification；copper-containing nitrite reductase

X55,Q89,Q938.1

A

1000-6923(2017)03-1082-07

賀 惠(1987-),女,山東日照人,博士,主要從事海洋生態(tài)學(xué)研究.發(fā)表論文4篇.

2016-07-08

國家自然科學(xué)基金資助項(xiàng)目(41620104001,41521064);中國科學(xué)院海洋生態(tài)與環(huán)境科學(xué)重點(diǎn)實(shí)驗(yàn)室、青島海洋科學(xué)與技術(shù)國家實(shí)驗(yàn)室海洋生態(tài)與環(huán)境科學(xué)開放課題(KLMEES201601)

* 責(zé)任作者, 副教授, mitiezhu@ouc.edu.cn