芘對(duì)土壤微生物氮轉(zhuǎn)化功能菌群的影響特征

胡 琴,張利蘭,2*,易美玲,楊 銳

芘對(duì)土壤微生物氮轉(zhuǎn)化功能菌群的影響特征

胡 琴1,張利蘭1,2*,易美玲1,楊 銳1

(1.重慶大學(xué),三峽庫區(qū)生態(tài)環(huán)境教育部重點(diǎn)實(shí)驗(yàn)室,重慶 400044；2.重慶大學(xué),煤礦災(zāi)害動(dòng)力學(xué)與控制國(guó)家重點(diǎn)實(shí)驗(yàn)室,重慶 400044)

通過構(gòu)建好氧降解微環(huán)境,分析環(huán)境濃度下的芘(12.09mg/kg)對(duì)土壤酶活性,氮轉(zhuǎn)化全過程以及相關(guān)功能微生物的影響.結(jié)果發(fā)現(xiàn),芘僅在降解第1d顯著促進(jìn)了脲酶活性,而在降解最初和后期均顯著刺激了脫氫酶活性.從細(xì)菌群落結(jié)構(gòu)分析可知,由于氨氧化菌()相對(duì)豐度的變化,導(dǎo)致芘在處理前期對(duì)其介導(dǎo)的好氧氨氧化,硝化功能表現(xiàn)為促進(jìn)作用,在后期表現(xiàn)為抑制作用,而對(duì)于固氮細(xì)菌(,和),尿素分解細(xì)菌()以及硝酸鹽還原細(xì)菌()則作用相反.與微生物群落結(jié)構(gòu)以及相關(guān)功能預(yù)測(cè)的變化不同,功能基因定量分析表明,芘雖在培養(yǎng)初期對(duì)固氮基因H表現(xiàn)為抑制作用,但H的豐度呈增長(zhǎng)趨勢(shì).結(jié)合土壤氨氧化和反硝化過程中關(guān)鍵酶活性及編碼基因的變化,芘在培養(yǎng)前期未促進(jìn)氨氧化過程,但在15d后明顯抑制了土壤氨氧化和反硝化過程,其中對(duì)氨氧化過程的抑制作用更為顯著.本研究闡明了芘對(duì)土壤微生物氮轉(zhuǎn)化過程的影響特征,為了解芘的環(huán)境風(fēng)險(xiǎn)提供重要參考價(jià)值.

芘；土壤酶活性；氮轉(zhuǎn)化細(xì)菌群落；氮轉(zhuǎn)化過程

多環(huán)芳烴(PAHs)是一類具有“三致”毒性的持久性有機(jī)污染物[1],由于其親脂性和惰性,可以通過生物富集等方式長(zhǎng)期存在于生態(tài)系統(tǒng)中,使其具有較大的生態(tài)環(huán)境風(fēng)險(xiǎn)[2-3].土壤是PAHs重要的賦存介質(zhì),土壤中PAHs濃度構(gòu)成主要受到人類活動(dòng)的影響.在城市,工業(yè)和農(nóng)業(yè)土壤中,交通尾氣,石油泄漏,燃煤和生物質(zhì)燃燒通常是造成表層土壤PAHs污染的主要原因,其濃度范圍分別為幾～幾千μg/kg[4-7],十幾～幾萬μg/kg[8-10]和幾～幾百μg/kg[11-13].其中高分子量多環(huán)芳烴(high molecular weight PAHs, HMW-PAHs)為主要成分,其濃度整體上略高于低分子量多環(huán)芳烴(low molecular weight PAHs, LMW-PAHs),可能由于HMW-PAHs揮發(fā)性差且較難被生物降解利用.土壤是自然界最復(fù)雜的生態(tài)系統(tǒng)之一,健康的土壤是保證糧食安全的關(guān)鍵.因此,土壤中PAHs的環(huán)境行為及生態(tài)效應(yīng)值得廣泛關(guān)注.

微生物群落作為土壤生態(tài)系統(tǒng)的骨架,在維持土壤健康方面發(fā)揮重要作用,其結(jié)構(gòu)的多樣性和穩(wěn)定性是保持土壤生態(tài)系統(tǒng)穩(wěn)定性和適應(yīng)性的基礎(chǔ)[14].微生物介導(dǎo)的土壤氮轉(zhuǎn)化是地球氮轉(zhuǎn)化系統(tǒng)的中樞環(huán)節(jié),對(duì)維持各個(gè)圈層的生態(tài)穩(wěn)定具有重要意義[15].在PAHs脅迫下,部分微生物通過降解代謝等途徑改變其群落組成或活性,進(jìn)而影響著土壤中的氮轉(zhuǎn)化.例如,LMW-PAHs的芴和菲對(duì)氮礦化細(xì)菌表現(xiàn)出毒性效應(yīng),且具有抑制土壤硝化潛勢(shì)的作用,抑制強(qiáng)度與PAHs的生物可利用性呈正相關(guān)[16];前期研究發(fā)現(xiàn),環(huán)境濃度下的菲(12.21mg/kg)增加了土壤中反硝化和固氮微生物的數(shù)量,并顯著抑制土壤氨氧化過程[17];相近濃度下HMW-PAHs的苯并[a]芘對(duì)反硝化和固氮微生物表現(xiàn)出毒性效應(yīng),抑制土壤的好氧氨氧化,固氮以及硝酸鹽還原過程[18].由此可知,不同結(jié)構(gòu)的PAHs對(duì)土壤氮轉(zhuǎn)化微生物產(chǎn)生的毒性效應(yīng)具有差異性.

芘作為典型的HMW-PAHs之一,由四個(gè)對(duì)稱的苯環(huán)組成,結(jié)構(gòu)與許多致癌的HMW-PAHs相似,比LMWPAHs的“三致”毒性更強(qiáng)[19].由于芘的理化性質(zhì)和生物可利用度等原因,可通過食物鏈累積在環(huán)境中持續(xù)存在,產(chǎn)生一定的生態(tài)毒性效應(yīng)[20-21].因此,芘通常用作研究HMW-PAHs環(huán)境行為及生態(tài)效應(yīng)的模型[22-23].已有研究證實(shí),低濃度的芘(1mg/kg)對(duì)沉積物中的固氮菌有一定的刺激作用,而濃度較高時(shí)(10和100mg/kg)則表現(xiàn)為明顯的抑制作用[24].此外,芘(0～500μg/g)會(huì)顯著改變氨氧化細(xì)菌和氨氧化古菌豐度的比值,且濃度越大,對(duì)氨氧化微生物的活性影響越大[25].基于目前芘對(duì)土壤氮轉(zhuǎn)化全過程以及相關(guān)微生物功能結(jié)構(gòu)的變化尚不清晰,本研究選擇以環(huán)境濃度下的芘作為目標(biāo)污染物,通過分析其對(duì)土壤酶活性,氮轉(zhuǎn)化過程以及相關(guān)微生物功能結(jié)構(gòu)隨時(shí)間變化的影響,揭示其對(duì)土壤微生物氮轉(zhuǎn)化功能菌群影響特征,為評(píng)估土壤中芘的微生態(tài)系統(tǒng)功能提供方法參考.

1 材料與方法

1.1 土壤樣品采集

1.2 室內(nèi)培養(yǎng)實(shí)驗(yàn)

本研究使用120mL帶丁基橡膠塞的棕色玻璃小瓶構(gòu)建好氧降解微環(huán)境,設(shè)置以下3個(gè)處理組:(1)向土樣中添加有機(jī)溶劑所溶解的芘(芘處理組,PYR),測(cè)得負(fù)載后的芘濃度為12.09mg/kg,此負(fù)載濃度接近環(huán)境濃度[26-28];(2)向土樣中添加等量有機(jī)溶劑(空白對(duì)照組,CK).(3)向經(jīng)過高溫高壓(0.1MPa,121℃)滅菌1h的土壤中負(fù)載等量芘(非生物對(duì)照組),并將土壤置于室溫下24h進(jìn)行復(fù)蘇,復(fù)蘇后用生理鹽水重懸土壤并取上清液進(jìn)行涂板,以確保滅菌后的土壤中無微生物活性.將所有處理后的土壤置于通風(fēng)櫥中,待有機(jī)溶劑充分揮發(fā)后分裝至棕色小瓶.每個(gè)瓶中分裝10g(干重)土樣,分裝好后置于22℃恒溫培養(yǎng)箱中培養(yǎng).每個(gè)處理組3個(gè)重復(fù).土壤含水率保持在田間持水量的50%～60%,培養(yǎng)過程中通過稱重監(jiān)測(cè)含水率變化.分別在培養(yǎng)0, 1, 3, 7, 15, 30, 60d后進(jìn)行破壞性取樣,以方便后續(xù)的提取和測(cè)量.

1.3 土壤芘的提取與測(cè)定

土壤中芘的提取采用溶劑超聲萃取法[29].以甲醇為提取劑提取土壤樣品中的芘:向2g(干重)土樣中加入20mL甲醇后進(jìn)行超聲提取(60Hz,50℃)1h,然后以8000r/min離心10min,上清液過0.22μm有機(jī)濾膜后使用高效液相色譜儀進(jìn)行芘濃度的測(cè)定.高效液相色譜儀配備SB-C18色譜分離柱和二極管陣列檢測(cè)器,流動(dòng)相為甲醇和水(芘90:10),流速為1mL/min,芘檢測(cè)波長(zhǎng)為240nm.超聲萃取提取土壤樣品中芘的回收率為80～93%.

1.4 酶活性測(cè)定方法

土壤酶來源于土壤微生物,植物等,是反映土壤健康和質(zhì)量的一項(xiàng)重要指標(biāo)[30-31].其中,土壤脲酶參與尿素水解,其活性反映了土壤氮素代謝的旺盛程度和無機(jī)氮供應(yīng)能力[32].而脫氫酶是反映有機(jī)物降解功能的酶,可作為微生物氧化還原活性的重要指標(biāo),指示土壤有機(jī)污染物的轉(zhuǎn)化速率[33].土壤脲酶活性測(cè)定采用S-UE試劑盒(索萊寶,中國(guó)),稱取過0.0425mm篩的0.05g土壤樣品于2mL離心管中,加入20μL甲苯溶液,在室溫下放置15min,再加入90μL尿素溶液和190μL緩沖液,37℃下培養(yǎng)24h后離心并加入顯色劑,于630nm處測(cè)定吸光值表示產(chǎn)生的氨氮含量,以每單位時(shí)間每克土產(chǎn)生的氨氮含量表示脲酶活性.土壤脫氫酶活性測(cè)定:向土壤中加入5-三苯基氯化四氮唑(TTC)溶液,在37℃培養(yǎng)24h后用甲醇提取產(chǎn)生的三苯甲酰亞胺(TPF),于485nm處測(cè)定吸光度.

氨單加氧酶(AMO),硝酸鹽還原酶(NAR)和亞硝酸鹽還原酶(NIR)作為氮轉(zhuǎn)化酶,其活性反映了土壤中的相關(guān)氮轉(zhuǎn)化速率.其中AMO作為催化土壤中NH4+轉(zhuǎn)化為NO2-的關(guān)鍵酶,在土壤硝化過程中發(fā)揮重要作用[34].土壤氨單加氧酶(AMO)活性測(cè)定主要采用Yang等[35](2020年)的方法:向2g(干重)土樣中加入20mL磷酸鹽緩沖溶液(PBS, pH7.4),于37℃黑暗震蕩培養(yǎng)4h (150r/min)后離心取上清液,測(cè)定上清液產(chǎn)生的亞硝酸鹽含量.另外,NAR和NIR分別催化土壤中NO3-和NO2-的還原過程,這是反硝化過程的前兩個(gè)限速步驟.基于硝酸鹽和亞硝酸鹽還原反應(yīng)原理,土壤硝酸鹽還原酶(NAR)和亞硝酸鹽還原酶(NIR)活性采用S-NR和S-NiR試劑盒(索萊寶,中國(guó))測(cè)定.

1.5 土壤DNA提取及功能基因定量分析

稱取0.25g土壤樣品,根據(jù)土壤DNA提取試劑盒(Qiagen,美國(guó))操作說明提取土壤微生物DNA.并用超微量分光光度計(jì)(Implen,德國(guó))測(cè)定DNA濃度與質(zhì)量.以提取的土壤微生物總DNA為模板,使用熒光定量PCR儀(Bio-Rad,美國(guó))對(duì)氮轉(zhuǎn)化功能基因AOAA, AOBA,H,G和S進(jìn)行定量分析,引物信息參照文獻(xiàn)[18].熒光定量PCR反應(yīng)體系體積為20μL:前/后引物各0.6μL,DNA模板0.8μL,無菌水8.0μL,2×SYBR Green Supermix10μL.各基因反應(yīng)條件和程序見參考文獻(xiàn)[23].

1.6 高通量測(cè)序

利用通用引物515F(5-GTGCCAGCMGCC- GCGGTAA-3)和806R(5-GGACTACHVGGGTWT- CTAAT-3)在Illumina MiSeq平臺(tái)(Majorbio, Shanghai, China)上擴(kuò)增土壤細(xì)菌16S rRNA基因V4區(qū)進(jìn)行測(cè)序分析.使用Qiime 2平臺(tái)對(duì)原始擴(kuò)增子進(jìn)行處理.首先使用DATA2對(duì)16S rRNA特定區(qū)域的高通量序列進(jìn)行降噪處理,以獲得每個(gè)樣本的擴(kuò)增子序列變體(Amplicon sequence variant, ASV).然后通過Bayes注釋方法,采用Silva 13注釋數(shù)據(jù)庫對(duì)獲得的16S rRNA ASV進(jìn)行分類.為最大程度降低采樣深度的影響,將ASV集進(jìn)行了抽平處理,以進(jìn)行下游分析.通過質(zhì)量篩選,共獲得了86960個(gè)16S rRNA高質(zhì)量序列,包含19860個(gè)細(xì)菌ASV.從19860個(gè)細(xì)菌ASV中篩選出40種與氮轉(zhuǎn)化相關(guān)的細(xì)菌屬.同時(shí),使用FAPROTAX對(duì)細(xì)菌氮轉(zhuǎn)化功能進(jìn)行注釋[36].

1.7 數(shù)據(jù)分析

芘在土壤中的好氧微生物降解動(dòng)力學(xué)擬合為零級(jí)動(dòng)力學(xué)方程(1).芘的半衰期由方程(2)計(jì)算.

0–C=(1)

50=0/2(2)

式中:0為芘的初始負(fù)載濃度,μg/g;C為采樣時(shí)芘的濃度,μg/g;為采樣時(shí)間,d;為生物降解速率常數(shù),d-1;50為芘的半衰期,d.

采用單因素方差分析(ANOVA)比較空白組(CK)和芘處理組(PYR)之間的差異.所有圖使用Origin 2021和R進(jìn)行繪制.

2 結(jié)果與討論

2.1 芘在土壤中的降解特征

與滅菌土壤相比,在非滅菌土壤環(huán)境中,微生物降解是PAHs最主要的衰減模式之一.PAHs在土壤中的降解行為與其結(jié)構(gòu)及理化性質(zhì)等方面息息相關(guān).本研究選擇在同一受試土壤中負(fù)載濃度相同且降解條件一致的菲(12.21mg/kg),芘(12.09mg/kg)和苯并[a]芘(8.11mg/kg)作為研究對(duì)象,3種PAHs的好氧生物降解如表1所示.菲和苯并[a]芘的好氧微生物降解均符合二級(jí)動(dòng)力學(xué),降解速率隨著培養(yǎng)時(shí)間的延長(zhǎng)而降低,而芘的降解符合零級(jí)反應(yīng)動(dòng)力學(xué)(擬合為:C=0-0.171,2=0.985),降解速率與采樣時(shí)間無關(guān),其降解半衰期和60d內(nèi)的降解率均介于菲和苯并[a]芘之間.

表1 菲,芘和苯并[a]芘的物理性質(zhì)及在土壤中的微生物降解特征

有研究發(fā)現(xiàn),PAHs的分子量與半衰期和疏水性呈正相關(guān),與降解效率和生物可利用性呈負(fù)相關(guān)[37-39].因此,降解半衰期和疏水性介于菲和苯并[a]芘的芘,其降解效率和生物可利用性也介于兩者之間.因此,芘對(duì)土壤的吸附作用介于菲和苯并[a]芘之間,導(dǎo)致其生物可利用性介于兩者之間.以上結(jié)果表明,結(jié)構(gòu)越復(fù)雜,疏水性越強(qiáng)的PAHs在土壤中停留時(shí)間越長(zhǎng),降解效率和生物可利用性越低.

2.2 芘對(duì)土壤酶活性的影響

通過分析脫氫酶和脲酶活性的變化來揭示芘對(duì)微生物氧化還原活性及無機(jī)氮供應(yīng)能力的影響.如圖1(a)所示,芘處理組中土壤脫氫酶活性在第0,30和60d均顯著高于空白組(0.05),促進(jìn)率為25.4%～49.3%.說明芘在降解第0d和降解后期(第30,60d)刺激了土壤脫氫酶活性.已有報(bào)道發(fā)現(xiàn),由于芘為土壤降解菌提供了代謝基質(zhì)的原因,50～ 200mg/kg的芘不僅可以增加土壤微生物代謝活力,還能刺激土壤脫氫酶活性[41-42].此外,脫氫酶可以參與PAHs的氧化,與PAHs降解速率呈顯著正相關(guān)[43-44].這說明脫氫酶活性的增強(qiáng)有可能是由于參與了芘的降解氧化過程.脲酶作為尿素水解的關(guān)鍵酶,是土壤氨氮的來源之一[45].如圖1(b)所示,與空白組相比,芘處理組僅在第1d顯著促進(jìn)了脲酶活性,促進(jìn)率為21.4%,說明芘在降解第1d對(duì)脲酶活性有一定的刺激作用,但在后期脲酶活性可能適應(yīng)了芘的脅迫.

圖1 空白組和芘處理組中脫氫酶和脲酶活性的變化

圖中不同小寫字母代表差異顯著(<0.05),下同

2.3 芘對(duì)土壤氮轉(zhuǎn)化細(xì)菌群落組成及功能的影響

微生物群落是執(zhí)行固氮,氨氧化等土壤氮轉(zhuǎn)化功能的主力軍,分析微生物群落結(jié)構(gòu)一定程度上可反映土壤潛在功能的變化,并預(yù)測(cè)外部壓力下的生態(tài)系統(tǒng)穩(wěn)定性.芘對(duì)土壤氮轉(zhuǎn)化相關(guān)細(xì)菌群落組成影響如圖2(a)所示,按細(xì)菌功能來分,與硝化過程相關(guān)的和固氮細(xì)菌是所有土壤樣品中最豐富的細(xì)菌屬,占總豐度的74.3%～ 94.4%.與空白組相比,處理組中的相對(duì)豐度由第0d的68.7%增長(zhǎng)到第3d的89.6%,隨后在第60d又回降至61.0%.屬于氨氧化古菌,參與土壤中的好氧氨氧化和硝化過程[46].有研究表明,氨氧化微生物對(duì)碳?xì)浠衔锏奈廴竞苊舾衃47],且在有機(jī)質(zhì)含量較低的堿性土壤中,是氨氧化過程的主要參與者,具有明顯的功能優(yōu)勢(shì)[48].因此相對(duì)豐度的變化潛在的表明了芘在最初促進(jìn)了好氧氨氧化和硝化過程,在后期則表現(xiàn)為抑制作用.固氮細(xì)菌,[49]和[49]在芘處理初期相對(duì)豐度顯著降低,在第3d降低至3.5%,隨著芘的降解,它們的總相對(duì)豐度在第60d又增加至21.0%.其中,屬于寡營(yíng)養(yǎng)型細(xì)菌,更傾向于生活在營(yíng)養(yǎng)物質(zhì)或有機(jī)碳有限的環(huán)境中[50].芘的添加可能提供了一定數(shù)量的有機(jī)碳,導(dǎo)致芘處理過程中的相對(duì)豐度呈現(xiàn)出先降低后增加的趨勢(shì).此外,[51]和[49]分別還具有尿素分解和硝酸鹽還原功能.對(duì)于參與尿素分解過程的[51]和硝酸鹽還原過程的[52],與空白組相比,其相對(duì)豐度在芘脅迫下的前3d由2.3%～2.7%降低至0.2%～0.6%,隨后在第60d又升高至1.5%～2.4%.這說明,芘在前期還有可能抑制土壤中的尿素分解和硝酸鹽還原過程,但在降解后期又對(duì)其表現(xiàn)為促進(jìn)作用.綜上,芘在降解過程中對(duì)氨氧化古菌的生長(zhǎng)表現(xiàn)為先促進(jìn)后抑制的作用,而對(duì)于固氮細(xì)菌,和以及尿素分解細(xì)菌和硝酸鹽還原細(xì)菌則作用相反.

圖2 空白組和芘處理組中土壤氮轉(zhuǎn)化細(xì)菌屬水平上的組成(a)及氮轉(zhuǎn)化相關(guān)功能預(yù)測(cè)結(jié)果(b)

根據(jù)上述土壤中氮轉(zhuǎn)化微生物群落結(jié)構(gòu)的變化,我們進(jìn)一步分析了芘對(duì)氮轉(zhuǎn)化功能強(qiáng)度的影響.如圖2(b)所示,土壤樣品中硝化功能(Nitrification)和好氧氨氧化功能(Aerobic_ammonia_oxidation)的細(xì)菌豐度最高,占總豐度的72.9%～96.1%.與空白組相比,芘在前3d促進(jìn)了氨氧化功能和硝化功能的細(xì)菌增長(zhǎng),促進(jìn)率分別為10.5%和10.3%.推測(cè)可能是土壤中富集了許多能代謝氨的氨氧化菌,其群落結(jié)構(gòu)的變化導(dǎo)致土壤中相關(guān)功能的變化.此外,固氮(Nitrogen_fixation),硝酸鹽還原(Nitrate_reduction)和尿素分解(Ureolysis)的功能強(qiáng)度在芘培養(yǎng)的第0d由3.6%～5.8%降低至第3d的0.9%～1.1%,但在第60d又增加至4.3%～7.3%.結(jié)合之前的討論,芘對(duì)土壤中氮轉(zhuǎn)化細(xì)菌的影響可能會(huì)直接導(dǎo)致對(duì)應(yīng)功能強(qiáng)度的變化,最終干擾土壤中的氮轉(zhuǎn)化過程.

2.4 芘對(duì)土壤氮轉(zhuǎn)化相關(guān)酶活性的影響

為了揭示芘對(duì)土壤中氮轉(zhuǎn)化相關(guān)酶活性的影響,進(jìn)一步定量分析了土壤氨氧化和反硝化過程中關(guān)鍵酶活性的變化.如圖3(a)所示,在所有采樣時(shí)間點(diǎn),芘處理組中AMO活性均顯著低于空白組(0.05).表明芘在處理過程中對(duì)AMO活性呈現(xiàn)抑制作用,且抑制作用在第7d最大,為56.1%.如圖3(b)所示,與空白組相比,芘在第7,30,60d對(duì)NAR活性呈顯著的抑制作用(0.05).對(duì)于NIR活性,芘在第0,1,15,60d對(duì)其表現(xiàn)出抑制作用,抑制率在14.0%～38.0%.以上結(jié)果表明,與2.3的討論結(jié)果不同,芘在降解前3d增強(qiáng)了土壤氨氧化功能強(qiáng)度,但在整個(gè)降解過程中顯著抑制了AMO活性,推測(cè)可能是由于其他氨氧化菌的減少.對(duì)于反硝化過程中的NAR和NIR活性,芘在培養(yǎng)第60d對(duì)其均表現(xiàn)為明顯的抑制作用.

2.5 芘對(duì)土壤氮轉(zhuǎn)化相關(guān)基因的影響

為了明確芘對(duì)土壤氮轉(zhuǎn)化相關(guān)基因的影響,進(jìn)一步分析了氮轉(zhuǎn)化過程編碼基因豐度隨時(shí)間的變化趨勢(shì).如圖4(a)所示,空白組和處理組中固氮基因H的豐度第0, 1, 3d有所上升,但到第7d開始下降,且芘對(duì)其抑制作用達(dá)到最大值,為27.8%,隨后抑制作用減弱.與2.3結(jié)論不同,土壤中的固氮功能強(qiáng)度在芘脅迫下的前3d呈現(xiàn)出降低的趨勢(shì),并未增加.編碼基因?yàn)锳OAA和AOBA分別由氨氧化古菌和氨氧化細(xì)菌進(jìn)行催化.如圖4(b),芘僅在第15, 30, 60d顯著降低了AOAA基因的豐度,抑制率為47.5%～55.7%.與前面的討論結(jié)果不同,氨氧化古菌的豐度在芘培養(yǎng)第15d和第30d無明顯變化.此外,芘在第15d和第60d顯著降低了AOBA的豐度,抑制率分別為44.5%和33.8%.說明氨氧化細(xì)菌在芘污染環(huán)境下也受到顯著的抑制作用.如圖4(d)所示,芘處理組中編碼NAR的G基因豐度僅在第3d顯著低于空白組(0.05),在其他時(shí)間點(diǎn)與空白組無顯著差異.另外,處理組中S基因的豐度在第7d顯著低于空白組.這表明芘對(duì)這兩個(gè)基因的豐度影響比較小.NAR可分為膜結(jié)合和周質(zhì)NAR,分別由G和編碼[53].本研究觀察到芘處理組中NAR活性顯著降低,而G基因豐度變化較小,推測(cè)可能是芘抑制了G和基因的表達(dá)量,這需要進(jìn)一步探究.NIR同樣也有兩種類型:可溶性含銅酶和Cu型NIR,分別由S和K編碼[53].上述的定量分析結(jié)果可知,芘在培養(yǎng)初期(第0, 1, 7d)和后期(第15, 30, 60d)分別顯著抑制了固氮基因和氨氧化基因的豐度,但對(duì)于反硝化基因G和S,芘對(duì)其抑制作用分別僅在第3d和第7d表現(xiàn)明顯.另外,本研究發(fā)現(xiàn)基因豐度的變化趨勢(shì)和功能活性不一致,推測(cè)可能是由于芘還影響了相關(guān)基因的表達(dá)量,未來還需要進(jìn)行深入探究.

圖4 空白組和芘處理組土壤微生物氮轉(zhuǎn)化編碼基因豐度的變化

3 結(jié)論

3.1 芘(12.09mg/kg)在土壤中的好氧微生物降解符合零級(jí)反應(yīng)動(dòng)力學(xué),降解半衰期為37d.因受其結(jié)構(gòu)和理化性質(zhì)的影響,其降解率,降解半衰期以及生物可利用性均介于菲和苯并[a]芘之間.

3.2 土壤酶活性分析結(jié)果顯示,芘僅在降解第1d刺激了脲酶活性,而在降解第0d和降解后期(第30, 60d)顯著刺激了土壤脫氫酶活性.這說明環(huán)境濃度的芘整體上促進(jìn)了微生物的代謝活力.

3.3 分析土壤中氮轉(zhuǎn)化相關(guān)微生物的結(jié)構(gòu)變化可知,由于氨氧化古菌相對(duì)豐度的變化,芘在培養(yǎng)前期(第0, 1, 3d)促進(jìn)了好氧氨氧化和硝化功能,在第60d則表現(xiàn)為抑制作用,而對(duì)于具有固氮功能的,以及,尿素分解細(xì)菌和硝酸鹽還原細(xì)菌則作用相反.因此,芘對(duì)土壤中氮轉(zhuǎn)化相關(guān)微生物群落結(jié)構(gòu)的影響可能會(huì)導(dǎo)致對(duì)應(yīng)功能的變化.

3.4 從功能基因方面定量分析芘對(duì)土壤氮轉(zhuǎn)化過程的影響,結(jié)果顯示:在芘處理前3d固氮基因H的豐度有所增加,但第7d芘對(duì)其表現(xiàn)出的抑制作用達(dá)到最大,隨后抑制作用減弱.根據(jù)土壤氨氧化和反硝化過程中關(guān)鍵酶活性及編碼基因的變化,芘在降解過程中顯著抑制了氨氧化過程,但對(duì)于氨氧化基因(AOAA和AOBA)僅在降解后期(第15, 30, 60d)起到抑制作用.此外,芘對(duì)反硝化過程也主要在降解第60d表現(xiàn)為抑制作用,但對(duì)其編碼的G和S的基因豐度則作用不明顯.

3.5 氮轉(zhuǎn)化基因定量分析結(jié)果與相關(guān)微生物的群落結(jié)構(gòu)和功能預(yù)測(cè)結(jié)果不同.因此,為了明晰污染物對(duì)元素轉(zhuǎn)化過程的影響特征,不僅需要定量分析相關(guān)酶活性以及基因,未來還需要進(jìn)一步定量分析基因的RNA表達(dá)量,才能準(zhǔn)確評(píng)估微生物群落結(jié)構(gòu)與功能活性的變化.

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Characterization of pyrene's impact on the soil functional microorganisms for nitrogen transformation.

HU Qin1, ZHANG Li-lan1,2*, YI Mei-ling1, YANG Rui1

(1.Key Laboratory of Three Gorges Reservoir Region’s Eco-environment, Ministry of Education, Chongqing University, Chongqing 400044, China；2.State Key Laboratory of Coal Mine Disaster Dynamics and Control, Chongqing University, Chongqing 400044, China)., 2023,43(10)：5574～5582

An aerobic degradation microenvironment was constructed to analyze the effects of pyrene (12.09mg/kg) at ambient concentration on soil enzyme activities, the whole process of nitrogen transformation and related functional microorganisms. The results showed that pyrene only significantly increased urease activity on the first day of degradation, but promoted the dehydrogenase activity at both the early and late phases of degradation. The analysis of the bacterial community structure revealed that the variation of the relative abundance of ammonia-oxidizing bacteria () promoted pyrene-mediated aerobic ammonia oxidation and nitrification in the early stages of treatment and inhibited that in the late stages. In contrast, the effects of nitrogen-fixing bacteria (,and), urea-degrading bacteria (), and nitrate-reducing bacteria () were opposite. The quantitative analysis of functional genes showed that, despite pyrene's repressive effect on the nitrogen-fixing geneH at the start of the culture, the abundance ofH showed an increasing trend, which was not consistent to the anticipated changes in microbial community structure and associated functions. Compared with changes in key enzyme activities and genes encoding the processes of ammonia oxidation and denitrification, pyrene did not significantly boost ammonia oxidation in the early stages of incubation, t severely hampered ammonia oxidation and denitrification after 15days, significantly inhibited the ammonia oxidation process. In this study, how pyrene influenced the microbial nitrogen transformation process in soil was reported, fundamental data on understanding the environmental hazard of pyrene were provided.

pyrene；soil enzyme activity；nitrogen cycling bacterial community；nitrogen cycling processes

X172,X171.5

A

1000-6923(2023)10-5574-09

2023-03-01

國(guó)家重點(diǎn)研發(fā)計(jì)劃(2019YFC1805500);國(guó)家自然科學(xué)基金資助項(xiàng)目(42177363)

* 責(zé)任作者, 副教授, lilanzhang@cqu.edu.cn

胡 琴(1997-),女,重慶忠縣人,重慶大學(xué)碩士研究生,主要研究方向?yàn)榭剐曰蚍肿由飳W(xué).zh3357@qq.com.

胡 琴,張利蘭,易美玲,等.芘對(duì)土壤微生物氮轉(zhuǎn)化功能菌群的影響特征 [J]. 中國(guó)環(huán)境科學(xué), 2023,43(10):5574-5582.

Hu Q, Zhang L L, Yi M L, et al. Characterization of pyrene's impact on the soil functional microorganisms for nitrogen transformation [J]. China Environmental Science, 2023,43(10):5574-5582.