基于非培養(yǎng)手段的多環(huán)芳烴降解微生物解析

陳亞婷,姜 博,邢 奕,張娜娜,連路寧,路 培

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基于非培養(yǎng)手段的多環(huán)芳烴降解微生物解析

陳亞婷,姜 博*,邢 奕*,張娜娜,連路寧,路 培

(北京科技大學(xué)能源與環(huán)境工程學(xué)院,工業(yè)典型污染物資源化處理北京市重點(diǎn)實(shí)驗(yàn)室,北京 100083)

本文對(duì)原位識(shí)別土壤未培養(yǎng)微生物的非培養(yǎng)手段進(jìn)行綜述,揭示其在PAHs降解中發(fā)揮的作用.首先對(duì)土壤中PAHs的降解微生物、降解原理以及功能基因進(jìn)行全面總結(jié);其次對(duì)熒光原位雜交技術(shù)(FISH)、穩(wěn)定同位素探針技術(shù)(SIP)、宏基因組學(xué)技術(shù)(Metagenomics)、轉(zhuǎn)錄組學(xué)技術(shù)(Transcriptomics)、單細(xì)胞技術(shù)(Single-cell technology)以及磁性納米材料技術(shù)(MMI)等非培養(yǎng)手段的原理、特點(diǎn)以及應(yīng)用進(jìn)行歸納總結(jié).重點(diǎn)討論這6種非培養(yǎng)手段在原位識(shí)別PAHs降解微生物中的應(yīng)用潛力.其中SIP在研究PAHs降解機(jī)制中最為成熟、穩(wěn)定,應(yīng)用最廣泛.MMI作為一種新型的非培養(yǎng)手段,能夠從復(fù)雜的生態(tài)環(huán)境中分離獲得活的功能微生物,為深入探究未培養(yǎng)的PAHs降解微生物提供解決途徑.

未培養(yǎng)微生物；多環(huán)芳烴降解；土壤；功能微生物；非培養(yǎng)方法

土壤的多孔結(jié)構(gòu)以及富含有機(jī)質(zhì)、沉積物等特點(diǎn),為PAHs的全球范圍遷移提供了儲(chǔ)存場所.這使得土壤中的PAHs含量高、難處理.尋找高效的土壤PAHs修復(fù)技術(shù)迫在眉睫.修復(fù)PAHs污染土壤的常用技術(shù)包括物理修復(fù)、化學(xué)修復(fù)以及生物修復(fù)[11].物理修復(fù)技術(shù)是根據(jù)污染物顆粒粒徑、密度、分布大小及表面性質(zhì),借助物理手段將污染物從土壤中分離或去除,包括熱修復(fù)法、換土法及電動(dòng)修復(fù)法等[12-13].物理法能耗高,受土壤性質(zhì)影響大,易破壞土壤結(jié)構(gòu)與性質(zhì),不能徹底清除污染物質(zhì)且容易造成二次污染.化學(xué)修復(fù)技術(shù)是利用土壤中污染物性質(zhì),向其中加入相應(yīng)化學(xué)物質(zhì)并經(jīng)過一系列化學(xué)反應(yīng),最終實(shí)現(xiàn)污染物改變、分離或固化,包括土壤淋洗法、電化學(xué)修復(fù)法、化學(xué)氧化法、化學(xué)還原法等[14-15].修復(fù)過程中污染物遷移擴(kuò)散、廢氣廢水處理、成本消耗以及二次污染等是制約化學(xué)修復(fù)技術(shù)發(fā)展的主要因素.相比之下,生物修復(fù)技術(shù)具有低成本、高效率、小污染、操作簡便等優(yōu)勢(shì),是土壤修復(fù)領(lǐng)域研究重點(diǎn)之一[16].

生物修復(fù)技術(shù)包括微生物修復(fù)技術(shù)和植物修復(fù)技術(shù),本文針對(duì)PAHs污染土壤微生物修復(fù)技術(shù)的作用原理、特點(diǎn)以及研究進(jìn)展進(jìn)行論述,對(duì)微生物修復(fù)技術(shù)中常用的分子生物學(xué)技術(shù)的優(yōu)缺點(diǎn)、發(fā)展現(xiàn)狀、適用范圍進(jìn)行討論,旨在為從事污染場地修復(fù)的研究人員,提供微生物修復(fù)技術(shù)的理論指導(dǎo).

1 多環(huán)芳烴污染場地的微生物降解

微生物修復(fù)技術(shù)[17]是微生物利用PAHs作為生長繁殖的碳源或能源,合成自身生命物質(zhì)的同時(shí)以共代謝以及礦化作用的方式降解土壤中的PAHs.參與PAHs降解的微生物包括細(xì)菌、真菌和藻類,其中對(duì)細(xì)菌的研究和報(bào)道較多,包括假單胞菌屬()[18]、紅球菌屬()[19]、氣單胞菌屬()[20]、分枝桿菌屬()[21]、微球菌屬()[22]、諾卡氏菌屬()[22]、黃桿菌屬()[23]、伯克氏菌屬()[24]、鞘氨醇單胞菌屬()[25]、芽孢桿菌屬()[28-29]等(表1).

PAHs的降解首先是羥化雙加氧酶對(duì)苯環(huán)鄰位C原子的羥基化,隨后在多種酶的作用下逐步催化降解.目前,對(duì)PAHs代謝途徑的推測(cè)主要依賴于識(shí)別和分析PAHs代謝產(chǎn)物.不同種類的PAHs代謝途徑不同,不同微生物對(duì)同種PAHs的代謝途徑也不相同,而同種微生物在代謝PAHs的過程中也會(huì)存在多種途徑.例如含有3個(gè)苯環(huán)的菲在sp. P1-1菌株降解過程中不僅發(fā)生3,4位C被羥基化,也會(huì)在1,2或者9,10位發(fā)生C被羥基化,在不同酶的作用下生成1,2-二羥菲或聯(lián)苯甲酸等[26].PAHs的代謝過程中涉及微生物多種酶的合成及催化,這些酶的合成及功能發(fā)揮受到多種功能基因的表達(dá)調(diào)控.如表2所示,已發(fā)現(xiàn)來源不同菌株的PAHs降解功能基因包括、、、及等基因,它們編碼的酶在PAHs代謝過程中發(fā)揮著不同的作用.如基因簇編碼的酶可以將菲轉(zhuǎn)化為順-3,4-二羥基-3,4-二氫菲(cis-3,4-dihydroxy-3, 4- dihydrophenanthrene),基因簇編碼的酶將1-羥基-2-萘酸轉(zhuǎn)化為鄰苯二甲酸酯[27];菌株NAH7質(zhì)粒攜帶的基因簇編碼的酶將萘轉(zhuǎn)化為水楊酸,基因簇編碼的酶將水楊酸轉(zhuǎn)化為乙酰輔酶A和丙酮酸[28].

表1 基于純培養(yǎng)方法分離的能夠降解PAHs的微生物

注:-表示產(chǎn)物暫不清楚.

然而,環(huán)境中已發(fā)現(xiàn)的微生物99%以上都無法在實(shí)驗(yàn)室純培養(yǎng)條件下得到.培養(yǎng)基富營養(yǎng)化、微生物生長因子缺乏以及對(duì)微生物間復(fù)雜聯(lián)系的忽視和破壞,都是導(dǎo)致傳統(tǒng)培養(yǎng)條件未培養(yǎng)微生物無法生長繁殖的主要原因[48].1982年Colwell實(shí)驗(yàn)室將這些微生物稱作活的但未培養(yǎng)狀態(tài)[30].未培養(yǎng)微生物分布極其廣泛,種類數(shù)量豐富多樣,且體內(nèi)含有多種活性化合物和新型基因,在物種進(jìn)化、自然界新成代謝中扮演重要角色,并成為各個(gè)領(lǐng)域極具研究價(jià)值的微生物資源[49].盡管分離、純化培養(yǎng)這些被定義為未培養(yǎng)的微生物存在著困難與挑戰(zhàn),但研究者認(rèn)為未培養(yǎng)微生物蘊(yùn)藏的基因、物種、功能多樣性信息具有十分重要的研究意義[50].關(guān)于未培養(yǎng)微生物的基因、群落結(jié)構(gòu)、功能、遺傳進(jìn)化等方面的研究受到了越來越多人的關(guān)注.Kim等[51]研究發(fā)現(xiàn)牛瘤胃中未培養(yǎng)細(xì)菌種類與可培養(yǎng)細(xì)菌種類同樣豐富,未培養(yǎng)細(xì)菌對(duì)牛瘤胃發(fā)酵纖維具有重要作用.Ouyang等[52]對(duì)3種不同制藥廢水處理系統(tǒng)的曝氣池內(nèi)污泥微生物群落進(jìn)行研究,發(fā)現(xiàn)未培養(yǎng)的念珠菌、厭氧繩菌和胚泡菌對(duì)廢水中銨態(tài)氮的去除均有貢獻(xiàn).為便于研究人員快速識(shí)別分類復(fù)雜的未培養(yǎng)微生物,2017年Konstantinidis等[53]提出采用一種新的基于基因組的分類方法,全面描述未培養(yǎng)微生物.研究未培養(yǎng)微生物在環(huán)境中的種類、群落結(jié)構(gòu)以及作用機(jī)制,有助于探究污染物PAHs在土壤中的原位降解機(jī)理,識(shí)別受污染土壤中降解PAHs的原位未培養(yǎng)微生物,最大限度的發(fā)揮復(fù)雜環(huán)境中PAHs微生物降解功能.

表2 PAHs降解功能基因

鑒于未培養(yǎng)微生物的復(fù)雜性,分離、提純培養(yǎng)分析技術(shù)會(huì)干擾和破壞原有微生物狀態(tài)、群落組成與結(jié)構(gòu),而原位表征未培養(yǎng)微生物技術(shù)則更加準(zhǔn)確和全面.熒光原位雜交技術(shù)、穩(wěn)定同位素探針技術(shù)、宏基因組學(xué)技術(shù)、轉(zhuǎn)錄組學(xué)、單細(xì)胞技術(shù)、磁性納米材料技術(shù)等基于非培養(yǎng)手段的研究方法,已經(jīng)被應(yīng)用于PAHs降解功能微生物的原位識(shí)別.非培養(yǎng)手段可以揭示在復(fù)雜的生態(tài)環(huán)境中,有哪些微生物參與污染物降解過程,這些微生物在降解過程中主要做什么,以及它們是如何發(fā)揮作用的[54].本文對(duì)這6種技術(shù)的原理、特點(diǎn)以及應(yīng)用進(jìn)行歸納總結(jié),以獲得較為全面的、能夠解析PAHs降解微生物群落的技術(shù)概況,為相關(guān)領(lǐng)域研究者提供參考依據(jù).

2 基于非培養(yǎng)手段的PAHs降解微生物的識(shí)別

2.1 熒光原位雜交技術(shù)(FISH)

熒光原位雜交技術(shù)是20世紀(jì)80年代末發(fā)展起來的一種利用非放射性熒光標(biāo)記的原位雜交技術(shù).其原理是應(yīng)用特殊的熒光標(biāo)記物標(biāo)記核酸探針,根據(jù)堿基互補(bǔ)配對(duì)原則,將標(biāo)記探針與目標(biāo)序列原位雜交,在熒光顯微鏡下直接觀察,或?qū)㈦s交后的標(biāo)記探針與熒光素分子耦聯(lián)復(fù)合物結(jié)合,進(jìn)行相對(duì)定性、定位、定量檢測(cè)目標(biāo)序列的雜交結(jié)果.利用標(biāo)記探針與特定環(huán)境基因組中DNA分子雜交,可以表征目標(biāo)微生物的豐度以及種群分布.該技術(shù)具備高靈敏度,高分辨率,可以同時(shí)檢測(cè)多種核苷酸序列,以及快速、安全、可靠的優(yōu)勢(shì).熒光原位雜交技術(shù)或與其他相結(jié)合,可以原位表征特定環(huán)境中降解PAHs的目標(biāo)微生物豐度、代謝過程以及種群分布.

Sanches等[55]從煉油化工廠廢水中富集厭氧微生物群落,探究其對(duì)菲和苊的生物降解能力.利用新一代測(cè)序技術(shù)和熒光原位降解技術(shù)表征降解PAHs的群落由附屬于和的細(xì)菌組成.Hesham等[56]利用酵母菌去除廢水中的PAHs,用活性污泥接種、構(gòu)建了3個(gè)不同的系統(tǒng),并通過PCR-DGGE技術(shù)和FISH技術(shù)分析對(duì)比3個(gè)系統(tǒng)中酵母菌的結(jié)構(gòu)以及豐度.結(jié)果表明,去除高分子量PAHs的酵母菌主要是來自其中兩種生物增強(qiáng)系統(tǒng)接種的5株酵母菌,這兩種接種了外來酵母菌的生物增強(qiáng)系統(tǒng)對(duì)高分子量PAHs的降解具有顯著作用.

2.2 穩(wěn)定同位素探針技術(shù)(SIP)

穩(wěn)定同位素探針技術(shù)(SIP)是一種基于同位素示蹤方法鑒別復(fù)雜群落中參與某種物質(zhì)代謝的功能微生物技術(shù),能夠?qū)⑽⑸锶郝浣Y(jié)構(gòu)與其功能相耦合.SIP方法的工作原理如圖1所示(以DNA-SIP為例).向環(huán)境樣品中原位添加同位素標(biāo)記(13C、15N、18O等)的化合物,經(jīng)過一段時(shí)間的培養(yǎng),樣品中的功能微生物同化標(biāo)記化合物為磷脂脂肪酸(PLFA)[57]、DNA[58]、RNA[59]、蛋白質(zhì)[60]等生物標(biāo)記物.分離微生物中的生物標(biāo)記物分子并對(duì)其中的重同位素進(jìn)行下游分析,即可獲得環(huán)境功能微生物信息,揭示其在環(huán)境化合物轉(zhuǎn)化中的群落以及功能.

圖1 DNA-SIP技術(shù)路線

1998年,PLFA-SIP技術(shù)首次應(yīng)用于研究甲烷氧化細(xì)菌[61].但當(dāng)時(shí)人們?nèi)狈?duì)目標(biāo)分子的充分了解,且PLFA-SIP技術(shù)分辨率低,導(dǎo)致構(gòu)建分類信息十分困難.到2000年,研究者成功利用DNA-SIP技術(shù)從含有13C-甲醇的土壤中,辨認(rèn)出-和在甲醇原位代謝過程中發(fā)揮主要作用[62].目前,SIP技術(shù)主要以DNA[58]、RNA[59]和蛋白質(zhì)[60]作為生物標(biāo)志物.其中,DNA分子比RNA更穩(wěn)定,作為一種信息量最為豐富的生物標(biāo)記物,DNA分子有最高的分類學(xué)分辨率,應(yīng)用最為廣泛.但是微生物基因組中的GC含量會(huì)增加未標(biāo)記DNA的浮力密度,影響標(biāo)記DNA的有效分離[63-64],且需要較長的培養(yǎng)時(shí)間.相比之下,RNA標(biāo)記獨(dú)立于細(xì)胞分裂,這使得RNA-SIP技術(shù)更靈敏.但從環(huán)境樣品中獲取的mRNA濃度低,離心不完全[65],分類精度低且不穩(wěn)定,以及cRNA缺乏完善的功能基因表達(dá)數(shù)據(jù)庫[63]等因素,限制了RNA-SIP技術(shù)的發(fā)展.蛋白質(zhì)-SIP技術(shù)也獨(dú)立于細(xì)胞分裂,具有靈敏度高、培養(yǎng)時(shí)間短以及快速標(biāo)記等特點(diǎn),但是相比于其他標(biāo)記技術(shù),蛋白質(zhì)-SIP技術(shù)的分類精度更低[63].

SIP技術(shù)與其他技術(shù)結(jié)合,已經(jīng)廣泛應(yīng)用于探究土壤、水、沉積物等環(huán)境介質(zhì)中參與生化反應(yīng)的功能微生物群落特征,包括廢水處理中硝化氧化微生物功能的識(shí)別[66-68],海洋微生物的遺傳特性及代謝機(jī)制研究[69-71],植物根際微生物與C元素循環(huán)關(guān)系解析[59,72],以及土壤環(huán)境中降解功能微生物的識(shí)別等[73-74].應(yīng)用SIP技術(shù)識(shí)別PAHs降解功能微生物的報(bào)道如表3所示.可以看出,目前的研究大多以13C標(biāo)記PAHs底物,采用DNA-SIP技術(shù)研究不同種類PAHs(萘、蒽、菲、熒蒽、芘等)的降解功能微生物.研究的環(huán)境介質(zhì)包括森林土壤、沉積物以及受污染土壤等,由于其含有不同的礦物質(zhì)、有機(jī)物以及腐殖質(zhì),具有完全不同的理化性質(zhì),因此發(fā)掘的微生物數(shù)量以及種類也各不相同. Song等[74]的研究,首次利用DNA-SIP技術(shù)發(fā)現(xiàn)森林土壤中相關(guān)細(xì)菌直接參與熒蒽的代謝. Rochman等[75]利用SIP技術(shù),在加拿大油砂尾礦庫表面檢測(cè)到家族和是降解萘的主要微生物.SIP方法實(shí)現(xiàn)了單一微生物向復(fù)雜微生物群落研究的轉(zhuǎn)變,為在群落整體水平,系統(tǒng)研究微生物在自然環(huán)境中重要生理過程的分子調(diào)控機(jī)制,定向發(fā)掘重要微生物資源和生物技術(shù)開發(fā)提供了關(guān)鍵技術(shù)支撐.

表3 SIP技術(shù)在降解PAHs中的應(yīng)用

2.3 基于高通量測(cè)序的宏基因組學(xué)技術(shù)(Metagenomics)

20世紀(jì)80年代基因組學(xué)問世,涵蓋基因組學(xué)、轉(zhuǎn)錄組學(xué)、蛋白質(zhì)組學(xué)及代謝組學(xué)的生物組學(xué)內(nèi)容逐漸豐富.1998年Handelsman等[83]第一次提出宏基因組學(xué)一詞.不同于其他生物組學(xué),宏基因組學(xué)直接從環(huán)境樣品中提取所有微生物的總DNA進(jìn)行研究,避開傳統(tǒng)分離、培養(yǎng)微生物技術(shù)的缺點(diǎn)和困難,全面分析特定環(huán)境中微生物群落的組成多樣性、遺傳多樣性、演替與進(jìn)化,探究全部微生物的種間聯(lián)系以及與環(huán)境的相互作用.宏基因組學(xué)技術(shù)包括環(huán)境總DNA提取,宏基因組文庫的構(gòu)建、篩選、測(cè)序以及分析(如圖2所示).

利用直接在土壤中裂解微生物細(xì)胞提取DNA或先分離細(xì)胞再提取DNA的方法,提取土壤中總微生物的DNA.根據(jù)所獲得DNA選擇合適的載體和宿主細(xì)胞,將DNA片段連接到載體上并導(dǎo)入宿主菌,構(gòu)建宏基因組文庫.對(duì)大量復(fù)雜的宏基因組文庫篩選有利于快速鑒定功能基因和生物活性物質(zhì).宏基因文庫的篩選分為序列驅(qū)動(dòng)型篩選和功能驅(qū)動(dòng)型篩選.序列驅(qū)動(dòng)型篩選利用已知基因作為分子探針通過PCR或分子雜交技術(shù)篩選克隆基因,以獲取某一類結(jié)構(gòu)或功能相似的分子和基因.功能驅(qū)動(dòng)型篩選則利用某種特殊手段的目標(biāo)活性物質(zhì),檢測(cè)其表達(dá)基因,發(fā)現(xiàn)新的生物活性物質(zhì).相比于功能驅(qū)動(dòng)型篩選,序列驅(qū)動(dòng)型篩選效率更高,但是其依賴于已知基因而不能發(fā)現(xiàn)新基因序列[84].

高測(cè)序通量及測(cè)序速度、低測(cè)序成本與污染以及生物信息技術(shù)的優(yōu)化推動(dòng)著宏基因組學(xué)測(cè)序技術(shù)不斷進(jìn)步[85-86].其中,基于高通量檢測(cè)技術(shù)的宏基因組學(xué)測(cè)序技術(shù)有2種,分別為高通量測(cè)序技術(shù)和基因芯片技術(shù).基因芯片技術(shù)是利用一塊固定含有大量已知序列分子探針的小型芯片進(jìn)行雜交測(cè)序,具有高微生物檢測(cè)深度、高通量性和定量性,但是基因芯片技術(shù)只能獲取樣品中已知物種基因信息而不能檢測(cè)出新基因或新物種[87].相比之下,高通量測(cè)序技術(shù)應(yīng)用更廣泛,它以羅氏454測(cè)序技術(shù)和Illumina測(cè)序技術(shù)為代表,也被稱作第二代測(cè)序技術(shù),具有準(zhǔn)確性高,信息識(shí)別真實(shí)可靠以及篩選新基因,發(fā)現(xiàn)新物種的特性;但其發(fā)現(xiàn)群落中豐度較低的微生物能力仍待提高[64,87-88].將測(cè)序所得數(shù)據(jù)進(jìn)行存儲(chǔ)和序列分析處理,通過比對(duì)、提煉信息,分析微生物的群落組成、結(jié)構(gòu)以及生態(tài)影響.

圖2 宏基因組學(xué)技術(shù)原理[86]

隨著與高通量測(cè)序技術(shù)應(yīng)運(yùn)而生的國際宏基因組大數(shù)據(jù)庫和分析平臺(tái)的建立和完善,使得宏基因組學(xué)技術(shù)的應(yīng)用更為廣泛[89].其對(duì)微生物多樣性的探索遍及海洋[90]、森林[91]、極端環(huán)境[92]、氣候變化[93]、農(nóng)業(yè)食品[94]、臨床醫(yī)學(xué)[95]、環(huán)境污染修復(fù)[96]等各個(gè)領(lǐng)域.尤其是將宏基因組學(xué)技術(shù)應(yīng)用到土壤、沉積物等復(fù)雜微生物環(huán)境中,揭示其中蘊(yùn)藏的巨大基因、物種、群落資源,為環(huán)境修復(fù)提供理論依據(jù).2014年,Mason等[97]采集墨西哥灣原油泄漏點(diǎn)2010年9月~10月期間的深海1500m處表層沉積物,提取樣品DNA構(gòu)建16S rRNA宏基因組文庫,并利用Illumina技術(shù)測(cè)序.最終再現(xiàn)了樣品碳?xì)浠衔锏慕到馔緩揭约疤囟ɑ衔锏慕到饣?發(fā)現(xiàn)了PAHs對(duì)表層沉積物中的微生物群落結(jié)構(gòu)有消極影響. Zafra等[98]向PAHs污染土壤中接種能夠降解PAHs的微生物群落,利用宏基因組學(xué)技術(shù)發(fā)現(xiàn)接種降解微生物群落能夠顯著提高土著微生物對(duì)PAHs的降解能力,降解基因豐度也發(fā)生明顯變化.Zhao等[99]從北京周邊的一個(gè)焦化廠土壤中采樣,利用高通量測(cè)序手段,探究了其中熒蒽的降解微生物群落、功能基因以及代謝產(chǎn)物,與其它技術(shù)結(jié)合發(fā)現(xiàn)該焦化廠土壤中的和相似豐度高于先前研究的受碳?xì)浠衔镂廴镜耐寥乐械南嗨曝S度,它們對(duì)原位降解PAHs發(fā)揮重要作用,其研究結(jié)果為復(fù)雜微生物群落構(gòu)建了第一個(gè)PAHs降解的協(xié)同代謝網(wǎng).

2.4 轉(zhuǎn)錄組學(xué)技術(shù)

中心法則闡述遺傳信息由DNA經(jīng)過轉(zhuǎn)錄傳遞給RNA,但這并不意味著DNA分子上的所有基因都會(huì)被轉(zhuǎn)錄,Hangauer等[100]研究證明有85%以上的基因會(huì)進(jìn)行轉(zhuǎn)錄,而剩下不到15%的基因都不會(huì)參與表達(dá).因此,基于RNA分子的研究在揭示基因表達(dá)水平、調(diào)控規(guī)律以及功能基因方面相比DNA分子研究更有優(yōu)勢(shì).隨著基因組學(xué)的建立,以某一狀態(tài)下特定組織或細(xì)胞轉(zhuǎn)錄出的RNA分子為研究對(duì)象的轉(zhuǎn)錄組學(xué)隨后誕生.其研究范圍不僅包括能夠編碼蛋白的mRNA也包括非編碼rRNA、tRNA等.目前,無法對(duì)RNA直接測(cè)序,所以將RNA轉(zhuǎn)化為DNA并對(duì)DNA測(cè)序分析是轉(zhuǎn)錄組學(xué)技術(shù)的主要思路.相比于微陣列測(cè)序技術(shù)對(duì)物種特性探測(cè)存在的偏差,基于高通量測(cè)序的RNA測(cè)序技術(shù)(RNA-Seq)具有更高的靈敏度和精度,承擔(dān)了轉(zhuǎn)錄組學(xué)技術(shù)的大部分測(cè)序任務(wù)[101],也是轉(zhuǎn)錄組學(xué)的研究熱點(diǎn).利用RNA-Seq技術(shù)不僅可以在全基因組的尺度上描述編碼RNAs和非編碼RNAs的轉(zhuǎn)錄差異,還有助于探究整個(gè)轉(zhuǎn)錄水平的基因表達(dá)與生物進(jìn)化的內(nèi)在聯(lián)系,識(shí)別不同生態(tài)環(huán)境下的基因轉(zhuǎn)錄表達(dá)差異以及基因?qū)Νh(huán)境變化的適應(yīng)潛力[101].

RNA-Seq技術(shù)包括實(shí)驗(yàn)設(shè)計(jì)、RNA分離、文庫準(zhǔn)備、高通量測(cè)序以及生物信息學(xué)分析幾個(gè)階段.其中,mRNA分離、富集以及測(cè)序深度、文庫大小是關(guān)系RNA-Seq技術(shù)成功進(jìn)行的主要因素[102].細(xì)胞中的總RNA分子90%以上由rRNA分子組成, mRNA分子只占1%~2%[102],而這極少的mRNA功能分子才是我們的研究重點(diǎn).目前,研究人員常用Oligo(dT)磁珠提取生物組織或細(xì)胞中的mRNA[103-104].再將mRNA片段化之后在引物作用下反轉(zhuǎn)錄為cDNA第一鏈、第二鏈等,之后擴(kuò)增cDNA獲得cDNA文庫,利用高通量技術(shù)測(cè)序并對(duì)數(shù)據(jù)結(jié)果進(jìn)行分析,即可得到樣品的基因表達(dá)水平.

轉(zhuǎn)錄組學(xué)技術(shù)廣泛應(yīng)用于臨床醫(yī)藥、生態(tài)環(huán)境、資源開發(fā)利用、健康風(fēng)險(xiǎn)評(píng)估等領(lǐng)域[101,105-106].對(duì)環(huán)境生態(tài)領(lǐng)域的探究在土壤[107]、水體[108]、極端環(huán)境[109]、氣候變化[110]等多方面均有涉足.利用轉(zhuǎn)錄組學(xué)技術(shù),對(duì)比基因表達(dá)水平在不同生態(tài)環(huán)境下的動(dòng)態(tài)變化,能夠揭示環(huán)境脅迫對(duì)動(dòng)植物或微生物體內(nèi)基因表達(dá)的影響.Singh等[107]從轉(zhuǎn)錄水平上論述了土壤中的重金屬對(duì)植物的脅迫作用.他們認(rèn)為土壤中重金屬脅迫會(huì)誘導(dǎo)大量的基因以及蛋白質(zhì)來連接傳遞信號(hào),這些基因中有一部分是負(fù)責(zé)編碼轉(zhuǎn)錄因子的調(diào)控基因,轉(zhuǎn)錄因子則負(fù)責(zé)在機(jī)體受到脅迫時(shí)調(diào)控各種反應(yīng)基因;另一部分功能基因則負(fù)責(zé)編碼代謝各種化合物的酶,這些基因共同操縱著植物在重金屬脅迫下的耐受性.在PAHs污染的土壤中,微生物為應(yīng)對(duì)環(huán)境因子的變化其基因表達(dá)也會(huì)受到影響,這種響應(yīng)可能體現(xiàn)在單個(gè)微生物個(gè)體的變化、微生物個(gè)體與個(gè)體間的變化、整個(gè)微生物群落結(jié)構(gòu)或功能的變化.目前,轉(zhuǎn)錄組學(xué)技術(shù)對(duì)PAHs的研究主要集中在對(duì)其生態(tài)毒性的探討以及對(duì)人體的健康風(fēng)險(xiǎn)評(píng)估[111-113],較少關(guān)注土壤中PAHs的生物降解過程.這可能是由于土壤相比于海洋、湖泊等其他介質(zhì)成分更為復(fù)雜,提取生物的mRNA更困難,且mRNA分子的穩(wěn)定性低于DNA分子.盡管如此,有研究者在幾年前就試圖從轉(zhuǎn)錄組學(xué)的角度闡明土壤中PAHs對(duì)微生物的基因表達(dá)的影響以及污染物代謝途徑.2012年Alexandre等[114]向低PAHs背景值的土壤中加入污染物菲,探究土壤樣品中的PAHs對(duì)土著微生物基因表達(dá)的影響.他們從菲完全去除的土樣中提取和分離mRNA、反轉(zhuǎn)錄為cDNA并進(jìn)行測(cè)序分析.結(jié)果顯示出與芳香類化合物代謝、呼吸作用以及應(yīng)激反應(yīng)相關(guān)的基因轉(zhuǎn)錄本都有不同程度的增加,與PAHs降解相關(guān)的微生物和數(shù)量增加.這是第一次利用轉(zhuǎn)錄學(xué)技術(shù)研究土壤微生物群落對(duì)污染物的原位響應(yīng),它成功的將PAHs的代謝與相應(yīng)微生物聯(lián)系起來,從基因表達(dá)的層面揭示微生物代謝PAHs的機(jī)理.這也表明,連接基因表達(dá)與微生物功能橋梁的轉(zhuǎn)錄組學(xué)技術(shù)在探究PAHs等污染物質(zhì)原位降解機(jī)制的巨大潛力,有助于深入探究微生物群落與功能的內(nèi)在聯(lián)系.2016年,Meckenstock等[115]應(yīng)用轉(zhuǎn)錄組學(xué)聯(lián)合其他生物化學(xué)方法揭示萘的生物降解途徑,這是第一次在生物化學(xué)和基因水平闡述萘的厭氧降解途徑,極大促進(jìn)了PAHs厭氧代謝以及代謝微生物的生態(tài)學(xué)研究.盡管土壤的復(fù)雜性給轉(zhuǎn)錄組學(xué)的應(yīng)用帶來阻礙,但其中包涵豐富的微生物及功能基因資源,借助于轉(zhuǎn)錄組學(xué)對(duì)基因轉(zhuǎn)錄—表達(dá)功能進(jìn)行深入探究,使得原位識(shí)別功能微生物群落結(jié)構(gòu)方面更準(zhǔn)確,也為土壤修復(fù)工程提供理論指導(dǎo).

2.5 單細(xì)胞技術(shù)(Sing-cell technology)

盡管宏基因組學(xué)技術(shù)和轉(zhuǎn)錄組學(xué)技術(shù)能夠分別在DNA分子和基因表達(dá)與調(diào)控的層面上揭示功能微生物在復(fù)雜生態(tài)環(huán)境下的響應(yīng)機(jī)制,但這兩種技術(shù)在發(fā)現(xiàn)新功能基因和物種豐度極小的未培養(yǎng)微生物方面仍有局限性[59],甚至不能提供明確的基因信息而模糊了特定微生物的代謝過程以及進(jìn)化歷程[116],單細(xì)胞技術(shù)的興起彌補(bǔ)了宏基因組學(xué)技術(shù)以及其他組學(xué)技術(shù)在微生物領(lǐng)域的認(rèn)識(shí)不足.

來源相同的細(xì)胞在分化過程中也會(huì)體現(xiàn)出不同細(xì)胞間的異質(zhì)性,生物學(xué)家們認(rèn)為生物學(xué)的最基本問題之一就是探究基因調(diào)控機(jī)制在細(xì)胞分化以及組織靈活、協(xié)調(diào)工作中是如何起作用的[117],而單細(xì)胞技術(shù)則是解決這一問題的新方法.它可以從微生物細(xì)胞中提供基因信息,確定在環(huán)境生物地球化學(xué)循環(huán)中占主導(dǎo)地位的未培養(yǎng)微生物的代謝潛力,開發(fā)細(xì)胞尺度上定量描述細(xì)胞特性的方法.如Rinke等[118]利用單細(xì)胞技術(shù)發(fā)現(xiàn)古生菌中有一種與肽聚糖合成有關(guān)的酶,由于古生菌中不含肽聚糖,他們推測(cè)這種酶可能是古生菌與其他細(xì)胞相互作用的結(jié)果.

單細(xì)胞基因組技術(shù)流程如圖3所示.包括單細(xì)胞的獲取、細(xì)胞裂解、全基因組擴(kuò)增、基因測(cè)序與數(shù)據(jù)分析.獲取單細(xì)胞的方式最常用的新方法是基于流式細(xì)胞術(shù)的熒光激活細(xì)胞分選法(FACS)和微流控芯片技術(shù).其中,FACS具有高通量、高靈活性、高純度的優(yōu)點(diǎn)被廣泛使用,但是FACS分離法對(duì)細(xì)胞存活率有一定影響[119];液滴微流控芯片技術(shù)需要樣品數(shù)量極少,價(jià)格低廉,污染小且高度自動(dòng)化、高通量[120].目前實(shí)現(xiàn)單細(xì)胞全基因組擴(kuò)增最新的方法是多重置換擴(kuò)增技術(shù)(MDA).Li等[121]實(shí)現(xiàn)了在微流控芯片上將MDA技術(shù)與芯片外PCR技術(shù)相結(jié)合,成功發(fā)現(xiàn)了細(xì)胞的異質(zhì)性.單細(xì)胞基因組技術(shù)現(xiàn)階段面臨的主要挑戰(zhàn)是將定量、綜合的基因組學(xué)與微觀的分辨率結(jié)合,提高從組織到細(xì)胞的分析精度[117].單個(gè)細(xì)胞的基因組與轉(zhuǎn)錄組存在一定的內(nèi)在聯(lián)系,而且在單個(gè)細(xì)胞內(nèi)同時(shí)測(cè)量多種分子類型比在多個(gè)細(xì)胞內(nèi)測(cè)量多種分子類型更有優(yōu)勢(shì),所以Macaulay等[122]提出在單個(gè)細(xì)胞中應(yīng)用多組學(xué)分析,有助于明確基因型與表現(xiàn)型的關(guān)系,同時(shí)了解從DNA到RNA再到蛋白質(zhì)以及細(xì)胞表型的調(diào)控機(jī)制.利用基因組序列構(gòu)建細(xì)胞譜系圖,同時(shí)利用轉(zhuǎn)錄組序列反映細(xì)胞的類型和狀態(tài),增強(qiáng)對(duì)細(xì)胞異質(zhì)性以及種群結(jié)構(gòu)、進(jìn)化的理解.

2009年,Stepanauskas[116]課題組建立了第一個(gè)專門從事單細(xì)胞基因組學(xué)研究的畢格羅實(shí)驗(yàn)室中心(SCGC),他們處理的微生物細(xì)胞來源涉及土壤、海洋、地下深層以及其他環(huán)境.單細(xì)胞技術(shù)的應(yīng)用十分廣泛,它可以從細(xì)胞水平闡述環(huán)境中PAHs等污染物的刺激對(duì)微生物細(xì)胞結(jié)構(gòu)、形態(tài)的影響以及微生物間復(fù)雜、微妙的相互作用.Chen等[123]利用流式細(xì)胞術(shù)探究電子垃圾場中的苯并[a]芘對(duì)降解微生物的細(xì)胞膜電位、細(xì)胞周期、細(xì)胞凋亡以及蛋白質(zhì)表達(dá)的影響.結(jié)果顯示,苯[a]芘是影響細(xì)胞膜電位以及破壞膜結(jié)構(gòu)的主要原因.此外,在污染物的脅迫下,微生物為適應(yīng)不良環(huán)境其參與污染物轉(zhuǎn)運(yùn)與代謝的酶以及蛋白質(zhì)表達(dá)有顯著變化.細(xì)胞膜作為細(xì)胞與污染物直接接觸的一道屏障,在接觸過程中發(fā)生著形態(tài)、結(jié)構(gòu)的改變:PAHs等污染物通過與細(xì)胞膜接觸,改變、破壞膜的脂質(zhì)結(jié)構(gòu),導(dǎo)致細(xì)胞膜空隙增大、細(xì)胞滲透性增強(qiáng)[124],這種改變?yōu)榧?xì)胞內(nèi)化污染物提供條件.雖然單細(xì)胞技術(shù)在探究土壤中PAHs生物降解的應(yīng)用較少,但該技術(shù)在研究功能微生物群落的進(jìn)化、結(jié)構(gòu)和功能方面,具有廣闊的前景.

圖3 單細(xì)胞基因組技術(shù)流程圖[118]

2.6 磁性納米材料技術(shù)

磁性納米材料(MMI)是一種利用磁性納米材料從復(fù)雜微生物群落中分離活的功能微生物的新型技術(shù),該技術(shù)的最大優(yōu)勢(shì)在于不依賴于對(duì)底物碳、氮源的標(biāo)記,能夠從復(fù)雜環(huán)境中分離活的功能微生物,這對(duì)準(zhǔn)確認(rèn)識(shí)功能微生物的生理生態(tài)特性和功能具有重要意義.

MMI技術(shù)流程示意如圖4所示.在一個(gè)復(fù)雜的微生物系統(tǒng)中,用磁性納米材料(MNPs)功能化系統(tǒng)中的所有細(xì)胞;向其中加入底物(如PAHs等),系統(tǒng)中能夠代謝底物為自身生長、繁殖提供物質(zhì)能源的磁性功能化細(xì)胞將會(huì)連續(xù)大量分裂、繁殖;分裂的細(xì)胞磁性會(huì)逐漸被稀釋直到完全失去磁性,而系統(tǒng)中另一部分不能代謝底物或代謝較慢的微生物細(xì)胞分裂、繁殖速率較慢,仍保留磁性;在外加磁場的作用下,系統(tǒng)內(nèi)帶有磁性的細(xì)胞和不帶磁性的細(xì)胞實(shí)現(xiàn)分離;收集得到活的功能微生物[125].

MNPs在物理、化學(xué)、生物各學(xué)科都有應(yīng)用研究[126-128],其優(yōu)點(diǎn)是尺寸小、有磁性且反應(yīng)活性高.在環(huán)境學(xué)領(lǐng)域的應(yīng)用主要集中在基于磁性納米材料高效分離、提取細(xì)胞內(nèi)核酸[128-131].利用MNPs分離復(fù)雜群落中的活細(xì)胞,前提是需要將MNPs有效吸附在細(xì)胞表面且保證細(xì)胞的活性與正常生理功能.Zhang等[132]的研究表明,納米顆粒與細(xì)胞的接觸、攝入過程中會(huì)發(fā)生物理、化學(xué)作用,其中納米顆粒的大小、形狀以及物化性質(zhì)都會(huì)影響這個(gè)過程,甚至產(chǎn)生毒性.2011年Zhang等[133]研發(fā)了一種具有生物相容性的MNPs功能化細(xì)胞的方法.該方法將聚烯丙基胺鹽酸鹽固定化的MNPs反復(fù)分離和純化,獲得具有良好生物相容性和細(xì)胞友好性的MNPs.利用這種方法功能化的細(xì)胞帶有磁性且功能化效率達(dá)到99.96%.2015年,Zhang等[125]利用這種方法,研究焦化廠廢水處理污泥中的微生物,發(fā)現(xiàn)未培養(yǎng)的spp.是負(fù)責(zé)原位降解苯酚的主要微生物,這是第一次將MMI應(yīng)用于原位識(shí)別污泥中的未培養(yǎng)功能微生物,并利用Biolog平板研究其對(duì)碳源和氮源的利用情況.2016年Wang等[134]將MMI技術(shù)應(yīng)用到原位識(shí)別受原油污染土壤中降解正烷烴的微生物,識(shí)別出和是降解正烷烴的主要微生物.

圖4 MMI技術(shù)流程(改自文獻(xiàn)[125])

3 前景與展望

環(huán)境中存在著大量數(shù)量龐大、功能多樣的未培養(yǎng)微生物,然而人們對(duì)它們的種類以及在地球生物化學(xué)循環(huán)中的作用還知之甚少.目前對(duì)PAHs降解微生物的研究還處于實(shí)驗(yàn)室純培養(yǎng)階段,但是復(fù)雜介質(zhì)中未培養(yǎng)微生物對(duì)PAHs的降解未忽視.FISH、SIP、宏基因組學(xué)、轉(zhuǎn)錄組學(xué)、單細(xì)胞以及磁性納米材料技術(shù),從基因和細(xì)胞的不同層次,提供了不依賴于純培養(yǎng)方法的識(shí)別PAHs降解微生物的解決途徑.目前,DNA-SIP技術(shù)已經(jīng)較為廣泛地應(yīng)用于對(duì)降解PAHs功能微生物的原位識(shí)別,其它技術(shù)也有少量應(yīng)用在污染物生物降解領(lǐng)域.雖然這些非培養(yǎng)手段避開了傳統(tǒng)培養(yǎng)方法的局限性,為我們?cè)涣私馕磁囵B(yǎng)微生物基因多樣性、群落多樣性、遺傳多樣性提供了可能性,但是在現(xiàn)有研究水平下這些技術(shù)仍有不足.如DNA-SIP技術(shù)依賴于底物的同位素標(biāo)記,組學(xué)技術(shù)容易忽略豐度低的微生物,磁性納米材料技術(shù)依賴于功能微生物在研究時(shí)間內(nèi)的大量分裂等.

為了更全面認(rèn)識(shí)未培養(yǎng)微生物的基因表達(dá)、遺傳進(jìn)化、群落結(jié)構(gòu)與功能,筆者認(rèn)為可以從以下幾方面完善非培養(yǎng)手段的不足.

3.1 多組學(xué)技術(shù)相結(jié)合.宏基因組學(xué)技術(shù)從DNA分子水平識(shí)別環(huán)境中未培養(yǎng)微生物,轉(zhuǎn)錄組學(xué)技術(shù)則從轉(zhuǎn)錄水平探究,單細(xì)胞技術(shù)更進(jìn)一步探究基因表達(dá)與功能的內(nèi)在聯(lián)系.將三者結(jié)合起來,同時(shí)進(jìn)行多組學(xué)分析則可以將遺傳信息在微生物體內(nèi)的所有參與過程整合起來,避免對(duì)微生物認(rèn)識(shí)的片面化.

3.2 高效利用最新測(cè)序技術(shù)和分析方法,加強(qiáng)數(shù)據(jù)存儲(chǔ)、分析平臺(tái)的建設(shè).近幾年來,測(cè)序成本不斷降低,讀取長度逐漸增加,高效利用最新的測(cè)序技術(shù)和分析手段會(huì)提高我們的數(shù)據(jù)精確度.同時(shí),大量的數(shù)據(jù)信息以及數(shù)據(jù)源的共享要求我們必須加強(qiáng)信息平臺(tái)的建設(shè).

3.3 對(duì)萌芽技術(shù)(MMI技術(shù))深入探究驗(yàn)證.MMI技術(shù)目前仍不夠成熟,對(duì)識(shí)別分裂能力較差的功能細(xì)胞能力有限,有待探究完善.

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致謝：感謝中國博士后科學(xué)基金(2017M620626)和中央高校基本科研業(yè)務(wù)費(fèi)(FRF-TP-16-063A1)對(duì)本研究的支持.

Identification and characterization on PAHs-degrading microorganisms via cultivation-independent approaches.

CHEN Ya-ting, JIANG Bo*, XING Yi*, ZHANG Na-na, LIAN Lu-ning, LU Pei

(Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, School of Energy and Environmental Engineering, University of Science & Technology Beijing, Beijing 100083, China)., 2018,38(9)：3562~3575

This review discussed the cultivation-independent approaches which have been developed to identify functional-yet- uncultivated soil microbesand revealed their functions in PAHs degradation. PAHs-degrading microbes, degradation mechanisms and functional genes were summarized. The principles, characteristics and applications of cultivation-independent methods, including Fluorescencehybridization (FISH), Stable-isotope probing (SIP), Metagenomics, Transcriptomics, Single-cell technology and Magnetic nanoparticle-Mediated Isolation technology (MMI) were also discussed. Examples were given on the application of these cultivation-independent approaches in identifying PAHs-degrading microbes. SIP technology is the most stable and widely used in the study of PAHs degradation mechanism. As a novel cultivation-independent approach, MMI technology provides a deep insight into exploring functional-yet-uncultivated PAHs-degrading microbes, which stands out for its ability to separate active functional microbes.

functional-yet-uncultivated microbes；PAHs biodegradation；soils；functional microorganisms；cultivation-independent approaches

X53

A

1000-6923(2018)09-3562-14

陳亞婷(1994-),女,新疆伊犁人,北京科技大學(xué)碩士研究生,主要研究污染場地的微生物修復(fù).發(fā)表論文2篇.

2018-03-09

中國博士后科學(xué)基金資助項(xiàng)目(2017M620626);中央高?；究蒲袠I(yè)務(wù)費(fèi)資助項(xiàng)目(FRF-TP-16-063A1)

* 責(zé)任作者, 姜博, 講師, jiangbo_seee@ustb.edu.cn; 邢奕, 教授, xingyi@ustb.edu.cn