申國(guó)蘭，李 利，陳 莎*

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微生物降解石油源多環(huán)芳香烴的研究進(jìn)展①

申國(guó)蘭1，李 利2，陳 莎2*

(1長(zhǎng)江大學(xué)地球科學(xué)學(xué)院，武漢 430100；2長(zhǎng)江大學(xué)生命科學(xué)學(xué)院，湖北荊州 434025)

石油源多環(huán)芳香烴是存在于石油中的一類致畸、致癌污染物，具有以低環(huán)(2 ~ 3環(huán)) 為主且取代基比例明顯高于其他來(lái)源PAHs的組分特征。石油泄露引發(fā)的PAHs污染，其降解主要依賴于微生物的活動(dòng)。本文對(duì)能夠降解PAHs的微生物種類、降解機(jī)理、代謝途徑及編碼基因進(jìn)行了概述。從PAHs作為碳源的角度將微生物降解機(jī)理劃分為能以PAHs為唯一碳源進(jìn)行生長(zhǎng)的降解機(jī)理和共代謝機(jī)理。對(duì)與PAHs有關(guān)的好氧和厭氧微生物降解途徑及對(duì)應(yīng)的編碼基因簇進(jìn)行了總結(jié)。自然界中細(xì)菌、放線菌、真菌及藻類都能夠降解PAHs，由加氧酶催化的苯環(huán)羥基化和還原酶介導(dǎo)的苯環(huán)脫芳烴化是好氧和厭氧降解途徑的關(guān)鍵步驟，與降解有關(guān)的,,,,和基因簇則分別調(diào)控好氧和厭氧降解過(guò)程。這些進(jìn)展有助于系統(tǒng)了解石油源PAHs的降解過(guò)程、微生物作用機(jī)理和分子遺傳機(jī)制，為進(jìn)一步利用微生物促進(jìn)環(huán)境生物修復(fù)提供理論依據(jù)。

石油源多環(huán)芳香烴；微生物降解；機(jī)理；代謝途徑；基因

石油是一種含有多種烴類及少量其他有機(jī)物的復(fù)雜混合物。根據(jù)烴類結(jié)構(gòu)特點(diǎn)和成分，可以將石油中的烴類物質(zhì)分為飽和烴、芳香烴、非烴和瀝青質(zhì)4種組分[1-2]。石油芳香烴物質(zhì)中以多環(huán)芳香烴(PAHs) 對(duì)環(huán)境污染威脅最大。PAHs是指兩個(gè)或兩個(gè)以上的苯環(huán)以線性、彎接或簇聚方式構(gòu)成的一類化學(xué)結(jié)構(gòu)穩(wěn)定、難于降解的烴類化合物，其中4環(huán)以上的PAHs容易被土壤或生物體富集而產(chǎn)生毒性，嚴(yán)重威脅到人類健康及生物安全[3-4]。PAHs有多種來(lái)源，不同來(lái)源的PAHs在組分特征上有所差異。石油來(lái)源的PAHs以2 ~ 3環(huán)為主，而煤炭、汽油、木材、天然氣等燃料不完全燃燒產(chǎn)生的PAHs以4 ~ 6環(huán)為主[5-6]。另外，石油來(lái)源的PAHs取代基比例明顯高于其他來(lái)源的PAHs，Buddziński等[7]發(fā)現(xiàn)石油中的單甲基菲含量明顯高于未取代菲，而燃料燃燒產(chǎn)生的PAHs中單甲基和二甲基取代物的含量遠(yuǎn)遠(yuǎn)小于未被取代的化合物。

石油來(lái)源的PAHs一旦進(jìn)入自然環(huán)境后，光照、雨水淋濾、揮發(fā)和微生物降解等環(huán)境因素會(huì)引發(fā)降解，這種降解行為類似于環(huán)境的自我凈化修復(fù)。但是微生物對(duì)石油源PAHs具有選擇性，Lamberts等[8]分離得到的29株菌株中有11株能夠利用甲基菲，并發(fā)現(xiàn)其中僅有1株鞘氨醇單胞菌能夠降解1-甲基菲和2-甲基菲，其他鞘氨醇單胞菌只能降解1-甲基菲。具有取代基的PAHs在降解開(kāi)始時(shí)取代基團(tuán)先被氧化，Nadali等[9]分析了2-甲基菲和9-甲基菲的微生物降解產(chǎn)物，發(fā)現(xiàn)2-甲基菲生成2-羥甲基菲和2-菲甲醛，推測(cè)甲基基團(tuán)的氧化是降解開(kāi)始的第一步。取代基基團(tuán)氧化后經(jīng)過(guò)脫甲基化變成單體PAHs，后續(xù)的降解過(guò)程與PAHs單體的降解途徑一致。Novakovi? 等[10]發(fā)現(xiàn)微生物修復(fù)后的土壤中單體菲/甲基菲的比例顯著升高，可能原因是微生物細(xì)胞表面的“活性中心”與甲基基團(tuán)相互作用促使甲基菲的脫甲基化。本文在前人研究的基礎(chǔ)上對(duì)能降解PAHs的微生物種類、降解機(jī)理、降解途徑及調(diào)控基因等方面展開(kāi)綜述，以期為石油來(lái)源的PAHs降解研究提供幫助。

1 降解PAHs化合物的微生物種類

微生物降解是去除環(huán)境中PAHs的最主要途徑[11]。能夠降解PAHs的微生物有細(xì)菌、放線菌、真菌和藻類[12]，其中細(xì)菌中常見(jiàn)有紅球菌 ()、假單胞菌 ()、棒桿菌 ()、微球菌 ()、產(chǎn)堿桿菌 ()、分支桿菌()以及鞘脂菌()等；放線菌常見(jiàn)的是諾卡氏菌()；PAHs降解真菌又分為木質(zhì)素降解真菌和非木質(zhì)素降解真菌，木質(zhì)素降解真菌常見(jiàn)有平革菌()、側(cè)耳()、云芝()等；非木質(zhì)素降解真菌常見(jiàn)有青霉()、曲霉()及小銀克漢霉()等。部分藻類也具有PAHs降解能力，已經(jīng)報(bào)道的有阿格門(mén)氏藻()、顫藻()、柵藻()及月牙藻()等(表1)。

表1 降解PAHs的微生物菌株及其底物

2 能夠以PAHs作為唯一碳源的微生物降解機(jī)理

2.1 細(xì)菌的降解機(jī)理

2.1.1 好氧細(xì)菌 細(xì)菌降解芳香烴化合物根據(jù)降解環(huán)境的氧氣含量可以分為有氧降解和無(wú)氧降解。在雙加氧酶作用下PAHs的羥基化是有氧降解的主要途徑。鄰位或間位引入羥基形成反式–二氫-二羥基化合物，提高芳香環(huán)活性，然后繼續(xù)氧化直至芳香環(huán)破裂生成不飽和的直鏈脂肪酸[58]，后續(xù)的降解進(jìn)入PAHs中心降解途徑與三羧酸循環(huán)中間產(chǎn)物相連[59]。以菲為例，菲是石油污染后在環(huán)境中存在量最大的PAHs之一。細(xì)菌有氧降解菲存在多種不同的開(kāi)環(huán)方式，根據(jù)加氧酶作用位點(diǎn)不同，菲一般在1,2位、3,4位和9,10位開(kāi)環(huán)(圖1)[31, 60-61]。細(xì)菌中不同的菌株對(duì)應(yīng)有一條或多條代謝途徑。節(jié)桿菌sp. P1-1和分支桿菌JS19b1T有3條降解菲的途徑，分別從1,2位、3,4位和9,10位開(kāi)環(huán)[25, 61]。伯克氏菌sp.C3降解菲有兩條途徑，分別從1,2位和3,4位開(kāi)環(huán)[60]。分支桿菌sp. Strains PYR-1、馬特爾氏菌sp. AD-3 降解菲也有兩條途徑，但是分別從3,4位和9,10位開(kāi)環(huán)[62-63]。假單胞菌NCIB 9816、鞘氨醇單胞菌sp. PheB4中菲僅有3,4位開(kāi)環(huán)一條途徑[64-65]。菲1,2位和3,4位環(huán)裂解有著共同的中間產(chǎn)物1,2-二羥萘，該產(chǎn)物對(duì)應(yīng)有兩條降解途徑。對(duì)于能夠利用萘的細(xì)菌，1,2-二羥萘轉(zhuǎn)變成水楊酸后經(jīng)過(guò)龍膽酸途徑降解，而不能利用萘的細(xì)菌，1,2-二羥萘通過(guò)原兒茶酸途徑降解(圖1)。菲也可以在9,10位形成二羥基，繼而生成2,2’-聯(lián)苯二酸。

圖1 菲好氧降解的可能途徑[31, 60-61]

2.1.2 厭氧細(xì)菌 厭氧細(xì)菌降解PAHs的起始步驟主要涉及羧基化和甲基化(圖2)。以萘為例，萘在厭氧環(huán)境中有兩條降解途徑，一條通過(guò)甲基化在2號(hào)位加上甲基，形成2-甲基萘；另一條通過(guò)羧基化也在2號(hào)位加上羧基生成2-萘甲酸。甲基化和羧基化產(chǎn)物進(jìn)一步降解需要輔酶的參與 (琥珀酰輔酶A或輔酶A)，再經(jīng)過(guò)還原酶作用生成烯酰輔酶A，烯酰輔酶A在水合酶作用下與輔酶A相連的芳香環(huán)被打開(kāi)，后者經(jīng)過(guò)類似于β-氧化的步驟進(jìn)一步完全降解為乙酰輔酶A和CO2，具體的降解過(guò)程見(jiàn)圖2[66]。研究發(fā)現(xiàn)PAHs的完全降解過(guò)程中，多種厭氧菌參與其中發(fā)揮著不同的作用。TSAI 等[67]發(fā)現(xiàn)硫酸鹽降解菌會(huì)將芴和菲厭氧降解成共同的中間產(chǎn)物苯酚。Fang 等[68]發(fā)現(xiàn)脫硫腸狀菌屬()和梭菌屬()在厭氧條件下將苯酚轉(zhuǎn)化為苯甲酸鹽，互養(yǎng)菌()能夠進(jìn)一步將苯甲酸鹽降解為乙酸鹽、H2和CO2，產(chǎn)甲烷菌()最后將乙酸鹽、H2和CO2轉(zhuǎn)化為甲烷。

2.2 真菌降解PAHs機(jī)理

2.2.1 木質(zhì)素降解真菌 自然界中有一類能夠分泌木質(zhì)素降解酶系(木質(zhì)素過(guò)氧化物酶、錳過(guò)氧化物酶和漆酶)的真菌，這些分泌到細(xì)胞外的非特異性酶作用底物范圍廣，能夠降解包括PAHs在內(nèi)的多種有機(jī)污染物，是真菌降解PAHs的獨(dú)特機(jī)制[69]。有機(jī)物的存在能夠誘導(dǎo)激活過(guò)氧化物酶和漆酶從而降解PAHs，例如在白腐真菌中添加草酸、丙二酸發(fā)現(xiàn)木質(zhì)素過(guò)氧化物酶的含量升高，含錳的有機(jī)物能夠刺激錳過(guò)氧化物酶活性提升。木質(zhì)素降解酶能夠在PAHs特定位點(diǎn)引入羥基。糙皮側(cè)耳()降解菲是從9,10位點(diǎn)形成二氫二醇，然后生成2,2’-聯(lián)苯二酸，最終降解為CO2，這個(gè)過(guò)程與好氧細(xì)菌降解菲中的9,10位點(diǎn)裂解途徑非常相似[70-71]。

圖2 萘的厭氧降解過(guò)程[66]

2.2.2 非木質(zhì)素降解真菌 有些真菌除了分泌過(guò)氧化物酶系和漆酶外，還可以產(chǎn)生類似細(xì)胞色素P450單加氧酶的酶系降解PAHs。PAHs在細(xì)胞色素P450單加氧酶的作用下首先形成不穩(wěn)定的芳烴氧化產(chǎn)物，然后在環(huán)氧化物酶作用下轉(zhuǎn)變成為反式-二氫二醇或者酚類物質(zhì)，繼續(xù)轉(zhuǎn)化為-葡萄糖苷、-葡萄糖醛酸苷、-硫酸酯、-木糖苷及-甲基進(jìn)一步分解(圖3)[72]。細(xì)胞色素P450單加氧酶對(duì)于不同PAHs的起始作用位點(diǎn)各異。糙皮側(cè)耳()中細(xì)胞色素P450單加氧酶的酶系降解芘和蒽分別生成反式-4,5-芘二醇和反式1,2-蒽二醇、9,10-蒽二醇，但催化芴則在脂肪橋上羥基化和酮基化，生成9-芴醇和9-芴酮[73]。

圖3 非木質(zhì)素降解真菌降解苯并芘的可能途徑[72]

2.3 放線菌降解PAHs的機(jī)理

放線菌降解PAHs的機(jī)理與好氧細(xì)菌相似。以苯并芘為例，苯并芘有多種起始羥基化位點(diǎn)(圖4)。Schneider 等[28]分離得到了4,5-屈二羧酸，推測(cè)分支桿菌(sp. strain RJGⅡ-135)中雙加氧酶作用于苯并芘的4,5- 位點(diǎn)。分支桿菌(PYR-1)降解苯并芘最初從4,5-, 9,10-, 11,12- 位點(diǎn)開(kāi)始羥基化[30]。PYR-1在苯并芘11,12羥基化生成順式和反式-11,12-二氫-11,12-二羥基苯并芘，推測(cè)PYR-1可能同時(shí)存在雙加氧酶和單加氧酶[30]。

2.4 藻類降解PAHs的機(jī)理

藻類降解PAHs的起始步驟也涉及羥基化。Cerniglia等[54]將阿格門(mén)氏藻()接入C14標(biāo)記的含萘培養(yǎng)基上，發(fā)現(xiàn)可以將萘轉(zhuǎn)化為1-萘酚，而且檢測(cè)到1,2-二羥基-1,2-二氫萘的存在，說(shuō)明萘的降解涉及羥基化。Safonova等[56]研究柵列藻()降解菲的代謝產(chǎn)物時(shí)也檢測(cè)到了1,2-二羥基- 1,2-二氫菲。Chan[57]認(rèn)為藻降解PAHs有單加氧酶和雙加氧酶的參與，該研究利用月牙藻()降解含菲、熒蒽及芘的混合物，發(fā)現(xiàn)4 d內(nèi)該藻可以降解96% 菲、100% 熒蒽和100% 芘。分析降解產(chǎn)物發(fā)現(xiàn)了4種不同的單羥基菲和3種羥基化的熒蒽和芘產(chǎn)物，分析單羥基產(chǎn)物的出現(xiàn)由單加氧酶途徑產(chǎn)生。同時(shí)產(chǎn)物中也檢測(cè)到了2種二羥基菲，推測(cè)雙加氧酶同時(shí)參與了降解過(guò)程。

圖4 放線菌降解苯并芘的可能途徑[30]

3 不能以PAHs為唯一碳源的(共代謝)降解機(jī)理

共代謝現(xiàn)象最早是Leadbetter和Foster[74]在甲烷假單胞菌()中發(fā)現(xiàn)的，該菌不能利用乙烷、丙烷和丁烷作為碳源生長(zhǎng)，但是添加外加碳源甲烷后該菌能夠氧化上述3種碳源。Leadbetter和Foster將此現(xiàn)象稱之為共氧化(Co-oxidation)，認(rèn)為在生長(zhǎng)基質(zhì)(甲烷)存在的情況下，在微生物的作用下非生長(zhǎng)基質(zhì)(乙烷、丙烷和丁烷) 發(fā)生氧化。隨后，Jesnsen[75]提出用共代謝(Co-metabolism)的概念來(lái)替代共氧化，認(rèn)為在生長(zhǎng)基質(zhì)存在時(shí)，微生物對(duì)非生長(zhǎng)基質(zhì)的轉(zhuǎn)化不僅有氧化，還有還原作用，都應(yīng)該屬于代謝的范疇?，F(xiàn)在PAHs共代謝是指在外加碳源情況下，難生物降解的PAHs有可能被微生物轉(zhuǎn)化甚至完全降解[76]。Gibson 等[77]發(fā)現(xiàn)盡管strain B-836不能利用苯并芘作為碳源生長(zhǎng)，但是有琥珀酸和聯(lián)苯的存在下，能夠?qū)⒈讲④叛趸啥涠蓟衔?。另外，有研究?bào)道某些真菌能夠利用PAHs作為生長(zhǎng)碳源，但是添加某些有機(jī)物后PAHs降解效率顯著提高，這些研究把它歸結(jié)為共代謝[78]。微生物以共代謝方式降解PAHs可能有以下幾種原因：①缺少進(jìn)一步降解的酶系。當(dāng)某種易降解物加入后，微生物在代謝易降解物過(guò)程中誘導(dǎo)產(chǎn)生某種專一性較差的酶，這種酶的作用導(dǎo)致了PAHs的降解。缺少這類酶時(shí)，降解反應(yīng)無(wú)法繼續(xù)進(jìn)行。②由于中間產(chǎn)物的抑制。③需要另外的基質(zhì)誘導(dǎo)代謝酶或提供細(xì)胞反應(yīng)中不能充分供應(yīng)的物質(zhì)[79]。有研究認(rèn)為土壤中微生物代謝產(chǎn)生的多酚氧化酶參與了共代謝降解PAHs的過(guò)程，劉世亮等[80]發(fā)現(xiàn)，當(dāng)苯并芘加入土壤7 d后，加有共代謝底物的組分(水楊酸、鄰苯二甲酸、琥珀酸鈉)中多酚氧化酶活性明顯高于對(duì)照組，到第35天，加有水楊酸和琥珀酸鈉的處理組多酚氧化酶活性明顯高于其他2個(gè)處理，與土壤中苯并芘的降解率相一致。

4 微生物降解PAHs 的途徑及其調(diào)控基因

PAHs的微生物降解是復(fù)雜的降解過(guò)程，好氧細(xì)菌及真菌分解依靠加氧酶等一系列酶催化PAHs生成一些關(guān)鍵中間代謝物(原兒茶酸、水楊酸、龍膽酸、鄰苯二酚)[81]。厭氧細(xì)菌則借助硫酸鹽、硝酸鹽、甲烷等電子受體將PAHs逐步降解為苯甲酸鹽。這些中間產(chǎn)物再通過(guò)相應(yīng)的降解途徑徹底分解。目前已知的中間產(chǎn)物主要有鄰苯二酚、3,4-二羥基苯甲酸、龍膽酸(1,5-二羥基苯甲酸)、1,2,4-苯三酚、6-氯-1,2,4-苯三酚、對(duì)苯二酚、氯代對(duì)苯二酚、苯甲酰輔酶A等，這些物質(zhì)主要通過(guò)β-酮己二酸途徑、苯乙酸途徑和龍膽酸途徑以及苯甲酰輔酶A途徑等進(jìn)行降解[82]。

4.1 β-酮己二酸途徑及其調(diào)控基因

β-酮己二酸(ketoadipate)途徑 (鄰位裂解途徑)是芳香烴降解的一條重要途徑，好氧細(xì)菌和真菌中都具有這條降解途徑。該途徑有鄰苯二酚和原兒茶酸(3,4-二羥基苯甲酸) 兩個(gè)中間產(chǎn)物，對(duì)應(yīng)著兩條并行的降解支路，兩條支路分別通過(guò)鄰苯二酚1,2-雙加氧酶和原兒茶酸3,4-雙加氧酶在鄰位羥基位點(diǎn)將芳香環(huán)打開(kāi)，然后經(jīng)過(guò)異構(gòu)、脫羧形成共同的代謝中間產(chǎn)物β-酮己二酸烯醇內(nèi)酯，再經(jīng)過(guò)水解、輔酶A轉(zhuǎn)移、硫解等步驟生成了乙酰輔酶A和琥珀酰輔酶A。β-酮己二酸途徑主要是受和基因簇調(diào)控，其中基因簇(調(diào)控原兒茶酸支路)存在于考克氏菌屬(spp.)、不動(dòng)桿菌屬(spp.)、棒桿菌屬(spp.)、鏈霉屬(spp.)、紅球菌屬(spp.)及假單胞菌屬(spp.)的一些菌株[83-87]?；虼?cái)?shù)量不同種屬的細(xì)菌中有所區(qū)別，即使相同屬的細(xì)菌基因簇?cái)?shù)量也有不同?？伎耸暇鶧C2201、不動(dòng)桿菌sp. ADP1、新月柄桿菌只有一個(gè)單獨(dú)的基因簇，而sp. strain RHA1存在兩個(gè)基因簇，分別由兩個(gè)不同的操縱子調(diào)控(JI和H-GBLRF)，KT2440中存在3個(gè)基因簇(RKFTBDCP、IJ和GH)[83,87-91]。多個(gè)基因簇有可能分布在一個(gè)染色體上，也有可能分布于不同染色體，和中多個(gè)基因簇分布在兩條不同染色體，而中基因簇則分布在一條染色體上[87]?；?(調(diào)控鄰苯二酚支路) 多集中在一個(gè)基因簇上[90]，但、、中有多個(gè)基因簇，且分布在不同染色體上[87]。基因簇中是轉(zhuǎn)錄調(diào)控子，調(diào)控相鄰基因的轉(zhuǎn)錄表達(dá)。節(jié)細(xì)菌屬中是LysR型轉(zhuǎn)錄調(diào)控子，通過(guò)-粘康酸誘導(dǎo)激活相鄰基因的轉(zhuǎn)錄，而紅球菌中是IclR型調(diào)控子，控制原兒茶酸的代謝調(diào)控[92-94]。

4.2 苯乙酸途徑(PAA) 及其調(diào)控基因

作為該途徑的中間代謝產(chǎn)物苯乙酸沒(méi)有采取脂肪烴降解方式降解為苯甲酸進(jìn)入β-酮己二酸途徑，而是先連接上輔酶A，形成苯乙酰輔酶A，然后在芳香環(huán)2,3位上引入羥基形成順式-二氫二醇，再經(jīng)過(guò)環(huán)裂解、水合、氧化硫酯、脫氫步驟分解為乙酰輔酶A和琥珀酰輔酶A，進(jìn)入TCA循環(huán)。苯乙酸途徑受基因簇調(diào)控?；虼氐臄?shù)量在不同種屬中有所不同，紅球菌PR4和假單胞菌KT 2440/U中有兩個(gè)基因簇，而鏈霉菌A3中基因簇?cái)?shù)量超過(guò)3個(gè)[82]。RHA1、PR4及KT 2440/U菌株中基因簇中均含有兩個(gè)連續(xù)的核心功能區(qū)域：GHIJK (編碼芳香環(huán)羥基化) 和( 編碼β-氧化)。多個(gè)基因簇在染色體上的位置不一定連續(xù)。PR4的兩個(gè)基因簇為連續(xù)分布，而RHA1的基因簇則存在2.6 kb的間隔，A3也有類似情況出現(xiàn)[82]。

4.3 龍膽酸途徑(GEN)及其調(diào)控基因

sp. Strain U2菌株中萘被降解為水楊酸后沒(méi)有轉(zhuǎn)化為兒茶酚為進(jìn)入β-酮己二酸途徑，而是繼續(xù)被氧化為龍膽酸，在龍膽酸1,2-雙加氧酶催化開(kāi)環(huán)，通過(guò)后續(xù)代謝進(jìn)入TCA循環(huán)，這條途徑被稱為龍膽酸途徑[95]。許多PAHs在分解過(guò)程中產(chǎn)生的萘、水楊酸、3-羥基苯甲酸和鄰氨基苯甲酸等產(chǎn)物都可以通過(guò)龍膽酸途徑轉(zhuǎn)變?yōu)楸岷脱雍魉?，進(jìn)入TCA循環(huán)[96]。該途徑受基因簇的調(diào)控，假單胞菌G7的NAH7質(zhì)粒中基因簇上游操縱子(AaAbAcAdBFCQED)負(fù)責(zé)編碼由萘轉(zhuǎn)為水楊酸的酶系，下游操縱子(GTHINLOMKJXY) 負(fù)責(zé)編碼水楊酸轉(zhuǎn)變?yōu)楸岷鸵胰?，操縱子R處于上、下游操縱子之間，是調(diào)節(jié)基因，調(diào)節(jié)上、下操縱子的表達(dá)，水楊酸可以誘導(dǎo)激活R，導(dǎo)致基因簇的高效表達(dá)[97]。假單胞菌屬的不同菌中均存在下游操縱子的部分序列(THINLOMKJ)[98]。

另外，在不同種屬的菌株中還發(fā)現(xiàn)一些基因與. putida G7的NAH7質(zhì)粒中基因簇非常相似，且高度保守，因此通常被稱為“經(jīng)典的基因”。這些編碼降解PAHs關(guān)鍵酶的基因有的位于質(zhì)粒上，有的位于染色體上，如.NCIB9816質(zhì)粒中基因簇ABC，sp.strain C18菌株中的基因簇ABDEFGHIJ，OUS82染色體中的基因簇AaAbAcAdBFCQED和PaK1菌株中的基因簇A1-A2A3A4BFCQED以及AN10菌株中的基因簇AaAbAcAdBFCED與G7的NAH7質(zhì)粒中基因簇非常相似[99-103]。

4.4 苯甲酰輔酶A降解途徑及其調(diào)控基因

Tsai等[68]發(fā)現(xiàn)硫酸鹽降解菌會(huì)將芴和菲厭氧降解成共同的中間產(chǎn)物苯酚。Fang等[68]發(fā)現(xiàn)脫硫腸狀菌屬()和梭菌屬()在厭氧條件下將苯酚轉(zhuǎn)化為苯甲酸鹽。PAHs的厭氧降解又需要輔酶的參與，因此推測(cè)苯甲酰輔酶A 是PAHs厭氧降解的中間產(chǎn)物。苯甲酰輔酶A的完全降解又分為上游降解途徑和下游降解途徑(圖5)。上游降解途徑是指從苯甲酰輔酶A經(jīng)過(guò)一系列酶促反應(yīng)催化降解為7-羧基-庚酰輔酶A的過(guò)程，整個(gè)上游降解途徑可分為兩個(gè)重要的降解步驟：一是脫芳烴化，脫芳烴化是指苯甲酰輔酶A在ATP和H供體的存在下，被苯甲酰輔酶A還原酶(BCR)催化下生成1,5環(huán)己二烯酰輔酶A。二是在1,5環(huán)己二烯酰輔酶A水合酶、脫氫酶和水解酶的作用下生成7-羧基-庚酰輔酶A或3-羥基-7羧基-庚酰輔酶A，這個(gè)過(guò)程類似β-氧化過(guò)程(圖5中的a、b)[104]。苯酰輔酶A降解途徑涉及到眾多降解基因或基因簇，其中兼性厭氧菌編碼苯環(huán)脫芳烴化的苯甲酰輔酶A還原酶(BCR)在陶厄氏菌 ()中已經(jīng)研究得比較清楚，該BCR酶由αβγδ四聚體組成，分別由基因編碼。BCR酶有兩個(gè)功能不同的結(jié)構(gòu)域：由編碼的αδ亞基上有兩個(gè)ATP結(jié)合位點(diǎn)和鐵硫聚合物的電子結(jié)合位點(diǎn)；βγ亞基由編碼，起到結(jié)合一個(gè)苯甲酰輔酶A和協(xié)調(diào)鐵硫聚合物的作用[105-107]。磁螺菌屬(spp.)的不同菌株中編碼BCR酶的基因也有與陶厄氏菌 ()相似的基因簇[108-109]。紅假單胞菌()中分離得到的BCR也由αβγδ四聚體組成，由基因編碼，但基因編碼的產(chǎn)物氨基酸序列與基因只有64% ~ 76% 的相似性[109-110]。固氮弧菌屬() 中BCR四聚體由基因編碼與和基因產(chǎn)物僅有22% ~ 43% 的相似性[111]。專性厭氧菌有著與兼性厭氧菌不同的脫芳烴化酶系，研究已經(jīng)證實(shí)專性厭氧菌中互養(yǎng)菌()和地桿菌()缺乏兼性厭氧菌中典型的BCR結(jié)構(gòu)[112-113]。地桿菌()中基因簇編碼苯甲酰輔酶A脫芳烴化的酶系，互養(yǎng)菌()中編碼苯甲酰輔酶A脫芳烴化的酶系也由基因簇控制，兩個(gè)基因簇有高度相似性(氨基酸水平＞50% 相似性)[113]。

圖5 苯和苯乙酰輔酶A的厭氧降解途徑[104]

苯甲酰輔酶A下游降解途徑是指從7-羧基-庚酰輔酶A或3-羥基-7羧基-庚酰輔酶A開(kāi)始經(jīng)過(guò)一系列酶促反應(yīng)最終降解為乙酰輔酶A和CO2的過(guò)程。固氮弧菌屬()、地桿菌()、陶厄氏菌 ()、磁螺菌屬(spp.)、互養(yǎng)菌() 中苯甲酰輔酶A經(jīng)過(guò)酶促反應(yīng)苯環(huán)開(kāi)鏈生成3-羥基-7羧基-庚酰輔酶A，而紅假單胞菌()的產(chǎn)物為7-羧基-庚酰輔酶A，在脫氫酶和水合酶的作用下，7-羧基-庚酰輔酶A羥基化生成3-羥基-7羧基-庚酰輔酶A。在有NAD+存在下，3-羥基-7羧基-庚酰輔酶A被還原生成3-羰基-7羧基-庚酰輔酶A，然后在CoA參與下脫去1分子乙酰輔酶A生成5-羧基-戊二酰輔酶A，在戊二酰輔酶A脫氫酶作用下脫去2H+和1 CO2生成巴豆酰輔酶A(丁烯酰輔酶A)，在3-羥基丁酰輔酶A脫氫酶和1 H2O催化下生成3-羥基丁酰輔酶A，在脫氫酶的作用下最終降解生成2分子乙酰輔酶A[104]。

5 展望

伴隨著經(jīng)濟(jì)全球化的進(jìn)程，石油及其產(chǎn)品已經(jīng)遍及全球各個(gè)角落。石油及其產(chǎn)品的開(kāi)采、煉制、儲(chǔ)運(yùn)和使用都可能會(huì)產(chǎn)生PAHs。PAHs具有高度穩(wěn)定性、耐降解性和環(huán)境毒性，給生態(tài)環(huán)境及人類生活帶來(lái)極大的威脅。利用微生物降解因石油泄露殘留在環(huán)境中的PAHs是綠色、安全、低耗能的辦法，已經(jīng)成為了世界性的研究課題，相關(guān)研究已經(jīng)在不同微生物中PAHs的降解途徑、功能酶系、編碼基因及信號(hào)調(diào)控方面展開(kāi)，其中單環(huán)芳香烴的開(kāi)環(huán)、好氧降解途徑以及相關(guān)的編碼基因已經(jīng)研究得比較清楚，低分子量(≤3環(huán))的PAHs好氧降解機(jī)理、代謝途徑以及編碼基因也逐漸明了，高分子量(≥4環(huán))PAHs的微生物降解盡管已成為當(dāng)前研究熱點(diǎn)，但相關(guān)降解途徑及編碼基因還不甚清楚。另外，PAHs的厭氧降解途徑的了解還十分有限，厭氧降解途徑的相關(guān)基因以及調(diào)控機(jī)理已經(jīng)成為目前的研究熱點(diǎn)。與細(xì)菌相比，真菌特別是非木質(zhì)素降解真菌對(duì)PAHs降解的機(jī)理目前也不清楚，這方面的研究也逐漸開(kāi)始成為未來(lái)PAHs降解的研究方向之一。

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Microbial Degradation of Polycyclic Aromatic Hydrocarbons from Crude Oils: A Review

SHEN Guolan1, LI Li2, CHEN Suo2*

(1 College of Geosciences, Yangtze University, Wuhan 430100, China; 2 College of Life Science, Yangtze University, Jingzhou, Hubei 434025, China)

Polycyclic aromatic hydrocarbons (PAHs) from crude oils is a kind of teratogenic and carcinogenic contaminant, in which the low aromatic nucleus ring (2–3 ring) are the dominated components and the substituent group ratio is significantly higher than those from other origins. The degradation of PAHs caused by oils leakage are mainly dependent on microbial activities. This paper summarizes the microbial species, degradation mechanisms, metabolic pathways and coding genes with relation to PAHs biodegradation. The degradation mechanisms are divided into co-metabolism mechanism and the mechanism in which PAHs could be acted as the only carbon source of microbial population from the perspective of carbon source. The degradation pathways of aerobic and anaerobic microorganisms associated with PAHs and corresponding encoding gene clusters are also elaborated in this paper. In natural environment, bacteria, actinomycetes, fungi and algae can degrade PAHs. The hydroxylation and dearomatization of benzene respectively catalyzed by oxygenases and reductases are the key steps in aerobic and anaerobic degradation pathways. Moreover,,,,,andgene clusters associated with degradation regulate the aerobic and anaerobic degradation process respectively. These advances can contribute to systematically understand the PAHs degradation process, the mechanism of microbial action and molecular genetic mechanisms, and thus can provide a theoretical basis for further utilization of microorganisms in environmental bioremediation.

Polycyclic aromatic hydrocarbons from crude oils; Microbial degradation; Mechanism; Degradation pathways; Genes

國(guó)家自然科學(xué)基金項(xiàng)目(31501453)資助。

(chensuo9803@126.com)

申國(guó)蘭 (1983— )，女，河北石家莊人，碩士研究生，主要從事地質(zhì)環(huán)境生態(tài)學(xué)方面研究。E-mail: 270968223@qq.com

10.13758/j.cnki.tr.2018.01.003

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