韓永和，何睿文，李超，向萍，羅軍，崔昕毅

南京大學(xué)環(huán)境學(xué)院 污染控制與資源化研究國家重點(diǎn)實(shí)驗(yàn)室，南京210046

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鄰苯二甲酸酯降解細(xì)菌的多樣性、降解機(jī)理及環(huán)境應(yīng)用

韓永和，何睿文，李超，向萍，羅軍，崔昕毅*

南京大學(xué)環(huán)境學(xué)院 污染控制與資源化研究國家重點(diǎn)實(shí)驗(yàn)室，南京210046

鄰苯二甲酸酯(phthalic acid esters, PAEs)是一類對(duì)人體內(nèi)分泌系統(tǒng)有干擾作用的持續(xù)性有機(jī)污染物(persistent organic pollutants, POPs)。PAEs在環(huán)境介質(zhì)如水體、底泥和土壤中長(zhǎng)期賦存會(huì)對(duì)生物體產(chǎn)生毒害效應(yīng)，其分布廣、濃度高和難降解等特點(diǎn)是限制有效環(huán)境治理的主要因素。作為環(huán)境的重要組成部分，微生物對(duì)污染物有很強(qiáng)的適應(yīng)能力和高效的降解能力，這為PAEs的生物修復(fù)提供了可能。與物理化學(xué)修復(fù)法相比，微生物修復(fù)技術(shù)具有可控性強(qiáng)、修復(fù)面廣和靈活性高等優(yōu)勢(shì)。本文綜述了已報(bào)道的大部分PAEs降解細(xì)菌的種類及其代謝機(jī)制，并分析了其在PAEs污染水體和土壤修復(fù)中的應(yīng)用現(xiàn)狀與前景，以期為PAEs環(huán)境行為與生物修復(fù)研究提供參考。

內(nèi)分泌干擾物；持久性有機(jī)污染物；鄰苯二甲酸酯；微生物；生物降解；健康風(fēng)險(xiǎn)

Received 7 November 2015 accepted 22 December 2015

鄰苯二甲酸酯(phthalic acid esters, PAEs)是一類以人工合成為主要來源的環(huán)境內(nèi)分泌干擾物，因其在塑料制品、醫(yī)療用品、家電和玩具等材料中的廣泛使用，可在土壤、水體和大氣中積累并直接或間接地影響人類健康[1-2]。作為最常見的單體塑化劑，環(huán)境中PAEs主要包括鄰苯二甲酸二(2-乙基)己酯(di-(2-ethylhexyl) phthalate, DEHP)、鄰苯二甲酸一丁酯(monobutyl phthalate, MBP)、鄰苯二甲酸二丁酯(dibutyl phthalate, DBP)、鄰苯二甲酸丁芐酯(butyl benzyle phthalate, BBP)和鄰苯二甲酸二乙酯(diethyl phthlate, DEP)等。國際癌癥研究機(jī)構(gòu)(IARC)統(tǒng)計(jì)表明，塑料成品中PAEs含量通常為10%～60%[3]。其中，小分子量PAEs(DBP、鄰苯二甲酸二甲酯(dimethyl phthlate, DMP)和DEP)主要添加至化妝品和個(gè)人護(hù)理產(chǎn)品中，起到香水保香和抑揮發(fā)及指甲油的抑脆裂作用，而大分子PAEs如BBP和DEHP是工業(yè)塑化制品如聚氯乙烯(polyvinylchloride, PVC)的主要原材料[1, 3]。在我國，90%以上PAEs被用于PVC的生產(chǎn)[2]。PAEs在合成材料中往往以非化學(xué)共價(jià)鍵的形式存在，可通過風(fēng)化作用等向環(huán)境釋放[1]。目前PAEs在市政固體廢棄物，室內(nèi)降塵，底泥和廢水及垃圾滲濾液中被頻繁檢出；同時(shí)，PAEs在土壤微生物、植物和動(dòng)物體內(nèi)的富集及生物鏈傳遞引發(fā)的人體健康風(fēng)險(xiǎn)也備受關(guān)注[1]。

我國塑料制品使用泛濫，由此引發(fā)的PAEs污染問題日趨嚴(yán)重[2, 4]。在農(nóng)業(yè)活動(dòng)中，PAEs污染問題尤為突出。He等[2]認(rèn)為，農(nóng)業(yè)中塑料薄膜的使用、市政固體廢物的排放、農(nóng)藥的使用和廢水灌溉是我國農(nóng)田PAEs污染的主要來源。對(duì)山東平原某蔬菜大棚PAEs污染的調(diào)查表明，37個(gè)采樣點(diǎn)共檢出16種PAEs，濃度均值為1.939～35.442 mg·kg-1，其中DBP、DMP、DEP和DEHP較高[4]。PAEs對(duì)農(nóng)田生態(tài)系統(tǒng)的危害不僅體現(xiàn)在對(duì)土壤微生物和酶活性的抑制作用，還體現(xiàn)在對(duì)農(nóng)作物產(chǎn)量和質(zhì)量方面的影響[5]。

環(huán)境中PAEs的治理技術(shù)通常包含3類：物理-化學(xué)治理、生物治理和氧化降解[1-2]。其中，物理-化學(xué)治理的吸附法和絮凝-聚沉法、生物治理的微生物降解和植物修復(fù)以及光/氧化降解等工藝效果顯著[1-2]。然而，物理-化學(xué)或光/氧化工藝無法在PAEs污染土壤修復(fù)中大規(guī)模應(yīng)用，還有可能導(dǎo)致二次污染。環(huán)境中PAEs的降解與分布在很大程度上取決于微生物的群落與功能差異，理解微生物介導(dǎo)的PAEs代謝機(jī)制可為PAEs環(huán)境歸趨研究與PAEs修復(fù)提供理論基礎(chǔ)[5]。本文以環(huán)境中最常見的PAEs(包括DBP、MBP、BBP、DEP和DEHP)為研究對(duì)象，綜述了目前國內(nèi)外報(bào)道的PAEs降解菌及其多樣性、PAEs降解機(jī)理及PAEs降解菌的環(huán)境應(yīng)用，擬從微生物學(xué)、分子生物學(xué)、生態(tài)學(xué)和環(huán)境化學(xué)等角度闡述PAEs降解菌在環(huán)境中的特性及其意義。

1 PAEs降解菌多樣性

微生物是環(huán)境的重要組成部分，其在地球上的出現(xiàn)可追溯至早太古代(～3.2 Ga)的河口沉積物中。極強(qiáng)的生命力、多樣性和適應(yīng)能力造就了環(huán)境中豐富多彩的微生物世界。PAEs作為一種人工合成塑化劑其在環(huán)境中出現(xiàn)的年代并不久遠(yuǎn)，但微生物很快對(duì)PAEs產(chǎn)生了適應(yīng)能力，主要體現(xiàn)在對(duì)多種PAEs的有效降解[5]。環(huán)境中PAEs難以通過水解或光降解去除，研究認(rèn)為微生物才是PAEs降解的主要參與者[1]。根據(jù)微生物分類學(xué)，PAEs降解菌以好氧細(xì)菌和部分真菌為主，具備厭氧降解功能的細(xì)菌較少[6]。另一方面，目前對(duì)PAEs降解菌群落結(jié)構(gòu)與功能的認(rèn)識(shí)還非常有限，關(guān)注PAEs降解的微生物群落效應(yīng)是今后研究的重點(diǎn)內(nèi)容。

2008年以前發(fā)現(xiàn)的PAEs降解細(xì)菌有節(jié)細(xì)菌Arthrobacter keyseri 12B(舊名為Micrococcus sp.)，假單胞菌Pseudomonas fluorescens、P. putida、P. acidovorans、P. testosterone和P. pseodoalcaligenes，以及紅球菌Rhodococcus rubropertinctus等[6]。雖然PAEs降解菌報(bào)道廣泛，對(duì)其進(jìn)行詳細(xì)的遺傳學(xué)分類較少。Liang等[6]統(tǒng)計(jì)表明，PAEs降解細(xì)菌主要分布在α、β和γ變形桿菌門(Proteobacteria)，厚壁菌門(Firmicutes，低G+C含量)，放線菌門(Actinobacteria，高G+C含量)，擬桿菌門(Bacteroidetes)和綠菌門(Chlorobi)等5個(gè)門類的29個(gè)屬。奇異球菌-棲熱菌門(Deinococcus-Thermus)的2個(gè)菌株在2010年[7]和2014年[8]首次得到報(bào)道。目前PAEs降解細(xì)菌已擴(kuò)充到了36個(gè)屬(表1和圖1A)，有超過80個(gè)PAEs降解菌株得到了詳細(xì)的研究與報(bào)道。

進(jìn)化分析顯示，具備PAEs降解能力的細(xì)菌集中在γ-Proteobacteria和Acinobacteria，其次是α和β-Proteobacteria，F(xiàn)irmicutes最少(圖1A)。其中，Sphingomonas、Comamonas、Pseudomonas、Arthobacter和Rodococcus是PAEs降解菌的主要屬[5]。這些微生物多數(shù)能以PAEs為唯一碳源和能源物質(zhì)進(jìn)行生長(zhǎng)繁殖，且有近一半菌株具備降解多種PAEs的能力(表1)。例如，Wu等[9]研究表明，農(nóng)桿菌Agrobacterium sp. JDC-49可同時(shí)降解DBP、DEP、DMP、鄰苯二甲酸二正辛酯(di-n-octyl phthalate, DOP)、鄰苯二甲酸二異壬酯(di-isononyl phthalate, DINP)和鄰苯二甲酸(phthalic acid, PA)等7種PAEs。類似的多功能菌株還包括Diaphorobacter sp. QH-6[10]，Rhodococcus sp. JDC-11[11]和鞘氨醇單胞菌Sphingobium sp. SM42[12]等。

表1 部分已報(bào)道的PAEs降解細(xì)菌及其相關(guān)信息

注：DEHP=鄰苯二甲酸二(2-乙基己)酯，DBP=鄰苯二甲酸二丁酯，MBuP=鄰苯二甲酸單丁酯，PA=鄰苯二甲酸，DEP=鄰苯二甲酸二乙酯，DMP=鄰苯二甲酸二甲酯，MMP=單甲基鄰苯二甲酸，MEHP=鄰苯二甲酸單-2-乙基己酯，BBP=鄰苯二甲酸丁芐酯，DOP=鄰苯二甲酸二辛酯，DiOP=鄰苯二甲酸二異辛酯，PCA=原兒茶酸，MBeP=鄰苯二甲酸單芐酯，DPRP=鄰苯二甲酸丙酯，DPEP=脫氧葉紅初卟啉，DiBP=鄰苯二甲酸二異丁酯，DPP=鄰苯二甲酸二丙酯，MEHP=鄰苯二甲酸單(2-乙基己基)酯，BA=苯甲酸，DEHA=己二酸二(2-乙基己)酯，MEHA=單乙基己基酯，EHA=乙基己醇，EHAA=乙基己酸，DMIP=間苯二甲酸二甲酯，DMTP=對(duì)苯二甲酸二甲酯，TA=對(duì)苯二甲酸。a未提及，bDOP：DEHP的別稱。
Note:DEHP = di-2-ethylhexyl phthalate, DBP = di-butyl phthalate, MBuP = mono-n-butyl phthalate, PA = phthalic acid, DEP = diethyl phthalate, DMP = dimethyl phthalate, MMP = monomethyl phthalate, DPP = dipropyl phthalate, MEHP = mono-2-ethylhexyl phthalate, BBP = butyl benzyl phthalate, DOP = dioctyl phthalate, DiOP = di-iso-octyl phthalate, PCA = protocatechuic acid, MBeP = mono-benzyl phthalate, DPRP = dipropyl phthalate, DPEP = deoxophylloerythroetioporphyrins, DiBP = di-iso-butyl phthalate, MEHP = mono-2-ethylhexyl phthalate, BA = benzoic acid, DEHA = di-2-ethylhexyl adipate, MEHA = mono-ethylhexyl adipate, EHA= 2-ethylhexanal, EHAA = 2-ethylhexanoic acid, DMIP = dimethyl isophthalate, DMTP = dimethyl terephthalate, TA = terephthalic acid.anot mentioned,bDOP: also named DEHP.

圖1 基于16S rRNA(A)和鄰苯二甲酸酯雙加氧酶基因(B)序列同源性構(gòu)建的細(xì)菌系統(tǒng)發(fā)育樹 注：圖示文獻(xiàn)報(bào)道且在NCBI數(shù)據(jù)庫同時(shí)提交了序列的鄰苯二甲酸酯降解菌的基因信息。圖1A中藍(lán)色框示該屬細(xì)菌的鄰苯二甲酸酯雙加氧酶基因已被提交到NCBI數(shù)據(jù)庫。Fig. 1 Rooted phylogenetic trees based on 16S rRNA (A) and phthalate dioxygenase gene (B) sequences Note: Figures show the gene information of PAEs-degrading bacteria those have been reported in the literature and whose sequences have been submitted to NCBI database. Blue boxes in Fig. 1A indicate that the phthalate dioxygenase genes in these bacteria have been submitted to NCBI database.

然而，目前對(duì)PAEs降解細(xì)菌的研究還比較基礎(chǔ)。在43株已報(bào)道且在NCBI數(shù)據(jù)庫提交了16S rRNA信息的細(xì)菌中只有35條序列的片段長(zhǎng)度1200 bp，據(jù)此構(gòu)建的系統(tǒng)進(jìn)化樹如圖1A所示。對(duì)應(yīng)PAEs降解菌的16S rRNA多樣性，只有Acinetobacter、Delftia、Sphingomonas、Rhdococcus和Gordonia等5個(gè)屬的10多個(gè)菌株其鄰苯二甲酸酯雙加氧酶基因(phthalate dioxygenase gene, PDOG)得到了克隆分析(圖1B)。除Delftia外，其他細(xì)菌均屬于Acinobacteria(圖1B)，而R. coprophilus G9和Mycobacterium vanbaalenii PYR-1的PDOG片段長(zhǎng)度只有420 bp和267 bp[13]。這說明，對(duì)于PAEs降解菌的分子生物學(xué)研究，未來還有很多工作值得開展。

2 PAEs對(duì)細(xì)菌群落的影響

環(huán)境中微生物資源豐富，但可培養(yǎng)微生物所占比例低于1%。如前所述，PAEs在環(huán)境中的廣泛分布會(huì)對(duì)微生物群落結(jié)構(gòu)和酶活性產(chǎn)生影響，相應(yīng)地，PAEs降解菌會(huì)對(duì)PAEs的環(huán)境歸趨產(chǎn)生直接或間接的作用[5]。

聚合酶鏈反應(yīng)-變性梯度凝膠電泳(polymease chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE)分析顯示，往土壤中添加DEP濃度高達(dá)10 mg·kg-1時(shí)，土壤微生物的種類降低到了個(gè)位數(shù)，主要包括Sphingomonas spp.、Pseudomonas spp.和Actinomycetes spp.[5]。Chen等[14]研究表明，DMP、DEP和DOP都會(huì)顯著降低土壤脲酶(urease)活性。有趣的是，即使DMP或DEP添加濃度高達(dá)500 mg·kg-1，脲酶活性也只降低了32%和31%。顯然，微生物對(duì)PAEs的抗性和降解緩和了污染物毒性[14]。類似地，Cartwright等[15]發(fā)現(xiàn)，DEP低至1 mg·kg-1即可在1 d內(nèi)顯著降低土壤可培養(yǎng)細(xì)菌總數(shù)(47%，BIOLOG法)，這可能與PAEs誘發(fā)的細(xì)菌細(xì)胞膜流動(dòng)性增加有關(guān)。不同的是，DEHP并未對(duì)土壤微生物造成任何影響。事實(shí)上，土壤微生物數(shù)量變化與DEP的有效降解并無顯著相關(guān)性(降解半衰期為0.75 d)；相反，DEHP在70 d后只減少了10%，說明微生物的數(shù)量并不能反映土壤微生物對(duì)PAEs的降解活性[15]。因此，PAEs誘導(dǎo)效應(yīng)可能主要體現(xiàn)在對(duì)群落結(jié)構(gòu)的調(diào)整以增加PAEs代謝菌群豐度，這與Kapane等[5]的結(jié)論相符。對(duì)土壤多種酶活性的檢測(cè)結(jié)果表明，添加DBP雖然降低了脲酶、纖維素酶(cellulose)和β-葡萄糖苷酶(β-glucosidase)活性，卻增強(qiáng)了脫氫酶(dehydrogenase)、催化酶(catalase)、蛋白酶(protease)和磷酸酶(phosphatase)活性[16]。這種現(xiàn)象與微生物總數(shù)降低卻依然能夠有效降解PAEs是一致的，即具備PAEs降解功能的細(xì)菌在污染物誘導(dǎo)下成為了優(yōu)勢(shì)菌群。例如，Wang等[17]發(fā)現(xiàn)，隨著DMP濃度從5 mg·kg-1升至20 mg·kg-1，微生物群落分類單元(operational taxonomic units, OTUs)隨之降低，但DMP降解效率卻隨之升高，說明優(yōu)勢(shì)菌的存在是DMP降解的主要貢獻(xiàn)者。

雖然環(huán)境中PAEs的微生物行為已有諸多報(bào)道，現(xiàn)有研究并未很好地跟蹤微生物群落變化的基本規(guī)律。這一方面與環(huán)境體系的復(fù)雜性有關(guān)，同時(shí)也與PAEs種類的多樣性及微生物降解PAEs的特異性及互作效用密切相關(guān)(見下文)。由于對(duì)PAEs降解菌種類及功能認(rèn)識(shí)的不足(表1和圖1)，PAEs降解基因的多樣性及其環(huán)境意義仍是今后研究的重點(diǎn)內(nèi)容。

3 微生物降解PAEs的特異性和降解效率

研究指出，某些降解菌可同時(shí)代謝多種PAEs，但已報(bào)道的80多個(gè)菌株中超過50%只能代謝1種底物(表1)。此外，具備多種PAEs代謝功能的微生物也存在底物特異性，這種現(xiàn)象早于2003年已在Arthrobacter sp.和S. paucimobilis中被觀察到。研究發(fā)現(xiàn)，Arthrobacter sp.可迅速將DMP降解為鄰苯二甲酸單甲酯(monomethyl phthalate, MMP)和PA，此后PA被繼續(xù)代謝為CO2和H2O；然而，該菌不能繼續(xù)代謝MMP，卻由S. paucimobilis完成這個(gè)過程[59]。最近研究也發(fā)現(xiàn)，Camelimonas sp. M11對(duì)DBP、DEP、鄰苯二甲酸二丙酯(dipropyl phthalate, DPP)和鄰苯二甲酸二戊酯(dipentyl phthalate, DNPP)有很強(qiáng)的降解能力，但不能降解DMP[32]。Vega等[59]認(rèn)為，DBP被Arthrobacter sp.和S. paucimobilis代謝存在DBP→PA和DBP→MMP兩種機(jī)制，并提出環(huán)境微生物協(xié)同完成復(fù)合污染物代謝的假設(shè)(見下文)。

鑒于微生物對(duì)PAEs的碳源利用特性，理論上它們具備較強(qiáng)的PAEs耐受能力和降解能力。一般而言，PAEs側(cè)鏈越短，微生物降解效率也越高。然而，PAEs降解酶活性不受側(cè)鏈長(zhǎng)度影響，而與側(cè)鏈產(chǎn)生的空間位阻有關(guān)[6]。Liang等[24]發(fā)現(xiàn)，不動(dòng)桿菌Acinetobacter sp. JDC-16在20 h內(nèi)(pH=8、35oC)可將500 mg·L-1DEP完全降解。動(dòng)力學(xué)擬合結(jié)果表明，該菌在500 mg·L-1DEP暴露下只需6.9 h即可達(dá)到穩(wěn)定期，DEP降解常數(shù)Rm高達(dá)54 mg·L-1·h-1[24]。多數(shù)PAEs降解菌對(duì)DBP的降解能力也很強(qiáng)。例如，Diaphorobacter sp. QH-6對(duì)500 mg·L-1底物的降解半衰期只需5.2 h[10]，而R. ruber DP-2和Enterobacter sp. T5對(duì)高達(dá)1 200和1 500 mg·L-1DBP的降解半衰期也只需近30 h[36, 53]。相較而言，細(xì)菌對(duì)結(jié)構(gòu)較復(fù)雜、分子量較大的BBP降解能力較差。例如，在最優(yōu)pH條件下，P. fluorescence B-1對(duì)10 mg·L-1BBP代謝速率低至0.03 mg·L-1·h-1[49]；Acinetobacter sp. FW略高，為2.1 mg·L-1·h-1[22]。類似地，雖然P. fluorescence FS1在60 d內(nèi)可將200 mg·L-1DEHP中的75%有效降解，但降解半衰期高達(dá)17 d[63]。有趣的是，B. subtilis No. 66對(duì)DEHP有較強(qiáng)的降解能力，但幾乎不能降解DBP或只能降解DEP、脫氧葉紅初卟啉(deoxophylloerythroetioporphyrins, DPEP)和鄰苯二甲酸丙酯(dipropyl phthalate, DPRP)的一小部分[64]。

值得注意的是，在污染物結(jié)構(gòu)特定的情況下，微生物對(duì)PAEs的降解能力往往取決于微生物本身[23]。例如，Yang等[23]發(fā)現(xiàn)，Acinetobacter sp. HS-B1和Arthrobacter sp. HS-B2對(duì)起始濃度為500 mg·L-1BBP的24 h降解率分別達(dá)28%和59%。當(dāng)培養(yǎng)基中添加1%LB時(shí)，HS-B1和HS-B2對(duì)BBP的去除率提高到了40%和75%，說明微生物對(duì)PAEs的代謝不僅是碳源利用，還可能存在純粹的酶學(xué)解毒過程[23]。此外，Gordonia sp. MTCC 4818和P8219對(duì)PAEs的高效降解也備受關(guān)注，其對(duì)80 μmol BBP的90 h降解率和1 300 mg·L-1DEHP的45 h降解率可達(dá)100%[39-40]。

4 微生物介導(dǎo)的PAEs降解機(jī)理

如表1所示，多數(shù)微生物可以PAEs為唯一碳源和能源物質(zhì)進(jìn)行代謝生長(zhǎng)。然而，微生物無法直接利用長(zhǎng)側(cè)鏈或帶苯環(huán)等復(fù)雜結(jié)構(gòu)的有機(jī)物大分子，將長(zhǎng)碳側(cè)鏈縮短、將雙側(cè)鏈降解成單側(cè)鏈、將苯環(huán)開環(huán)并進(jìn)一步代謝成CO2和H2O是微生物代謝PAEs的主要步驟[6]。雖然目前報(bào)道的PAEs降解酶基因信息很少(圖1B)，通過代謝產(chǎn)物分析技術(shù)如氣相色譜-質(zhì)譜聯(lián)用(Gas Chromatograph-Mass Spectrometer, GC-MS)和液相色譜-質(zhì)譜聯(lián)用(Liquid Chromatography-Mass Spectrometer, LC-MS)等可推測(cè)幾種比較經(jīng)典的微生物代謝路徑。

4.1 PAEs側(cè)鏈的降解

側(cè)鏈代謝是PAEs微生物降解的首要步驟，包括β-氧化作用(β-oxidation)、轉(zhuǎn)酯化(trans-esterificaiton)或去烷基化作用(de-alkylation)和脫脂化作用(de-esterificaiton)[6]。對(duì)于側(cè)鏈雙酯基碳數(shù)大于2的PAEs，β-氧化介導(dǎo)的長(zhǎng)鏈降解是一種很重要的代謝機(jī)制，該過程涉及雙鏈乙烷基的同時(shí)脫落。Amir等[65]研究表明，在堆肥中添加DBP后，底泥中檢測(cè)到了DEP和DMP，說明DBP→DEP的代謝過程屬于β-氧化(圖2)。然而，目前關(guān)于PAEs的β-氧化作用并未在純菌體系中得到研究。對(duì)于PAEs單側(cè)鏈的降解，則以轉(zhuǎn)酯化或脫脂化作用為主。如圖2所示，DEP→EMP→DMP屬于單側(cè)鏈的烷基脫落，這種側(cè)鏈酯基發(fā)生變化的過程又稱為轉(zhuǎn)酯基作用。該路徑最早由Cartwright等[66]于2000年提出，與β-氧化作用類似，其后續(xù)研究并未得到充分的開展。目前報(bào)道的唯一一種具備轉(zhuǎn)酯化作用的微生物是A. lwoffii R-3[21]。脫脂化作用是研究最透徹的PAEs降解路徑，其與側(cè)鏈烷基變化無關(guān)，而是將PAEs一側(cè)或雙側(cè)酯基水解，產(chǎn)物為帶苯環(huán)的酸類物質(zhì)。例如，某些微生物可將DEHP一側(cè)酯基脫落形成MEHP，MEHP酯基可被進(jìn)一步水解成PA(圖2)。這類微生物有G. polyisoprenivorans p8219[40]、Microbacterium sp. CQ0110Y[41]、P. fluorescens FS1[63]和M. luteus等[42](表1)。類似地，DBP→MBuP，BBP→MBzP，MBzP、DEP、DMP和MMP→PA都屬于微生物介導(dǎo)的脫脂化降解，相關(guān)微生物包括Gordonia sp. MTCC 4818[39]、Atrhrobacter sp. WY[22]、Paenibacillus sp. S-3/H-2[47-48]和P. fluorescens B-1[49]等(表1)。研究發(fā)現(xiàn)，脫脂化作用在厭氧和好氧條件下都可發(fā)生，該過程由酯酶介導(dǎo)完成[40, 59-60]。

如圖2所示，PA是PAEs的代謝“中轉(zhuǎn)站”，PA的進(jìn)一步代謝與環(huán)境中含氧量有關(guān)。在好氧條件和3,4或4,5-鄰苯二甲酸酯雙加氧酶作用下，PA可被降解成3,4或4,5-雙羥基鄰苯二甲酸酯，進(jìn)而形成PCA[6]。在厭氧條件下，PA將被轉(zhuǎn)化成苯甲酸(benzoic acid, BAc)，其中一部分BAc可轉(zhuǎn)化成PCA并進(jìn)入下一步代謝[6](圖2)。由此可見，除PA外，PCA是PAEs降解的另一重要“中轉(zhuǎn)站”。

需要指出的是，BBP的微生物降解產(chǎn)物還包括苯甲醇(benzyl alcohol, BAl)和1-丁醇(1-butanol)。與PA厭氧代謝類似，BAl可在相同條件下被代謝成BAc，從而進(jìn)入下一個(gè)代謝路徑(圖2)。在Acinetobacter sp. FW和Gordonia sp. MTCC 4818等菌株中，1-丁醇的代謝主要由β-氧化作用完成[22, 39]。β-氧化作用的產(chǎn)物CO2和H2O將進(jìn)入三羧酸循環(huán)(TCA cycle)，為微生物生長(zhǎng)提供碳源物質(zhì)(圖2)。

圖2 微生物降解鄰苯二甲酸酯的可能路徑 注：實(shí)線和虛線分別表示PAEs的主要和次要代謝路徑。Fig. 2 Possible pathways involved in microbes-mediated PAEs degradation Note: Solid and dotted lines indicate the major and minor pathways of PAEs degradation respectively.

4.2 PAEs苯環(huán)的降解

苯環(huán)降解是實(shí)現(xiàn)PAEs碳源利用的重要步驟，該過程以PA、PCA和BAc為主要代謝中間產(chǎn)物。在好氧條件下，革蘭氏陽性菌可將PA轉(zhuǎn)變成順-3,4-雙氫-3,4-雙羥基鄰苯二甲酸酯(cis-3,4-dihydro-3,4-dihydroxyphthalate)，而革蘭氏陰性細(xì)菌則在苯環(huán)的4,5位發(fā)生雙氧化，產(chǎn)生順-4,5-雙氫-4,5-雙羥基鄰苯二甲酸酯[6](圖2)。例如，R. ruber DP-2同時(shí)含有酚水解酶基因(phenol hydroxylase gene, pheu)和3,4-鄰苯二甲酸酯雙加氧酶基因(pht)，其對(duì)600～1 200 mg·L-1DBP的降解時(shí)間只需15.8～27.8 h[53]。DBP經(jīng)進(jìn)一步還原和脫羧基作用，可被轉(zhuǎn)化成另一種重要的代謝中間產(chǎn)物PCA。若PCA發(fā)生鄰位(ortho-)或間位(meta-)解環(huán)，可分別產(chǎn)生β-酮基己二酸(β-ketoadipate)和4-羧基-2-雙羥基粘康半醛(4-carboxy-2-hydroxymuconic semialdehyde)。經(jīng)一系列代謝后，產(chǎn)生的草酰乙酸(oxaloacetate)和丙酮酸(pyruvate)將進(jìn)入TAC循環(huán)[6](圖2)。Eaton和Ribbons[67]認(rèn)為，Micrococcus sp. 12B對(duì)DBP的降解屬于這一代謝路徑。以PCA為代謝中間產(chǎn)物，PAEs的完全降解還可通過β-羧基-順,順-粘康酸(β-carboxy-cis, cis-muconate)→γ-羧基-粘康酸內(nèi)酯(γ-carboxy muconolactone)→TCA路徑實(shí)現(xiàn)，這種代謝機(jī)制已在Arthrobacter sp. WY中得到了詳細(xì)研究[22]。

除PCA外，BAc是PAEs代謝的另一“二級(jí)中轉(zhuǎn)站”。如前所述，有部分PA在好氧條件下可通過BAc路徑實(shí)現(xiàn)完全降解，而BAl→BAc轉(zhuǎn)化發(fā)生在厭氧條件下[6](圖2)。除BAc→PCA代謝路徑外，目前還發(fā)現(xiàn)了另外3種典型的降解機(jī)制。第1種是BAc→1-環(huán)己烯羧酸(1-cyclohexene carboxylic acid)→2-羥基環(huán)己烷羧酸(2-hydroxy-cyclohexane carboxylic acid)→己二酸(adipic acid)路徑。例如，Chatterjee和Dutta[39]報(bào)道顯示，Gordonia sp. MTCC 4818不能將PA轉(zhuǎn)化成PCA，但可實(shí)現(xiàn)BAc的己二酸代謝。第2種是BAc→2-羥基苯甲酸(benzenecarboxylic acid)→苯酚(phenol)→鄰苯二酚(catechol)→順,順-己二烯二酸(cis, cis-muconic acid)→粘康酸內(nèi)酯(muconolactone)路徑。P. fluorescence FS1是該降解路徑的代表菌株[50, 63]。在某些情況下，微生物還可將BAc轉(zhuǎn)化成不穩(wěn)定的中間產(chǎn)物如順-1,6-雙羥基-2,4-環(huán)己二烯-1-羧酸(cis-1,6-dihydroxy-2,4-cyclohenadiene-1-carboxylic acid)，此后進(jìn)入類似于路徑二的代謝過程(圖2)。Chatterjee和Dutta[22]驗(yàn)證了Acinetobacter sp. FW中該代謝路徑的存在。

4.3 PAEs的協(xié)同代謝機(jī)制

微生物介導(dǎo)的PAEs降解是一個(gè)復(fù)雜系統(tǒng)，不同微生物或同一微生物在不同條件下，其代謝路徑都可能存在較大差異(圖2)。某些微生物代謝PAEs的能力很強(qiáng)。例如，P. fluorescence FS1可將DEHP進(jìn)行逐級(jí)降解，產(chǎn)生終產(chǎn)物CO2和H2O[50, 63]。具備完全降解功能的微生物還包括Flavobacterium sp. A-1/A-9[37-38]、B. thuringiensis HD-1[30]和Variovorax sp. BS1[62]等(表1)。通常而言，具備產(chǎn)生CO2和H2O的菌株屬于PAEs完全代謝菌。然而，環(huán)境中某些微生物不具備完全代謝PAEs的能力，該過程往往需要多種微生物的協(xié)同作用。例如，Vega和Bastide[59]研究發(fā)現(xiàn)，Arthrobacter sp.可將DMP代謝成MMP和PA，但該菌不能進(jìn)一步代謝MMP。當(dāng)該菌與篩自同一土壤的微生物S. paucimobilis (MMP降解菌)混合培養(yǎng)后，DMP和MMP都未在培養(yǎng)基中被檢測(cè)到，說明二者共存時(shí)可協(xié)同代謝PAEs。由此可見，實(shí)際環(huán)境中PAEs的降解往往是多種微生物共同作用的結(jié)果[22]。

5 PAEs降解菌的環(huán)境應(yīng)用

5.1 微生物修復(fù)PAEs污染水體/底泥

固定化技術(shù)是實(shí)現(xiàn)微生物負(fù)載及污染物環(huán)境治理的常規(guī)手段。為提高PAEs降解菌的活性持留，微生物固定化技術(shù)在PAEs廢水中的應(yīng)用也逐漸得到了推廣[1]。在固定化微生物治理PAEs的實(shí)踐中，清華大學(xué)王建龍教授課題組做出了突出貢獻(xiàn)。1995年，作者率先嘗試以聚乙烯醇(polyvinyl alcohol, PVA)固定化基質(zhì)包埋微生物對(duì)DBP的降解研究[69]。結(jié)果表明，菌株A固定化小球可在40 h內(nèi)將100 mg·L-1DBP完全降解，且固定化細(xì)胞的降解活性比游離細(xì)胞更高。目前已報(bào)道的固定化菌還包括B. subtilis[28]，Bacillus sp.[26]、Micrococcus sp.[43]和Variovorax sp. BS1[62]等。為實(shí)現(xiàn)PAEs污染水體的有效治理，混合包埋不同功能的PAEs降解菌是今后研究的主要方向。

我國部分地區(qū)的底泥PAEs濃度高達(dá)1 250 mg·kg-1[2]?？紤]到微生物固定化技術(shù)在底泥修復(fù)中應(yīng)用的局限性，越來越多研究將重點(diǎn)轉(zhuǎn)向土著微生物的篩選與回用。如表1所示，目前已得到深入研究的PAEs降解菌超過34種來源于底泥或沉積物。利用活性污泥中微生物群落的群感效應(yīng)，可實(shí)現(xiàn)廢水中PAEs的有效治理[1, 6]。Chang等[70]發(fā)現(xiàn)，好氧條件下，微生物對(duì)底泥中50 mg·L-1DEP、DBP、BBP和DEHP的降解半衰期只需2.7、1.8、2.1和3.8 d。底泥中PAEs在厭氧條件下的降解速率也很高。例如，10 mg·L-1DMP和DBP的降解半衰期低至1.0和1.4 d，但DOP的降解半衰期比較長(zhǎng)，為19.4 d[71]。以上結(jié)論說明PAEs的微生物降解與其結(jié)構(gòu)密切相關(guān)[1, 6]。因此，在長(zhǎng)鏈PAEs濃度較高的廢水中，有必要采取微生物-物理/化學(xué)(如高級(jí)氧化技術(shù))相結(jié)合的手段實(shí)現(xiàn)其有效治理[1]。此外，研究發(fā)現(xiàn)，當(dāng)往底泥中添加壬基酚(nonylphenol)或多環(huán)芳烴(polycyclic aromatic hydrocarbons, PAHs)時(shí)，Sphigomonas sp. DK4和Corynebacterium sp. O18對(duì)PAEs的降解能力會(huì)受到顯著抑制[34]。鑒于PAEs污染廢水可能還包含其他有毒有害重(類)金屬和有機(jī)污染物，篩選具備多種污染物抗性的菌株及其生理生化研究是工程應(yīng)用的基礎(chǔ)。

5.2 微生物修復(fù)PAEs污染土壤

土壤微生物已被證明具備降解多種PAEs的能力(表1)，利用這些微生物回土修復(fù)是一種理想的方法。Wang等[55]從DEHP污染土壤篩得一株高效降解菌Rhodococcus sp. WJ4，在液體培養(yǎng)基中該菌對(duì)200 mg·L-1DEHP的去除率高達(dá)96.4%。土壤修復(fù)實(shí)驗(yàn)表明，WJ4對(duì)1 g·kg-1DEHP污染土壤的21 d修復(fù)率達(dá)55%，顯示出了較好的應(yīng)用潛力。然而，類似的PAEs污染土壤的微生物修復(fù)實(shí)例未見報(bào)道?，F(xiàn)有研究表明，生物泥漿和固相反應(yīng)器(bioslurry and solid-phase bioractors, BSSB)修復(fù)污染土壤是比較可行的技術(shù)，目前已得到了一些推廣與應(yīng)用[2, 6]。例如，利用BSSB修復(fù)技術(shù)，Di Gennaro等[72]將5.51 mg·g-1DEHP降至0.63 mg·g-1，76 d內(nèi)總?cè)コ矢哌_(dá)89%。這種方法的可行性取決于微生物培養(yǎng)條件的可控程度及營養(yǎng)物質(zhì)的供給效率[72]。若土壤中添加堆肥的粒徑很小，也有助于提高PAEs的降解效率[2]。

由此可見，將微生物進(jìn)行回土修復(fù)或在泥漿相中進(jìn)行強(qiáng)化修復(fù)都是可行的。Liang等[6]認(rèn)為，生物泥漿法修復(fù)PAEs污染土壤的優(yōu)勢(shì)主要體現(xiàn)在1) 營養(yǎng)物質(zhì)、末端電子受體和底物的均勻分布；2) 提高了微生物與污染物的有效接觸。不足的是，生物泥漿法需要反復(fù)挖掘土壤，因此限制了其修復(fù)能力和實(shí)際應(yīng)用[2]。

6 結(jié)論與展望

多數(shù)PAEs都具有內(nèi)分泌干擾作用且廣泛存在于各種環(huán)境介質(zhì)中，實(shí)施PAEs的土壤和水體修復(fù)是保證人體健康的重要舉措。微生物對(duì)多種PAEs都具備較強(qiáng)的降解能力。目前已有6個(gè)門類、30多個(gè)屬的80多個(gè)PAEs降解菌株得到了詳細(xì)研究，通過GC-MS和LC-MS等檢測(cè)技術(shù)或代謝路徑的模型擬合[54]提出了PAEs代謝的微生物學(xué)機(jī)制。然而，已報(bào)道的PAEs微生物降解酶基因數(shù)量有限，這限制了從生物化學(xué)和分子生物學(xué)水平認(rèn)識(shí)PAEs代謝的機(jī)理，同時(shí)也不利于環(huán)境中PAEs降解菌的群落結(jié)構(gòu)分析和功能研究。雖然PAEs污染水體(底泥)的微生物修復(fù)已取得一定成效，土壤修復(fù)仍是當(dāng)前PAEs污染治理的主要內(nèi)容。

基于PAEs微生物代謝的重要性及當(dāng)前研究的不足，今后的工作可從以下幾方面展開：1) 在現(xiàn)有基礎(chǔ)上繼續(xù)篩選PAEs降解菌并研究其降解能力；2) 深入闡釋現(xiàn)有PAEs降解菌代謝PAEs的分子生物學(xué)機(jī)制，推動(dòng)對(duì)新獲菌株的全面研究；3) 利用高通量分析技術(shù)等實(shí)現(xiàn)對(duì)環(huán)境中PAEs降解菌多樣性的充分認(rèn)識(shí)；4) 繼續(xù)探討PAEs降解菌在土壤和水體污染治理中的應(yīng)用。

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◆

Phthalic Acid Esters-degrading Bacteria: Biodiversity, Degradation Mechanisms and Environmental Applications

Han Yonghe, He Ruiwen, Li Chao, Xiang Ping, Luo Jun, Cui Xinyi*

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, China

Phthalic acid esters (PAEs) are typical persistent organic pollutants (POPs) that can disrupt endocrine systems in humans. Long-term exposure to PAEs can induce toxic effects to living organisms in various environmental matrixes, including water body, sediment and soil. PAEs are persistent and frequently detected with high concentrations in environment, which consequently limit their effective removal from contaminated sites. With high tolerance for contaminants and outstanding ability to biodegrade contaminants, microbes show great potentials to bio-remediate PAE-contaminated sites. Compared to physical and chemical approaches, microbial remediation technology has several advantages, including easy manipulation, large-scale application in remediation and high flexibility. The aims of this review are to summarize most of the reported bacterial species, metabolic mechanisms mediated by PAE-degrading bacteria, and the applications of these bacteria in remediation of PAEs-polluted water and soil. This review can provide some new information for further studies on the bio-remediation of PAEs.

endocrine disrupter; persistent organic pollutants; phthalic acid esters; microbes; biodegradation; health risk

10.7524/AJE.1673-5897.20151107002

江蘇省自然科學(xué)基金青年科學(xué)基金項(xiàng)目(BK20130558)；國家自然科學(xué)基金青年科學(xué)基金項(xiàng)目(21307055)

韓永和(1986-)，男，博士研究生，研究方向?yàn)槲廴疚锏沫h(huán)境微生物行為，E-mail：hanyonghe0423@163.com

*通訊作者(Corresponding author)， E-mail: lizzycui@nju.edu.cn

2015-11-07 錄用日期：2015-12-22

1673-5897(2016)2-037-13

X171.5

A

簡(jiǎn)介：崔昕毅(1983-)，女，環(huán)境工程博士，副教授，主要研究有機(jī)污染物的環(huán)境行為、生態(tài)風(fēng)險(xiǎn)評(píng)價(jià)及修復(fù)和痕量有機(jī)物的環(huán)境分析監(jiān)測(cè)，發(fā)表SCI論文20余篇。

韓永和, 何睿文, 李超, 等. 鄰苯二甲酸酯降解細(xì)菌的多樣性、降解機(jī)理及環(huán)境應(yīng)用[J]. 生態(tài)毒理學(xué)報(bào)，2016, 11(2): 37-49

Han Y H, He R W, Li C, et al. Phthalic acid esters-degrading bacteria: Biodiversity, degradation mechanisms and environmental applications [J]. Asian Journal of Ecotoxicology, 2016, 11(2): 37-49 (in Chinese)