劉金香,蒲亞帥,謝水波,劉迎九,陳子庚,李仕友*

滅活與非滅活條件下植物乳桿菌去除U(VI)的機(jī)理

劉金香1,2,蒲亞帥1,謝水波2,劉迎九1,陳子庚1,李仕友1*

(1.南華大學(xué)土木工程學(xué)院,湖南 衡陽(yáng) 421001；2.南華大學(xué)污染控制與資源化技術(shù)湖南省高校重點(diǎn)實(shí)驗(yàn)室,湖南 衡陽(yáng) 421001)

在不同時(shí)間,pH值和生物量濃度條件下,進(jìn)行了滅活與非滅活植物乳桿菌去除水中鈾的對(duì)比試驗(yàn),探討了二者去除水中鈾的機(jī)理,通過(guò)SEM-EDS、FTIR、XPS及XRD分析了鈾與菌體表面的微觀作用機(jī)理以及菌體表面沉積物的特征.結(jié)果表明:植物乳桿菌經(jīng)滅活后,其吸附鈾的能力得到顯著的提高,當(dāng)U(VI)初始濃度為10mg/L、pH值為6.0、37℃條件下,120min內(nèi)滅活菌體對(duì)U(VI)的去除率為94.7%,而活菌體的去除率為88.9%.滅活菌體具有更高的鈾吸附容量,在生物量濃度為0.06~0.24mg/L,pH值(3.0~7.0)條件下,滅活菌體與活菌體的U(VI)累積容量比均大于1. SEM-EDS、FTIR分析結(jié)果表明,活細(xì)胞和滅活細(xì)胞都可通過(guò)細(xì)胞表面的羥基、?；棒然裙倌軋F(tuán)吸附、配位絡(luò)合U(VI). XRD分析表明,活菌體可生物磷酸礦化水中的U(VI).活菌體的XRD譜圖在2(18.023,25.492,27.343,40.813°處)有4個(gè)明顯的磷酸鈾酰晶體峰,而滅活菌體的XRD譜圖顯示為非晶態(tài).XPS結(jié)果表明,活菌體可生物還原U(VI).活菌體能譜圖中U4f7/2和U4f5/2 軌道出現(xiàn)了結(jié)合能為380.20eV和390.65eV的U(VI)分裂峰,而滅活菌體的能譜圖中沒(méi)有出現(xiàn)U(IV)的分裂峰.

植物乳桿菌；U(VI)；生物吸附；礦化機(jī)理；生物還原

鈾礦開(kāi)采尤其是地浸采鈾過(guò)程中,產(chǎn)生了大量的鈾廢渣、尾礦及放射性廢水,放射性核素通過(guò)地下水向環(huán)境擴(kuò)散,造成更大范圍的污染,對(duì)人類(lèi)的健康構(gòu)成嚴(yán)重威脅.與傳統(tǒng)的物理化學(xué)法相比,微生物法因具有環(huán)境友好、成本低、去除效率高等優(yōu)點(diǎn)成為研究熱點(diǎn)[1-2].微生物可以通過(guò)物理靜電吸附除鈾或利用細(xì)胞壁上的官能團(tuán)通過(guò)化學(xué)離子交換、絡(luò)合作用等將鈾結(jié)合到細(xì)胞壁表面.微生物對(duì)鈾的去除能力與細(xì)胞活性有關(guān)[3],在生物吸附去除重金屬應(yīng)用研究中,關(guān)于活菌體和滅活菌體選擇一直存在爭(zhēng)議.研究表明,滅活的釀酒酵母細(xì)胞比活細(xì)胞具有更好的鈾吸附能力[4],然而,存在共存離子時(shí),滅活的微生物對(duì)目標(biāo)重金屬離子的吸附缺乏選擇性.微生物活細(xì)胞具有解毒功能,能通過(guò)代謝作用與鈾形成鈾酰磷酸鹽礦物或者將U(VI)還原為難溶的U(IV),降低鈾的遷移能力,減小鈾對(duì)環(huán)境的危害[5-6].以往的研究主要關(guān)注的是微生物活性對(duì)吸附能力的影響,關(guān)于微生物活性對(duì)重金屬尤其是放射性重金屬的不同去除機(jī)制鮮有研究.

植物乳桿菌()是食品級(jí)益生菌,屬于革蘭氏陽(yáng)性菌,兼性厭氧.最新研究發(fā)現(xiàn)乳桿菌具有吸附鉛、鎘、鉻、鈾等多種毒性重金屬的特性[7-9],同時(shí)具有釋磷沉淀重金屬的功能[10-11].本研究對(duì)滅活及非滅活條件下植物乳桿菌去除水中U(VI)進(jìn)行了對(duì)比實(shí)驗(yàn)研究,探討了兩種情況下乳桿菌去除鈾的不同機(jī)理,以期為鈾污染水的治理及利用乳桿菌減緩鈾的毒性提供理論基礎(chǔ),同時(shí)可為鈾污染水體的修復(fù)策略提供一定的理論指導(dǎo).

1 材料和方法

1.1 主要試劑與分析測(cè)試方法

主要試劑:基準(zhǔn)八氧化三鈾U3O8(分析純),標(biāo)準(zhǔn)鈾溶液采用GBW04201方法配制;其他試劑均為分析純,實(shí)驗(yàn)用水為超純水.

U(Ⅵ)的測(cè)定:采用5-Br-PADAD可見(jiàn)光分光光度法測(cè)定微量鈾[12].

pH值的測(cè)定:采用上海精密科學(xué)儀器有限公司PHS-3C型pH計(jì).

1.2 植物乳桿菌培養(yǎng)和滅活

植物乳桿菌:購(gòu)自中國(guó)菌種保藏中心,編號(hào)ACCC11095.

MRS培養(yǎng)基:酪蛋白胨,10g/L;牛肉浸取物, 10g/L;酵母提取液,5g/L;葡萄糖,20g/L;乙酸鈉,5g/L;檸檬酸二胺,2g/L;吐溫80,1g/L;磷酸氫二鉀,2g/L;七水硫酸鎂,0.2g/L;七水硫酸錳,0.05g/L.

將植物乳桿菌在MRS液體培養(yǎng)基中培養(yǎng)20h, 8000r/min離心收集細(xì)胞,用無(wú)菌去離子水洗滌3次.將洗滌的細(xì)胞重新懸浮于滅菌的去離子水中得到生物質(zhì)懸浮液.取一定體積的懸浮液置于105℃的干燥箱內(nèi)烘干24h,確定生物質(zhì)濃度.經(jīng)高壓滅活(121℃,20min)后的細(xì)胞懸浮液與未滅活的細(xì)胞懸浮液均保存于4℃的冰箱內(nèi).

1.3 U(VI)去除試驗(yàn)

1.3.1 接觸時(shí)間對(duì)U(VI)去除的影響 取一定體積的細(xì)胞懸浮液加入100mL血清瓶中,再加入一定體積的鈾儲(chǔ)備液,然后加入無(wú)菌去離子水定容為100mL,使得溶液中生物質(zhì)干重為0.15g/L,U(VI)的初始濃度為10mg/L,pH值調(diào)至6.0.靜置于37℃的生化培養(yǎng)箱內(nèi),定時(shí)取樣,12000r/min離心取上清液測(cè)定溶液中U(VI)的剩余濃度. 所有實(shí)驗(yàn)組均設(shè)置3個(gè)平行實(shí)驗(yàn),且取其數(shù)據(jù)的平均值作為實(shí)驗(yàn)結(jié)果.

1.3.2 pH值與生物量濃度對(duì)U(VI)去除的影響 改變?nèi)芤旱膒H值及細(xì)胞懸浮液的體積,重復(fù)上述實(shí)驗(yàn)步驟.37℃的生化培養(yǎng)箱內(nèi)靜置6h后,取樣測(cè)定.

U(VI)去除率(%)和生物累積量(mg/g)按下式計(jì)算:

式中:0和e分別是鈾細(xì)胞混合液中U(VI)的初始濃度和剩余濃度,mg/L;是細(xì)胞對(duì)U(VI)的生物吸附量,mg/L;是植物乳桿菌的濃度,mg/L;H和L分別是滅活細(xì)胞和活細(xì)胞對(duì)U(VI)的累積容量,mg/L;是U(VI)的去除率;H和L分別是熱滅活細(xì)胞和活細(xì)胞對(duì)U(VI)的去除率;是滅活細(xì)胞與活細(xì)胞對(duì)于U(VI)的累積容量比或去除率比.

1.4 表征試驗(yàn)

1.4.1 掃描電鏡-能譜(SEM-EDS)表征 試驗(yàn)中U(VI)濃度為100mg/L,植物乳桿菌活細(xì)胞及熱滅活細(xì)胞的濃度為2.7g/L.溫度為37℃,接觸時(shí)間6h. 8000r/min離心10min收集細(xì)胞,用0.1mol/L氯化鈉溶液(非磷酸鹽緩沖液以避免外界磷的添加形成鈾酰-磷酸鹽沉淀)洗滌3次去除細(xì)胞雜質(zhì)并保持細(xì)胞滲透壓,用2.5%戊二醛在4℃下(配置使用生理鹽水而非磷酸鹽緩沖液)固定2h.再次用0.1mol/L氯化鈉溶液洗滌,之后每次在4℃分級(jí)乙醇(20%,50%,80%)中脫水15min,100%乙醇脫水30min,用純乙酸異戊酯處理樣品2h.再將樣品分別置于-20,-50,-80℃各12h,于冷凍干燥器中干燥12h.將樣品置于掃描電鏡(德國(guó)蔡司公司,Sigma)與X射線能譜儀(德國(guó)布魯克公司,Oxford-AZTECx-Max80)下觀察細(xì)胞形態(tài)變化和細(xì)胞表面元素變化.

1.4.2 紅外光譜(FTIR),X射線衍射(XRD),光電子能譜(XPS)表征分析 將上述方法離心收集的細(xì)胞用0.1mol/L氯化鈉溶液洗滌3次后,置于-50℃冰箱12h,冷凍干燥器干燥12h.制成樣品. 利用傅里葉紅外光譜儀(美國(guó)賽默飛世爾科技公司Nicolet-460),通過(guò)峰位、峰強(qiáng)及峰形的變化分析滅活與非滅活植物乳桿菌結(jié)合鈾過(guò)程中細(xì)胞表面的主要作用官能團(tuán);利用X射線衍射儀(德國(guó)布魯克公司D8- ADVANCE),通過(guò)圖譜中晶體峰的間距比照分析活菌體與滅活菌體表面鈾沉積物類(lèi)別;利用X射線光電子能譜儀(美國(guó)賽默飛世爾科技公司Escalab 250Xi),通過(guò)分峰軟件分析沉積物中鈾元素的價(jià)態(tài).

2 結(jié)果與討論

2.1 時(shí)間對(duì)滅活與非滅活植物乳桿菌去除U(VI)的影響

如圖1所示,反應(yīng)初期,活細(xì)胞與滅活細(xì)胞均能快速去除溶液中的U(VI),20min 內(nèi),滅活菌體對(duì)U(VI)的去除率達(dá)92.1%,而活菌體的去除率為82.3%.由于活細(xì)胞在高壓滅菌過(guò)程中細(xì)胞產(chǎn)生形變,表面變粗糙,細(xì)胞與鈾酰離子的接觸面積增大,有效作用位點(diǎn)增加,因此反應(yīng)初期滅活細(xì)胞對(duì)鈾的吸附效果明顯優(yōu)于活細(xì)胞.隨著接觸時(shí)間的延長(zhǎng),滅活菌體對(duì)U(VI)的吸附速率在120min時(shí)平衡,吸附去除率達(dá)94.7%;而活性菌體對(duì)鈾的去除速率隨著時(shí)間的延長(zhǎng)一直慢速增加,在360min時(shí)平衡,U(VI)的去除率達(dá)94.1%.這是由于滅活細(xì)胞對(duì)鈾的去除是一個(gè)代謝獨(dú)立的快速吸附過(guò)程,主要通過(guò)菌體表面的物理靜電作用和細(xì)胞壁的官能團(tuán)與金屬離子的配位絡(luò)合、離子交換等物理化學(xué)吸附作用[2].而活菌體是一個(gè)依賴(lài)于代謝的緩慢過(guò)程,需要菌體細(xì)胞代謝調(diào)控系統(tǒng)的參與,主要通過(guò)細(xì)胞內(nèi)酶作用進(jìn)行重金屬的胞內(nèi)轉(zhuǎn)移,生物胞內(nèi)累積.此外有些微生物可以通過(guò)緩慢代謝作用對(duì)U(VI)進(jìn)行礦化和還原[5].

圖1 吸附時(shí)間對(duì)滅活和非滅活植物乳桿菌去除U(VI)的影響

為進(jìn)一步探討活細(xì)胞與滅活細(xì)胞對(duì)U(VI) 的吸附機(jī)理,運(yùn)用3種動(dòng)力學(xué)模型(準(zhǔn)一級(jí)動(dòng)力學(xué)模型,準(zhǔn)二級(jí)動(dòng)力學(xué)模型和顆粒內(nèi)擴(kuò)散模型)對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行了擬合,擬合結(jié)果見(jiàn)表1.活細(xì)胞與滅活細(xì)胞對(duì)U(VI)的吸附均符合準(zhǔn)二級(jí)動(dòng)力學(xué)方程(2>0.99).說(shuō)明活細(xì)胞和滅活細(xì)胞對(duì)U(VI)的吸附以化學(xué)吸附為主,鈾主要通過(guò)化學(xué)鍵合作用結(jié)合在細(xì)胞表面,涉及鈾酰離子與菌體表面活性位點(diǎn)的電子共用和電子轉(zhuǎn)移.顆粒內(nèi)擴(kuò)散模型擬合結(jié)果常數(shù)項(xiàng)不為零,表明顆粒內(nèi)擴(kuò)散不是主要的速控步驟,活細(xì)胞和滅活細(xì)胞對(duì)U(VI)的吸附過(guò)程均為多種機(jī)理共同作用的結(jié)果.

表1 滅活與非滅活植物乳桿菌吸附U(VI)的動(dòng)力學(xué)模型參數(shù)

2.2 pH值對(duì)滅活與非滅活植物乳桿菌去除U(VI)的影響

溶液的pH值不僅影響活細(xì)胞和死細(xì)胞表面電荷位點(diǎn)分布和U(VI)在水中的水解及其絡(luò)合形態(tài),還會(huì)影響生物吸附劑表面官能團(tuán)的結(jié)構(gòu)與性能,進(jìn)而影響到U(VI)的去除率[8].本實(shí)驗(yàn)研究了滅活與非滅活2種條件下,溶液初始pH值(3~7)對(duì)植物乳桿菌去除U(VI)的影響.結(jié)果如圖2.pH值對(duì)活細(xì)胞和滅活細(xì)胞去除U(VI)的效果均有不同程度的影響,當(dāng)溶液中pH值從3.0增至6.0時(shí),活細(xì)胞對(duì)U(VI)的吸附率從60.0%提高到81.9%,滅活細(xì)胞對(duì)U(VI)的吸附率從64.6%提高至85.7%;當(dāng)pH值繼續(xù)增至7.0時(shí), 活細(xì)胞對(duì)U(VI)的吸附率從81.9%降至72.8%,而滅活細(xì)胞僅從86.7%降至83.2%.pH值為3~7時(shí),滅活細(xì)胞對(duì)U(VI)的吸附去除率均高于活細(xì)胞,且活細(xì)胞對(duì)pH值的變化更敏感,這可能是由于pH值的改變導(dǎo)致活細(xì)胞的活性及代謝水平的降低,對(duì)活細(xì)胞內(nèi)的重金屬還原酶(如細(xì)胞色素C、還原酶等)和礦化酶(酸性磷酸酶)的活性產(chǎn)生不利影響,減緩了U(VI)的礦化和還原作用,進(jìn)而對(duì)U(VI)的去除產(chǎn)生更加顯著的影響[13-14].

圖2 pH值對(duì)滅活和非滅活植物乳桿菌去除U(VI)的影響

2.3 生物量對(duì)滅活與非滅活植物乳桿菌去除U(VI)的影響

由圖3可知,在生物量濃度分別為0.06,0.12, 0.24mg/L條件下,改變?nèi)芤褐械膒H(3.0~7.0),滅活細(xì)胞與活細(xì)胞的U(VI)累積容量比均大于1,說(shuō)明植物乳桿菌滅活細(xì)胞對(duì)U(VI)的累積容量比活細(xì)胞的高.這是因?yàn)榫w在熱滅活過(guò)程中損壞了細(xì)胞膜,增強(qiáng)了細(xì)胞膜通透性,細(xì)胞產(chǎn)生形變,表面變粗糙,細(xì)胞與鈾酰離子的接觸面積增大,有效作用位點(diǎn)增加,從而提高了滅活細(xì)胞的U(VI)吸附容量.由圖3可看出,生物量濃度由0.06mg/L增加至0.12mg/L時(shí),值差異明顯,繼續(xù)增至0.24mg/L時(shí),值差異明顯減小.由于菌體濃度增加,失去活性的滅活細(xì)胞更易發(fā)生團(tuán)聚,影響滅活細(xì)胞與U(VI)結(jié)合的有效作用位點(diǎn),U(VI)吸附量增加較少,而活細(xì)胞生物量的增加,與U(VI)作用的菌體數(shù)量逐漸增加,U(VI)吸附量增加較多,因此,隨著生物投量的增加,滅活細(xì)胞與活細(xì)胞對(duì)U(VI)的累積容量比值差異變小[6,13].在菌體濃度較低時(shí),滅活細(xì)胞比活細(xì)胞更適合于處理低濃度含鈾廢水.

在pH值較低時(shí),滅活細(xì)胞與活細(xì)胞對(duì)U(VI)的吸附量比差異較小,表明滅活細(xì)胞和活細(xì)胞的U(VI)吸附能力相差不明顯.在中度酸性(pH4.0~5.0)條件下,滅活細(xì)胞與活細(xì)胞對(duì)U(VI)的累積容量比相差顯著,表明滅活細(xì)胞的U(VI)吸附能力比活細(xì)胞的更強(qiáng).一方面可能是由于pH值的改變導(dǎo)致了植物乳桿菌活細(xì)胞的代謝發(fā)生變化,與U(VI)的作用機(jī)理發(fā)生變化,另一方面也可能與pH值改變后溶液中U(VI)存在形態(tài)的改變有關(guān)[8].說(shuō)明在pH值為4.0~5.0時(shí),滅活細(xì)胞比活細(xì)胞更適合處理低濃度含鈾廢水.

圖3 不同pH值和生物量下植物乳桿菌活細(xì)胞與滅活細(xì)胞對(duì)U(VI)的累積容量比

2.4 SEM-EDS結(jié)果分析

圖4為植物乳桿菌吸附U(VI)前后的掃描電鏡圖,其中活細(xì)胞(圖4a)表面平滑,整體呈桿狀;而經(jīng)高溫高壓滅菌處理后,滅活細(xì)胞(圖4c)表面變粗糙,有絮狀毛層,比表面積增大,這是由于植物乳桿菌細(xì)胞表面具有胞外聚合物,主要由多糖和蛋白質(zhì)組成還具有少量的磷脂,在高溫環(huán)境中蛋白質(zhì)發(fā)生變性[15-16].

植物乳桿菌與水中U(VI)接觸6h后,無(wú)論是活細(xì)胞還是滅活細(xì)胞表面都有明顯的鈾沉積物 (圖4b和圖4d),且滅活細(xì)胞(圖4d)表面U(VI)的累積量明顯多于活細(xì)胞(圖4b)表面U(VI)的累積量,這證實(shí)了活細(xì)胞的U(VI)累積容量比滅活細(xì)胞的低.活細(xì)胞表面出現(xiàn)了納米級(jí)球形顆粒狀的鈾沉淀(圖4b中紅色圈)及不規(guī)則的凝膠塊狀鈾沉淀,而滅活的植物乳桿菌死細(xì)胞表面只出現(xiàn)了凝膠塊狀的鈾沉積物,鈾沉積形態(tài)的差異說(shuō)明植物乳桿菌活細(xì)胞對(duì)水中U(VI)的去除作用可能包括非吸附機(jī)理(可能為U(VI)的礦化或還原).

圖4 植物乳桿菌活細(xì)胞及滅活細(xì)胞吸附U(VI)前后的掃描電鏡圖

(a)吸附U(VI)前的活細(xì)胞 (b)吸附U(VI)后的活細(xì)胞 (c)吸附U(VI)前的滅活細(xì)胞 (d)吸附U(VI)后的滅活細(xì)胞

(a)活細(xì)胞吸附U(VI)后; (b)滅活細(xì)胞吸附U(VI)后

滅活與非滅活兩種條件下,細(xì)胞吸附U(VI)后的EDS能譜圖如圖5,可以看出,滅活細(xì)胞EDS光譜中的鈾峰比活細(xì)胞更明顯,說(shuō)明滅活細(xì)胞表面有更高的鈾結(jié)合量(滅活細(xì)胞鈾結(jié)合量重量百分比高達(dá)17.31,而活細(xì)胞鈾結(jié)合量?jī)H為4.29),說(shuō)明細(xì)胞滅火活后對(duì)U(VI)的吸附能力更強(qiáng),適合作為生物吸附劑.

2.5 FTIR結(jié)果分析

植物乳桿菌活細(xì)胞吸附U(VI)前(圖6b)后(圖6c)及滅活細(xì)胞吸附U(VI)前(圖6a)后(圖6d)的紅外光譜如圖6所示.活細(xì)胞與U(VI)作用后,部分峰的峰位、強(qiáng)度及峰形發(fā)生了變化.3430.51cm-1處的羥基(—OH)的振動(dòng)峰[16-17],1650.60cm-1處的蛋白質(zhì)酰胺I帶(C=O,C—N)振動(dòng)峰,1541.85cm-1處的蛋白質(zhì)酰胺II帶(N—H,C—N)的振動(dòng)峰[17]與1234.70cm-1處的蛋白質(zhì)酰胺III帶中羧基中的(C—O—C)的彎曲振動(dòng)和(C—PO32-)中的(P=O)的伸縮振動(dòng)峰[13,19]的峰位均向低波位數(shù)移動(dòng),峰強(qiáng)增加,峰形變寬;1059cm-1處的脂羰基(C—O—C)的伸縮振動(dòng)、脂肪族胺(C—N)的伸縮振動(dòng)及磷酸酯基的(P—O—C)的反對(duì)稱(chēng)伸縮振動(dòng),峰位未發(fā)生變化,但振動(dòng)峰強(qiáng)增加,峰型變寬[20].550~1000cm-1范圍內(nèi)峰的位置和強(qiáng)度的變化可歸因于鈾酰離子的不對(duì)稱(chēng)伸縮振動(dòng)和U(VI)與O締合后的伸縮振動(dòng)[21].其中915.91cm-1處出現(xiàn)的新峰歸因于鈾酰離子的不對(duì)稱(chēng)拉伸振動(dòng)[1,13].峰位、強(qiáng)度和峰形的變化表明活細(xì)胞表面的羥基、酰胺、羧基、脂羰基、磷酸酯基等官能團(tuán)在鈾結(jié)合過(guò)程中起重要作用.

滅活細(xì)胞與U(VI)作用后,羥基、酰胺、羧基等基團(tuán)特征峰的峰位、峰強(qiáng)及峰寬均發(fā)生了變化. 3422.04,1650.54,1541.09,1232.75cm-1處的特征峰移動(dòng)至3406.03,1652.94,1538,1233.47,917.45cm-1出現(xiàn)的新峰為鈾酰離子的不對(duì)稱(chēng)伸縮振動(dòng).與活細(xì)胞的特征峰(羥基3430.51cm-1,酰胺II帶1541.85cm-1,酰胺III帶1234.70cm-1)相比,滅活細(xì)胞特征峰(羥基3422.04cm-1,酰胺II帶1541.09cm-1,酰胺III帶1232.75cm-1)的峰位和強(qiáng)度發(fā)生了改變.這表明細(xì)胞滅活過(guò)程中蛋白質(zhì)結(jié)構(gòu)受到了破壞.由于滅活細(xì)胞比活細(xì)胞能吸附更高量的鈾,與活細(xì)胞的振動(dòng)峰(圖6c)相比,相應(yīng)的滅活細(xì)胞振動(dòng)峰(圖6d)強(qiáng)度更高.

圖6 植物乳桿菌滅活細(xì)胞和活細(xì)胞吸附U(VI)前后的紅外光譜圖

(a)滅活細(xì)胞吸附U(VI)前; (b)活細(xì)胞吸附U(VI)前; (c)活細(xì)胞吸附U(VI)后; (d)滅活細(xì)胞吸附U(VI)后

2.6 XRD結(jié)果分析

從圖7中觀察到,活細(xì)胞與鈾作用前(圖7c)后(圖7d)的XRD圖譜差異明顯;活細(xì)胞的XRD圖譜顯示為非晶態(tài),而接觸鈾后活細(xì)胞的XRD圖中發(fā)現(xiàn)有明顯的晶體峰.相反,滅活細(xì)胞與鈾作用前(圖7a)后(圖7b)的XRD圖譜均顯示為非晶態(tài),未出現(xiàn)顯著的晶體峰.活細(xì)胞接觸鈾后的XRD圖譜(圖7d)在2(18.023, 25.492,27.343,40.813°)處顯示出4個(gè)不同的峰,分別對(duì)應(yīng)于4.9178,3.491,3.259,2.209?的晶面間距值.這些間距值與已知化合物數(shù)據(jù)文件(與ICDD數(shù)據(jù)庫(kù)(ICDD,2003)匹配比對(duì), PDF)的比較顯示為磷礦物[PDF#35-0194,磷酸鈾酰水合物(UO2)3(PO4)2×H2O, PDF#29-0670,氫鈾云母H2(UO2)2(PO4)2×8H2O, PDF#29-1284,變鈉磷鈾云母Na[(UO2)(PO4)]×3H2O] (圖8a~c).這說(shuō)明植物乳桿菌活細(xì)胞與U(VI)作用可能形成了晶態(tài)磷酸鈾?；衔?在相同條件下的滅活細(xì)胞與U(VI)接觸后,表面卻沒(méi)有檢測(cè)到鈾的結(jié)晶峰,表明植物乳桿菌對(duì)U(VI)的生物礦化作用依賴(lài)于活細(xì)胞的代謝.植物乳桿菌可以產(chǎn)生磷酸酶,且能在降解有機(jī)物的同時(shí)釋放結(jié)合的磷酸鹽和聚磷酸鹽[10,22]. Choudhary等[23]發(fā)現(xiàn)銅綠假單胞菌J007將U(VI)礦化為磷酸鈾?；衔?Nedelkova等[24]發(fā)現(xiàn),在鈾協(xié)迫下微桿菌屬(spp.)能釋放磷酸鹽形成磷酸鈾酰沉淀以抵抗鈾的毒性.因此推測(cè)植物乳桿菌具有類(lèi)似的功能,當(dāng)環(huán)境中存在鈾時(shí),植物乳桿菌活細(xì)胞內(nèi)的磷酸酶發(fā)揮作用,細(xì)胞代謝產(chǎn)生磷酸鹽,促進(jìn)U(VI)的礦化沉淀,降低環(huán)境中的U(VI)濃度.

圖7 植物乳桿菌活細(xì)胞與滅活細(xì)胞吸附U(VI)前后的X射線衍射譜圖

(a)滅活細(xì)胞吸附U(VI)前; (b)滅活細(xì)胞吸附U(VI)后; (c)活細(xì)胞吸附U(VI)前; (d)活細(xì)胞吸附U(VI)后

圖8 吸附U(VI)的植物乳桿菌活細(xì)胞的晶體峰對(duì)應(yīng)的磷酸鈾酰化合物PDF卡片

2.7 XPS結(jié)果分析

在無(wú)氧條件下奧奈達(dá)希瓦氏菌可以將U(VI)還原為U(IV)[25],微氧條件下釀酒酵母菌可以將U(VI)還原為U(IV)[13],植物乳桿菌可以將Fe3+還原為Fe2+[26],然而植物乳桿菌能否將U(VI)還原為U(IV),目前還未見(jiàn)報(bào)道.運(yùn)用XPS對(duì)植物乳桿菌表面沉積物中鈾的價(jià)態(tài)進(jìn)行分析,結(jié)果如圖9所示.由XPS手冊(cè)得知,381.90,392.65eV附近的強(qiáng)峰分別對(duì)應(yīng)U(VI)的自旋軌道(L-S)分裂峰U4f7/2和U4f5/2[27-28].而在380.20,390.95eV附近則發(fā)現(xiàn)了U(IV)的自旋軌道U4f7/2和U4f5/2的分裂峰[29-30].圖9a、圖9b分別為非滅活和滅活條件下的植物乳桿菌與鈾接觸后的能譜圖,從圖9可知,植物乳桿菌活細(xì)胞表面的沉積物中存在四價(jià)鈾和六價(jià)鈾兩種價(jià)態(tài),而滅活植物乳桿菌細(xì)胞表面只有六價(jià)鈾.這表明植物乳桿菌活菌對(duì)U(VI)具有還原作用,而滅活的植物乳桿菌對(duì)U(VI) 沒(méi)有還原作用.植物乳桿菌還原U(VI)的特性對(duì)于阻止U(VI)在環(huán)境中的遷移具有重要意義.

圖9 植物乳桿菌活細(xì)胞與滅活細(xì)胞吸附U(VI)后的XPS分析圖 Fig.9 XPS analysis spectra of live and killed Lactobacillus plantarum after adsorption of U(VI)(a)活細(xì)胞吸附U(VI)后U元素的能譜圖; (b)滅活細(xì)胞吸附U(VI)后U元素的能譜圖; (c)活細(xì)胞吸附U(VI)后C元素的能譜圖; (d)滅活細(xì)胞吸附U(VI)后C元素的能譜圖

表2 植物乳桿菌活細(xì)胞和滅活細(xì)胞吸附的U和C元素的參數(shù)

續(xù)表2

滅活(圖9d)與非滅活(圖9c)植物乳桿菌吸附U(VI)后C元素的精細(xì)XPS譜圖(C1s),數(shù)據(jù)分析結(jié)果見(jiàn)表2.活細(xì)胞的C1s光譜的峰1,2和3對(duì)應(yīng)于284.6eV(C—O C—H)、285.3eV(C—C C—H)、286.25eV (C—O C—N),這些峰歸因于醇、胺或酰胺;4對(duì)應(yīng)于287.6eV(C=O O—C—O)歸因于羧酸酯和酰胺;5對(duì)應(yīng)于288.5eV(O=C—CH O=C—O—[R])歸因于羧基或脂官能團(tuán)[13-14,31].活細(xì)胞和滅活細(xì)胞能譜圖的不同是峰值2,3,4(C1s)的面積百分比的變化,這些峰的相對(duì)強(qiáng)度和位置的變化表明,官能團(tuán)如酰胺基或羧酸酯官能團(tuán)對(duì)熱滅活細(xì)胞的高吸附能力具有重要影響.

2.8 滅活與非滅活植物乳桿菌對(duì)U(VI)的生物去除機(jī)制

活細(xì)胞對(duì)水中U(VI)的去除是有代謝作用的吸附.植物乳桿菌表面的靜電作用及官能團(tuán)(如酰胺基,羥基,羧基、磷脂基等)的絡(luò)合配位等作用與鈾酰離子結(jié)合,活細(xì)胞通過(guò)代謝釋放磷酸鹽沉淀水溶液中的U(VI)或通過(guò)細(xì)胞表面的還原酶將可溶解態(tài)的U(VI) 還原為難溶態(tài)的U(IV),在菌體表面形成了鱗片狀的磷酸鈾酰沉淀,從而減緩鈾對(duì)細(xì)胞的毒性.植物乳桿菌的這種代謝富集機(jī)制對(duì)于阻止水體中放射性核素的遷移具有重要意義.

滅活細(xì)胞對(duì)U(VI)的去除是非代謝性的生物吸附作用.滅活菌體通過(guò)細(xì)胞表面的靜電吸附,細(xì)胞表面蛋白質(zhì)或多糖的羥基、蛋白質(zhì)酰胺基以及羧基、磷酸脂基等絡(luò)合配位作用與鈾酰離子結(jié)合.細(xì)胞表面結(jié)合的鈾呈凝膠塊狀沉淀.滅活過(guò)程導(dǎo)致蛋白質(zhì),核酸等大分子物質(zhì)的破壞,細(xì)胞形狀發(fā)生變化,表面粗糙,增大了比表面積,從而增加了熱滅活植物乳桿菌的U(VI)生物吸附能力.滅活植物乳桿菌是一種合適的鈾生物吸附劑.

3 結(jié)論

3.1 滅活與非滅活條件下,植物乳桿菌均可以去除U(VI),滅活細(xì)胞吸附U(VI)的能力要高于活細(xì)胞.在U(VI)的初始濃度為10mg/L、pH值為6.0、37℃, 120min條件下,滅活菌體的U(VI)去除率達(dá)94.7%,活菌體為88.9%.溶液的pH值對(duì)活菌體和滅活菌體去除鈾的效果有顯著影響.pH值為3.0~6.0時(shí),活菌體與滅活菌體對(duì)鈾的去除率與pH值呈正相關(guān), pH6.0~7.0時(shí),呈負(fù)相關(guān).

3.2 與活菌體相比,滅活菌體具有更高的鈾吸附容量.在生物量濃度為0.06~0.24mg/L,pH值(3.0~7.0)條件下,滅活菌體與活菌體的U(VI)累積容量比均大于1.

3.3 植物乳桿菌活細(xì)胞和滅活細(xì)胞表現(xiàn)出不同的U(VI)去除機(jī)理.活細(xì)胞可以通過(guò)吸附、配位絡(luò)合、生物礦化、生物還原等多種機(jī)制去除水中的U(VI);而滅活細(xì)胞僅通過(guò)單純的物理化學(xué)吸附去除水中的U(VI).

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Mechanism of U (VI) removal byunder inactivated and non-inactivated conditions.

LIU Jin-xiang1,2, PU Ya-shuai1, XIE Shui-bo1,2, LIU Ying-jiu1, Chen Zi-geng1, Li Shi-you1*

(1.School of Civil Engineering, University of South China, Hengyang 421001, China；2.Hunan Province Key Laboratory of Pollution Control and Resource Reuse Technology, University of South China, Hengyang 421001, China)., 2019,39(7)：2880~2888

The uranium removal tests of inactivated and non-inactivatedwere carried out under different pH and biomass concentration conditions, and the mechanism of uranium removal bywas discussed. Based on SEM-EDS, FTIR, XPS, and XRD, the microscopic mechanism of the interaction between uranium and microbial cell surface and the characteristics of sediments on the cell surface were analyzed. The ability ofto adsorb uranium was significantly improved after heat inactivation. With the pH 6.0, 37℃ and the 10mg/L U(VI), the removal efficiency of U(VI) by heat-killed cells was up to 94.7% during 120min, while the removal efficiency was only 88.9% by live cells. The inactivated bacteria had higher uranium adsorption capacity. At the biomass concentration of 0.06~0.24mg/L and pH value of 3.0~7.0, the rate() of accumulative capacity of U(VI) of inactivated bacteria to that of living bacteria is greater than 1. SEM-EDS、FTIR result illustrated the U(VI) could be adsorbed or coordinated on the surface of the active and inactivated cells through functional groups such as hydroxyl, acyl and carboxyl groups. There were 4 distinct crystal peaks of uranyl phosphate compound at 2(18.023, 25.492, 27.343 and 40.813°) in the XRD spectrum of living bacteria, while no crystal peaks in the spectrum of inactivated bacteria. XRD result indicated U(VI) can be precipitated with the form of uranyl phosphate by biological metabolism of live cells. There were the peaks attributed U(IV) at U 4f 7/2 with binding energy of 381.20eV and U 4f 5/2 with 390.95eV in the XPS energy spectrum of living bacteria. While There was no the peaks attributed U(IV) in the spectrum of inactivated bacteria. XPS result indicated that U(VI) can be induced to U(IV) by living bacteria.

；U(VI)；biosorption；mineralization mechanism；bioreduction

X172

A

1000-6923(2019)07-2880-09

劉金香(1972-),女,湖南常德人,教授,博士,主要從事(放射性)污染控制與修復(fù)、水處理理論與技術(shù)等方面的研究.發(fā)表論文50余篇.

2018-11-20

國(guó)家自然科學(xué)基金資助項(xiàng)目(11475080);湖南省南華大學(xué)博士科研啟動(dòng)基金項(xiàng)目(2016XQD06)

* 責(zé)任作者, 副教授, lsy730723@163.com