菌株Cupriavidus sp. DT-1對(duì)液體和土壤中TCP的降解

陸 鵬,周 慧,袁 夢

菌株sp. DT-1對(duì)液體和土壤中TCP的降解

陸 鵬*,周 慧,袁 夢

(安徽師范大學(xué)生命科學(xué)學(xué)院,安徽省分子酶學(xué)與重大疾病機(jī)理研究重點(diǎn)實(shí)驗(yàn)室,安徽 蕪湖 241000)

采用液質(zhì)聯(lián)用(HPLC-MS)的方法檢測菌株Cupriavidus sp. DT-1降解2-羥基吡啶(2-HP)的代謝產(chǎn)物.并用三親結(jié)合、熒光定量PCR (q-PCR)方法評(píng)價(jià)降解菌對(duì)3,5,6-三氯-2-吡啶酚(TCP)污染土壤的修復(fù)效果.結(jié)果表明,菌株可以進(jìn)一步降解2-HP,依次生成尼古丁藍(lán)、馬來酰胺酸和反丁烯二酸,直至轉(zhuǎn)化成菌株DT-1生長的碳源.接種菌株DT-1對(duì)污染土壤中TCP的降解起到較大的促進(jìn)作用,2組試驗(yàn)土壤中TCP(50mg/kg)降解率分別為94.4%和86.7%,未接種菌株的土壤中TCP降解率僅為20.4%和28.4%.帶有綠色熒光蛋白基因gfp標(biāo)記的菌株DT-1-gfp可在土壤中存活35d以上,并對(duì)TCP污染土壤的細(xì)菌群落豐度有顯著的恢復(fù)作用.

sp. DT-1；3,5,6-三氯-2-吡啶酚(TCP)；2-羥基吡啶(2-HP)；生物修復(fù)

3,5,6-三氯-2-吡啶酚 (TCP)是一種典型的有機(jī)氯污染物,在自然環(huán)境中半衰期長達(dá)65～360d[1].它是毒死蜱降解的主要產(chǎn)物,由于其具有比母體化合物更強(qiáng)的水溶性和流動(dòng)性,因此可引起水環(huán)境和土壤更廣泛的污染[2-8].2-羥基吡啶 (2-HP)是一種-雜環(huán)類有機(jī)污染物,具有一定的生物毒性和抗生物降解特性[9].此類污染物水溶性強(qiáng),容易滲入地下水和土壤,對(duì)生態(tài)環(huán)境和人類健康構(gòu)成威脅[10-11]. TCP和2-HP兩種物質(zhì)在結(jié)構(gòu)上具有很大相似性,均以吡啶作為母體化合物,其區(qū)別僅在于氯原子數(shù)目的不同.同時(shí)2-HP也是TCP、尼古丁等-雜環(huán)類化合物降解的主要中間代謝產(chǎn)物[12-13].因此,2-HP與TCP的降解是緊密聯(lián)系的,去除環(huán)境中2-HP的殘留不僅可消除其污染,同時(shí)對(duì)修復(fù)其前體劇毒化合物TCP造成的生態(tài)破壞具有重要意義.微生物降解具有效率高、成本低、無二次污染等特點(diǎn),是消除TCP殘留的一種環(huán)境友好型方法[14-15].目前各國學(xué)者已經(jīng)從不同屬微生物中分離獲得多種具有降解TCP能力的菌株,包括[5]、、、、、、、、、、等[16-27].然而,TCP的完整生物降解途徑仍然是未知的.多個(gè)研究表明3,6-二羥基吡啶-2,5-二酮是TCP降解過程中常見的中間代謝物[5,20,28].sp. DT-1是課題組前期分離獲得的TCP降解菌株,與其他菌株不同,DT-1具有獨(dú)特的TCP降解途徑,以2-HP為主要中間代謝產(chǎn)物,并對(duì)其進(jìn)一步礦化,達(dá)到完全降解TCP的效果[12].前期已推導(dǎo)出TCP降解為2-HP的代謝途徑,然而2-HP的后續(xù)降解過程尚不清楚.

本研究探究了菌株DT-1在液體培養(yǎng)基中對(duì)TCP和2-HP的降解,鑒定出2-HP降解的代謝產(chǎn)物,結(jié)合前期研究成果,推導(dǎo)出菌株DT-1降解TCP的完整代謝途徑.同時(shí)通過熒光標(biāo)記,構(gòu)建基因工程菌,結(jié)合qPCR的方法,研究了菌株DT-1對(duì)土壤中TCP的降解及其存活能力,評(píng)價(jià)降解菌對(duì)TCP污染土壤的生物修復(fù)效果.

1 材料與方法

1.1 試驗(yàn)材料

TCP (純度≥99%)、2-HP (純度≥99%)購自一基實(shí)業(yè)有限公司(中國上海),用無菌水配制成10g/L的濃縮原液.甲醇(HPCL級(jí))和其他試劑(AR級(jí))均購自上?；瘜W(xué)試劑有限公司(中國上海).

菌株sp. DT-1為本實(shí)驗(yàn)室分離獲得;SM10 (pir) pUT- mini-Tn5-(Ampr),HB101pRK2013 (Kmr),PowerSoil DNA Isolation Kit試劑盒,qPCR相關(guān)試劑購自南京金斯瑞生物科技有限公司(中國南京).

LB培養(yǎng)基(g/L):蛋白胨10.0,酵母膏5.0, NaCl 10.0, pH值7.0.基礎(chǔ)鹽培養(yǎng)基(MSM)(g/L):NH4NO31.0, K2HPO41.5, KH2PO40.5, NaCl 0.5, MgSO40.2, pH值7.0.配制好的培養(yǎng)基于121.3℃滅菌30min.

1.2 菌液制備

菌株DT-1在LB培養(yǎng)基(30℃,180r/min)中培養(yǎng)至指數(shù)期,于室溫6000離心5min收集菌體.用滅菌的MSM洗滌細(xì)胞2次,并重懸至濃度約2× 108CFU/mL.在液體培養(yǎng)基中進(jìn)行降解試驗(yàn)時(shí),細(xì)胞接種濃度約為1×107CFU/mL.

1.3 菌株DT-1對(duì)TCP和2-HP的降解

降解試驗(yàn)分別在含50mg/L TCP和500mg/L 2-HP的100mL MSM中進(jìn)行.試驗(yàn)組接種降解菌后于30℃,180r/min培養(yǎng)24h,每2h收集3mL樣品,測定TCP和2-HP濃度.所有樣本均做3組重復(fù).

1.4 菌體生長量測定

采用上述方法培養(yǎng)菌體,定時(shí)取樣1mL,10倍梯度稀釋獲得10-4～10-1稀釋液,取0.2mL涂布在LB平板上,30℃恒溫培養(yǎng)48h,選擇菌落數(shù)在30～300的平板進(jìn)行計(jì)數(shù).

1.5 化合物提取和檢測

采用高效液相色譜法(HPLC)檢測樣品中TCP的濃度.樣品(3mL)在15000下離心10min,上清液過0.2μm微孔濾膜.冷凍干燥后,溶解于2mL色譜純甲醇,用配備SPD- M20A紫外檢測器(190～800nm)和Agilent C-18柱(250mm×4.6mm, 5μm)的島津LC-201A高效液相色譜儀進(jìn)行分析.流動(dòng)相為甲醇/水(80:20,/),在柱溫25℃下以1.2mL/min的流速輸送.檢測波長230nm.在0.1～100mg/L濃度區(qū)間建立標(biāo)準(zhǔn)曲線,確定TCP的濃度.檢測限(LOD)為0.016mg/L,定量限(LOQ)為0.057mg/L.在0.1～ 100mg/L濃度范圍內(nèi),回收率為96.5%～101.5%,相對(duì)標(biāo)準(zhǔn)偏差(RSD)為1.10%～2.84%.用同樣的方法檢測2-HP濃度.濃度在0.1～100mg/L之間建立標(biāo)準(zhǔn)曲線,回收率和RSD分別為94.6%～103.8%和1.37%～ 2.66%,LOD為0.021mg/L,LOQ為0.075mg/L.

以500mg/L 2-HP為唯一碳源培養(yǎng)菌株DT-1,定時(shí)從培養(yǎng)物中收集樣品,15000離心10min,上清經(jīng)0.2μm纖維過濾器過濾后,冷凍干燥,溶解于1mL色譜純甲醇中,HPLC-MS分析(Finnigan TSQ Quantum Ultra, Thermal, USA),利用電子噴霧進(jìn)行負(fù)離子質(zhì)譜電離,在質(zhì)量范圍為30～400/的條件下檢測,鑒定2-HP降解產(chǎn)物.

1.6 菌株DT-1對(duì)土壤中TCP的降解

試驗(yàn)所用的土壤樣本分別來自安徽師范大學(xué)校園和一處此前未接觸TCP的農(nóng)田土壤.樣品風(fēng)干,篩至2mm均勻顆粒.取20g樣品,105℃干燥24h,測定樣品的含水量.采用凱氏定氮法測定土壤總氮和硝酸鹽氮[29];采用重鉻酸鉀容量法測定有機(jī)質(zhì)[30],土壤樣品的部分特性見表1.

表1 土壤樣品部分理化性質(zhì)

玻璃燒杯(200mL)中加入100g土壤,添加TCP 至終濃度50mg/kg.試驗(yàn)組加入含菌株DT-1的MSM 培養(yǎng)基4.0mL,終濃度約為1×107細(xì)胞/g土壤.對(duì)照組則加入不含DT-1的MSM培養(yǎng)基(4.0mL).土壤樣品在30℃無菌條件下培養(yǎng),定期稱重,重量損失通過添加無菌水來補(bǔ)償.每5d收集5g土壤樣品進(jìn)行TCP濃度分析,共培養(yǎng)35d.

1.7 菌株DT-1的gfp標(biāo)記

編碼熒光蛋白的基因是可用于監(jiān)測目的細(xì)胞最有用的生物標(biāo)記物之一,在污染土壤的生物修復(fù)中有良好的應(yīng)用前景[31-33].將菌株DT-1、SM10 (pir) pUT- mini-Tn5-和HB101pRK2013分別在LB培養(yǎng)基中培養(yǎng)12h.6000室溫離心5min收集細(xì)胞,無菌水洗滌2次并重懸.將3種菌懸液各取5mL,6000離心5min,用20μL LB培養(yǎng)基重懸,將混合菌懸液鋪在濾膜上,置于LB平板內(nèi)30℃培養(yǎng)24h,用生理鹽水洗滌并重懸細(xì)胞,涂布于含有100mg/L氨芐青霉素和卡那霉素的LB平板上,30℃培養(yǎng)24h后,在紫外燈下觀察.

1.8 土壤中重組菌株計(jì)數(shù)

將1g土壤溶解在9mL無菌水中制成懸浮液,依次梯度稀釋至10-4,取0.2mL稀釋液涂布于LB平板上,30℃培養(yǎng)48h,置于紫外燈下檢測,統(tǒng)計(jì)發(fā)出綠色熒光的菌落數(shù).

1.9 熒光定量PCR分析

qPCR是分析土壤細(xì)菌豐度的有效手段[34],為了解接種外源微生物DT-1-對(duì)土壤細(xì)菌數(shù)量的影響,提取土壤總DNA,進(jìn)行熒光定量PCR分析.取0.25g冷凍干燥后的土壤樣品,采用PowerSoil DNA Isolation Kit試劑盒提取土壤總DNA.1.0%瓊脂糖凝膠電泳檢測,并用微量分光光度計(jì)(ND-1000, NanoDrop Technologies,美國)測定其濃度和純度后保存于-20℃.qPCR反應(yīng)體系如下:2 × SYBR Premix Ex Taq 10μL,10μmol/L上下游引物各0.5μL,模板DNA 1.0μL,無菌雙蒸水8μL.標(biāo)準(zhǔn)曲線及每個(gè)樣品重復(fù)3次,同時(shí)設(shè)置空白對(duì)照.qPCR反應(yīng)所用引物338F (5’-ACTCCTACGGGAGGCAGC AG-3’)和518R (5’-ATTACCGCGGCTGCTGG-3’),反應(yīng)條件94℃預(yù)變性2min,94℃變性30s,55℃退火30s,72℃預(yù)延伸30s,35個(gè)循環(huán)[35].

1.10 數(shù)據(jù)處理

試驗(yàn)數(shù)據(jù)使用SPSS 18.0進(jìn)行統(tǒng)計(jì)分析,采用單因素方差分析(one-way ANOVA)和鄧肯(Duncan)法多重比較檢驗(yàn)各處理間的差異顯著性(<0.05).

2 結(jié)果與討論

2.1 液體培養(yǎng)基中TCP和2-HP的降解

如圖1所示,接種后2h,兩種物質(zhì)的濃度均沒有明顯變化,這是由于菌株需要適應(yīng)新的環(huán)境,而負(fù)責(zé)降解的酶還沒有合成.2～10h,TCP的濃度急劇下降,而2-HP的濃度迅速上升,說明菌株DT-1開始表現(xiàn)出以2-HP為主要產(chǎn)物的TCP降解活性.然而2-HP的總濃度小于減少的TCP濃度,表明2-HP被菌株DT-1進(jìn)一步降解(圖2).在10～18h,TCP被完全降解, 2-HP的濃度也隨之迅速下降,直到消失.

圖1 菌株DT-1對(duì)TCP和2-HP的降解

◆ TCP濃度; ■ 2-HP濃度

圖2 菌株DT-1利用2-HP作為唯一碳源的生長降解

◆ 2-HP濃度; ■ 細(xì)胞濃度

菌株DT-1在10h內(nèi)可降解500mg/L高濃度的2-HP,并以其作為生長的唯一碳源.菌株生長和2-HP降解主要發(fā)生在接種后2～8h (圖2).本研究中TCP的最佳初始降解濃度較低,因?yàn)楦邼舛萒CP對(duì)微生物有較大毒性.而2-HP對(duì)微生物的毒性較低,因此本研究采用較高初始濃度的2-HP作為降解底物,以獲取高濃度的代謝產(chǎn)物,便于后續(xù)鑒定.

研究表明,菌株sp. T6、sp. P2和X1T可降解TCP并產(chǎn)生3,6-二羥基吡啶-2,5-二酮,這是一種可以進(jìn)一步礦化分解的中間代謝物[1,5,20].由于未能檢測到后續(xù)降解產(chǎn)物,因此完整的降解途徑尚不清楚.Wang等[8]發(fā)現(xiàn)了一種能夠在厭氧條件下降解TCP的微生物菌群,推測了TCP從脫氯到吡啶環(huán)裂解的完整降解途徑.前期研究表明菌株DT-1能夠在好氧環(huán)境中通過三步脫氯將TCP降解為2-HP[12].這是一種新型的TCP生物降解途徑.為完善該途徑,2-HP的后續(xù)降解過程尚需研究.

2.2 2-HP的降解途徑研究

近幾十年來,2-HP的生物降解途徑已有了較深入的研究.其一是2-HP首先轉(zhuǎn)化為二羥基吡啶,再進(jìn)一步裂解吡啶環(huán).不同微生物產(chǎn)生不同的最終代謝物,如反丁烯二酸[36-37]、5-氨基-5-氧-2-戊烯酸、琥珀酸半醛[38].其二是2-HP首先轉(zhuǎn)化為三羥基吡啶,產(chǎn)生藍(lán)色物質(zhì)[39].在不同微生物作用下,最終代謝產(chǎn)物為延胡索酸[40]、丁二醛[41]和α-酮戊二酸[42].本研究通過HPLC檢測了2-HP及其降解的代謝產(chǎn)物.結(jié)果見圖3.

圖3 2-HP及其降解產(chǎn)物的液相檢測圖譜

A.接種0h (RT= 3.55min); B.接種6h (RT= 2.19, 2.68和3.53min); C.接種10h (RT= 2.20和3.05min)

a. 2-HP MS圖譜; b. 藍(lán)色化合物MS圖譜; c. 保留時(shí)間2.19min化合物的MS圖譜; d. 保留時(shí)間3.05min化合物的MS圖譜

2-HP的保留時(shí)間為3.55min (圖3中A峰).接種菌株DT-1 6h后,培養(yǎng)基顏色變藍(lán),液相檢測到3種化合物,保留時(shí)間分別為2.19, 2.68和3.53min (圖3B).其一為2-HP (保留時(shí)間3.53min),另外兩種應(yīng)為其降解產(chǎn)物.在接種10h的樣品中檢測到兩種化合物,一種保留時(shí)間為2.20min,與6h收集的樣品中檢測到的化合物相同,另一種新的代謝產(chǎn)物保留時(shí)間為3.05min (圖3中C峰).

質(zhì)譜結(jié)果顯示,保留時(shí)間為3.55min的物質(zhì)脫質(zhì)子化離子/= 94.08 [M-H]-,與2-HP的分子離子相匹配(圖4a).保留時(shí)間為2.68min(/=249.10 [M-H]-)的化合物(圖4b)被鑒定為尼古丁藍(lán),它使培養(yǎng)基由無色變?yōu)樗{(lán)色.這說明2-HP的降解產(chǎn)生了2,3,6-三羥基吡啶(2,3,6-THP),并進(jìn)一步在有氧環(huán)境下氧化成尼古丁藍(lán),這是一個(gè)可逆反應(yīng).因此保留時(shí)間為2.19和3.05min的物質(zhì)應(yīng)為2,3,6-THP的降解產(chǎn)物,分別為馬來酰胺酸(/= 114.08[M-H]-) (圖4c)和延胡索酸(115.10[M-H]-)(圖4d).該結(jié)果表明菌株DT-1對(duì)2-HP的降解途徑與菌株sp. (PNO)降解2-HP的途徑相同[35],這是首次在革蘭氏陰性菌中發(fā)現(xiàn)此途徑.結(jié)合前期結(jié)論,推測菌株DT-1降解TCP的完整代謝途徑如圖5所示.

圖5 菌株DT-1降解TCP的代謝途徑

研究表明,能夠?qū)?-HP轉(zhuǎn)化為藍(lán)色物質(zhì)的細(xì)菌均為革蘭氏陽性菌.本研究檢測了革蘭氏陰性細(xì)菌DT-1降解2-HP的代謝產(chǎn)物,并提出降解途徑.發(fā)現(xiàn)2-HP降解產(chǎn)生的主要中間代謝物是一種藍(lán)色物質(zhì)尼古丁藍(lán).這是一個(gè)新的發(fā)現(xiàn),揭示了菌株DT-1降解2-HP的機(jī)制.

2.3 菌株DT-1對(duì)土壤中TCP和2-HP的降解

如圖6所示,在含50mg/kg TCP的校園土壤中,接種菌株DT-1,培養(yǎng)35d后TCP濃度下降到2.8mg/kg (降解率94.4%),大部分降解發(fā)生在10～ 30d之間.降解產(chǎn)生的2-HP濃度很低,最高僅有9.8mg/kg.表明2-HP產(chǎn)生后被立即降解.在未接種的土壤中,約20.4%的TCP在35d內(nèi)自然降解,沒有檢測到2-HP.農(nóng)田土壤中TCP的整體降解趨勢與校園土壤相似.但接種降解菌后的降解率(86.7%)略低于校園土壤(94.4%).未接種的農(nóng)田土壤中TCP的降解率(28.4%)高于未接種DT-1的校園土壤(20.4%).這可能是由于農(nóng)田土壤中營養(yǎng)物質(zhì)豐富,使得土著微生物更加活躍,具有更強(qiáng)自凈能力,但這也競爭性地抑制了菌株DT-1的繁殖.該結(jié)果表明菌株DT-1在不同類型的土壤環(huán)境中都具備降解TCP的能力.

a. 校園土; b. 農(nóng)田土. ● 對(duì)照組土壤中TCP濃度; ◆ 接種DT-1后土壤中TCP濃度; ▲ 接種DT-1后土壤中2-HP濃度; ■ 對(duì)照組

土壤中2-HP濃度

2.4 菌株DT-1的gfp標(biāo)記和示蹤

為了觀察菌株DT-1在土壤中的存活情況,將熒光標(biāo)記基因?qū)刖陜?nèi),構(gòu)建基因工程菌,命名為DT-1-.圖7為菌株DT-1和DT-1-在紫外光下的菌落形態(tài),對(duì)照株DT-1無光澤,而DT-1-發(fā)出綠色熒光,說明基因在菌株DT-1-中高效表達(dá).

圖7 菌株DT-1和DT-1-gfp在紫外燈下的菌落照片

(A) DT-1; (B) DT-1-

工程菌在普通LB平板上無選擇壓力連續(xù)培養(yǎng)20代,在熒光顯微鏡下觀察到同樣的效果,表明基因在菌株DT-1中穩(wěn)定遺傳.降解試驗(yàn)結(jié)果表明,菌株DT-1-的特性與菌株DT-1一致.因此,基因工程菌在功能上與原菌株相當(dāng),可用于TCP污染土壤的生物修復(fù).表2為接種后菌株DT-1-在土壤中的存活情況.接種的初始細(xì)胞濃度為1×107CFU/g土壤,前7d由于土壤中支持細(xì)胞生長的營養(yǎng)物質(zhì)較為豐富,細(xì)菌數(shù)量略有增加.7～21d,由于土壤中本地微生物的增殖競爭性抑制了DT-1-的繁殖,目的細(xì)菌總量急劇減少,且農(nóng)田土壤的抑制作用強(qiáng)于校園土壤.21～35d,目的細(xì)菌種群保持穩(wěn)定,沒有顯著下降,說明土壤微生物群落達(dá)到了平衡狀態(tài).結(jié)果表明,菌株DT-1-能夠適應(yīng)復(fù)雜的土壤環(huán)境,并可存活足夠的時(shí)間來發(fā)揮其生物修復(fù)作用.

表2 菌株DT-1-gfp在校園和農(nóng)田土壤中的含量(′106CFU/g)

2.5 降解菌對(duì)土壤細(xì)菌數(shù)量的影響

添加TCP的兩種土壤中細(xì)菌16S rRNA拷貝數(shù)在不同培養(yǎng)階段均始終低于原始土壤(表3),表明TCP對(duì)土壤微生物具有明顯的毒性;添加TCP+ DT-1-處理的16S rRNA拷貝數(shù)在兩種土壤的培養(yǎng)前期(7d)均顯著低于對(duì)照,說明TCP的毒性作用依然存在;隨著DT-1-在土壤中的定殖和對(duì)TCP降解作用,添加了DT-1-的土壤中細(xì)菌數(shù)量逐漸上升,在35和56d均與對(duì)照無顯著差異,該結(jié)果表明TCP的毒性因?yàn)镈T-1-的降解作用而逐漸減弱,初步證實(shí)降解菌施加對(duì)污染土壤中微生物生態(tài)有顯著的恢復(fù)作用.然而降解菌的添加對(duì)土壤微生物群落結(jié)構(gòu)的影響目前尚未探明,后續(xù)將以此為中心逐步開展研究,以期提高降解菌的應(yīng)用潛力,并評(píng)價(jià)其生態(tài)安全性.

表3 TCP及菌株DT-1-gfp對(duì)土壤細(xì)菌數(shù)量的影響

注:同一列數(shù)據(jù)后不同字母表示不同處理間差異達(dá)到5%顯著水平.

由于對(duì)高毒有機(jī)磷農(nóng)藥的限制和禁止,以毒死蜱為代表的低毒有機(jī)磷農(nóng)藥的市場需求不斷增加.這不可避免地增加了TCP在環(huán)境中積累.TCP的積累強(qiáng)烈地抑制了微生物的生長,而微生物生長的抑制又加劇了TCP的積累,不僅阻礙了TCP本身的降解,還抑制了母體化合物毒死蜱以及其他有機(jī)化合物的降解[15,20,43],使農(nóng)藥殘留對(duì)環(huán)境造成的污染進(jìn)一步加劇.

基于微生物降解的污染土壤生物修復(fù)策略的發(fā)展逐漸被各國學(xué)者所重視.生物修復(fù)作為一種高效、廉價(jià)的生物技術(shù)手段,對(duì)其研究逐漸深入,并將大規(guī)模應(yīng)用于污染環(huán)境的治理[44-48].然而,TCP在土壤中的生物降解研究報(bào)道較少,多數(shù)研究都集中在毒死蜱污染土壤的生物修復(fù)方面.本研究以TCP作為污染底物,研究了不同環(huán)境中微生物對(duì)其降解作用,結(jié)果表明菌株DT-1能夠有效降解水體和土壤中的TCP,在TCP污染農(nóng)田的生物修復(fù)中具有很大的應(yīng)用潛力.

3 結(jié)論

3.1 菌株DT-1可利用2-HP為唯一碳源進(jìn)行生長,并降解濃度為500mg/L的2-HP.HPLC-MS檢測到3種代謝產(chǎn)物尼古丁藍(lán)、馬來酰胺酸和反丁烯二酸. TCP在菌株DT-1作用下經(jīng)過3步脫氯反應(yīng)生成2-HP,進(jìn)而依次代謝產(chǎn)生尼古丁藍(lán),馬來酰胺酸和反丁烯二酸,最終被徹底礦化分解.

3.2 菌株DT-1可降解土壤中濃度為50mg/kg的TCP,在兩類不同的土壤中35d內(nèi)的降解率分別為94.4%和86.7%,并能對(duì)代謝產(chǎn)物2-HP進(jìn)一步降解,大部分降解發(fā)生在10～30d之間.而作為對(duì)照的未接種菌株DT-1的土壤中,自然狀態(tài)下降解的TCP不產(chǎn)生2-HP.

3.3 基因工程菌DT-1-的降解特性、效果與原始菌株相同,并能在土壤中長時(shí)間存活,且添加降解菌后對(duì)TCP污染土壤細(xì)菌群落豐度有一定的恢復(fù)作用,表明該菌株具備應(yīng)用于TCP污染土壤生物修復(fù)的潛力.

[1] Fang L C, Shi T Z, Chen Y F, et al. Kinetics and catabolic pathways of the insecticide chlorpyrifos, annotation of the degradation genes and characterization of enzymes TcpA and Fre inX1T[J]. Journal of Agricultural and Food Chemistry, 2019,67(8):2245-2254.

[2] 陳詩卉,姜錦林,張換朝,等.毒死蜱在我國水稻上登記現(xiàn)狀及水生態(tài)風(fēng)險(xiǎn)評(píng)估 [J]. 中國環(huán)境科學(xué), 2020,40(8):3585-3594.

Chen S H, Jiang J L, Zhang H C, et al. Registration status of chlorpyrifos products for use on rice and its risk assessment for aquatic ecosystem in China [J]. China Environmental Science, 2020,40(8): 3585-3594.

[3] Yang L, Zhao Y H, Zhang B X, et al. Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium [J]. FEMS Microbiology Letters, 2005,251(1):67-73.

[4] Kim J R, Ahn Y J. Identification and characterization of chlorpyrifos- methyl and 3,5,6-trichloro-2-pyridinol degradingsp. strain KR100 [J]. Biodegradation, 2009,20(4):487-497.

[5] Li J Q, Liu J, Shen W J, et al. Isolation and characterization of 3,5,6-trichloro-2-pyridinol degradingsp. strain T6 [J]. Bioresource Technology, 2010,101(19):7479-7483.

[6] Rayu S, Nielsen U N, Nazaries L, et al. Isolation and molecular characterization of novel chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacteria from sugarcane farm soils [J]. Frontiers Microbiology, 2017,8:518.

[7] Dores E F G C, Spadotto C A, Weber O L S, et al. Environmental behavior of chlorpyrifos and endosulfan in a tropical soil in central Brazil [J]. Journal of Agricultural and Food Chemistry, 2016,64(20): 3942-3948.

[8] Wang S H, Zhang C, Lv Z W, et al. Degradation of 3,5,6- trichloro-2-pyridinol by a microbial consortium in dryland soil with anaerobic incubation [J]. Biodegradation, 2019,30(2/3):161-171.

[9] Lataye D H, Mishra I M, Mall I D. Removal of pyridine from aaqueous solution by adsorption on bagasse fly ash [J]. Industrial& Engineering Chemistry Research, 2006,45(11):3934-3943.

[10] Petkevi?ius V, Vaitekūnas J, Stankevi?iūt? J, et al. Catabolism of 2-hydroxypyridine bysp. strain MAK1: a 2-hydroxypyridine 5-monooxygenase encoded bycatalyzes the first step of biodegradation [J]. Applied and Environ mental Microbiology, 2018,84(11):e00387-18.

[11] Chu L, Yu S, Wang J. Degradation of pyridine and quinoline in aqueous solution by gamma radiation [J]. Radiation Physics and Chemistry, 2018,144:322-328.

[12] Lu P, Li Q F, Liu H M, et al. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol bysp. DT-1 [J]. Bioresource Technology, 2013,127:337-342.

[13] Yu H, Tang H Z, Zhu X Y, et al. Molecular mechanism of nicotine degradation by a newly isolated strain,sp. strain SJY1 [J]. Applied and Environmental Microbiology, 2015,81(1):272-281.

[14] Wang S H, Zhang C, Yan Y C. Biodegradation of methyl parathion and p-nitrophenol by a newly isolatedsp. strain Yw12 [J]. Biodegradation, 2012,23(1):107-116.

[15] 曹 禮,徐 琳.微生物降解3,5,6-三氯-2吡啶醇的研究進(jìn)展 [J]. 微生物學(xué)通報(bào), 2015,42(6):1158-1164.

Cao L, Xu L. Research progress in microbial degradation of 3,5,6- trichloro-2-pyridinol [J]. Microbiology China, 2015,42(6):1158-1164.

[16] Feng Y, Racke K D, Bollag J M. Isolation and characterization of a chlorinated-pyridinol-degrading bacterium [J]. Applied and Environ mental Microbiology, 1997,63(10):4096-4098.

[17] Xu G M, Zheng W, Li Y Y, et al. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a newly isolatedsp. strain TRP [J]. International Biodeterioration & Biodegradation, 2008, 62(1):51-56.

[18] Anwar S, Liaquat F, Khan Q M, et al. Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol bystrain C2A1 [J]. Journal of Hazardous Materials, 2009,168(1): 400-405.

[19] Singh D P, Khattar J I S, Nadda J, et al. Chlorpyrifos degradation by the cyanobacteriumsp. Strain PUPCCC 64 [J]. Environmental Science and Pollution Research, 2011,18(8):1351- 1359.

[20] Cao L, Liu H M, Zhang H, et al. Characterization of a newly isolated highly effective 3,5,6-trichloro-2-pyridinol degrading strainP2 [J]. Current Microbiology, 2012,65(3):231- 236.

[21] Abraham J, Silambarasan S. Biodegradation of chlorpyrifos and its hydrolyzing metabolite 3,5,6-trichloro-2-pyridinol bysp. JAS3 [J]. Process Biochemistry, 2013,48(10):1559-1564.

[22] Silambarasan S, Abraham J. Ef?cacy ofsp. JAS4in bioremediation of chlorpyrifos and its hydrolyzing metabolite TCP from agricultural soil [J]. Journal of Basic Microbiology, 2014, 54(1):44-55.

[23] Jabeen H, Iqbal S, Anwar S. Biodegradation of chlorpyrifos and 3,5,6- trichloro-2 -pyridinol by a novel rhizobial strainsp. HN3 [J]. Water and Environment Journal, 2015,29(1):151-160.

[24] Abraham J, Silambarasan S. Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol using a novel bacteriumsp. JAS2: A proposal of its metabolic pathway [J]. Pesticide Biochemistry and Physiology, 2016,126:13-21.

[25] Bempelou E D, Vontas J G, Liapis K S, et al. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeastsand[J]. Ecotoxicology, 2018,27(10):1368-1378.

[26] Bhardwaj A, Verma N. Proficient biodegradation studies of chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol byNJ11strain [J]. Research Journal of Microbiology, 2018,13(1): 53-64.

[27] Aswathi A, Pandey A, Sukumaran R K. Rapid degradation of the organophosphate pesticide-chlorpyrifos by a novel strain ofAR-3 [J]. Bioresource Technology, 2019, 292:122025.

[28] Bhuimbar M V, Kulkarni A N, Ghosh J S. Detoxification of chlorpyriphos byNCIM 2103,NCIM 2010 andNCIM 2036 [J]. Research Journal of Environmental Earth Sciences, 2011,3(5):614-619.

[29] Bulluck L R, Brosius M, Evanylo G K, et al. Organic and synthetic fertility amendments in?uence soil microbial, physical and chemical properties on organic and conventional farms [J]. Applied Soil Ecology, 2002,19(2):147-160.

[30] Ciavatta C, Govi M, Antisari L V, et al. Determination of organic carbon in aqueous extracts of soils and fertilizers [J]. Communications in Soil Science and Plant Analysis, 1991,22(9/10):795-807.

[31] 魏明寶,方呈祥,張甲耀,等.綠色熒光蛋白標(biāo)記在生物修復(fù)中的應(yīng)用[J]. 中國環(huán)境科學(xué), 2004,24(3):290-293.

Wei M B, Fang C X, Zhang J Y, et al. The application of EGFP marker in bioremediation [J]. China Environmental Science, 2004,24(3):290- 293.

[32] Errampalli D, Leung K, Cassidy M B, et al. Applications of the green fluorescent protein as a molecular marker in environmental microorganisms [J]. Journal of Microbiological Methods, 1999, 35(3):187-199.

[33] Elvang A M, Westerberg K, Jernberg C, et al. Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity ofA6cells during degradation of 4-chlorophenol in soil [J]. Environmental Microbiology, 2001,3(1): 32-42.

[34] 陳 娜,劉 毅,黎 娟,等.長期施肥對(duì)稻田不同土層反消化細(xì)菌豐度的影響[J]. 中國環(huán)境科學(xué), 2019,39(5):2154-2160.

Chen N, Liu Y, Li J, et al. Effects of long-term fertilization on the abundance of the key denitrifiers in profile of paddy soil profiles [J]. China Environmental Science, 2019,39(5):2154-2160.

[35] Fierer N, Jackson J A, Vilgalys R, et al. Assessment of soil microbial community structure by use of taxon-specific quantitative pcr assays [J]. Applied and Environmental Microbiology, 2005,71(7):4117-4120.

[36] Zhao S X, Hu C H, Guo L Z, et al. Isolation of a 3-hydroxypyridine degrading bacterium,sp. DW-1, and its proposed degradation pathway [J]. AMB Express, 2019,9(1):65-73.

[37] Stankevi?iūt? J, Vaitekūnas J, Petkevi?ius V, et al. Oxyfunctionalization of pyridine derivatives using whole cells ofsp. MAK1 [J]. Scientific Reports, 2016,6(1):39129.

[38] Zefirov N S, Agapova S R, Terentiev P B, et al. Degradation of pyridine byandstrains [J]. FEMS Microbiology Letters, 1994,118(1/2):71-74.

[39] 胡春輝,徐 青,于 浩.sp. 2PR降解2-羥基吡啶動(dòng)力學(xué)及降解特性研究[J]. 中國生物工程雜志, 2017,37(8):31-38.

Hu C H, Xu Q, Yu H. Characteristics and kinetic study of 2-hydroxypyridine degradation by a novel bacteriumsp. 2PR [J]. China Biotechnology, 2017,37(8):31-38.

[40] Shukla O P, Kaul S M. Microbiological transformation of pyridine N-oxide and pyridine bysp [J]. Canadian Journal of Microbiology, 1986,32(4):330-341.

[41] Khasaeva F, Vasilyuk N, Terentyev P, et al. A novel soil bacterial strain degrading pyridines [J]. Environmental Chemistry Letters, 2010,9(3): 439-445.

[42] Vaitekūnas J, Gasparavi?iūt? R, Rutkien? R, et al. A 2- hydroxypyridine catabolism pathway instrain PY11 [J]. Applied and Environmental Microbiology, 2016, 82(4):1264-1273.

[43] Singh B K, Walker A, Morgan J A W, et al. Biodegradation of chlorpyrifos bystrain B-14and its use in bioremediation of contaminated soils [J]. Applied and Environmental Microbiology, 2004,70(8):4855-4863.

[44] 王海蘭,臧海蓮,成 毅,等.氯嘧磺隆降解菌的篩選及對(duì)污染土壤的生物修復(fù) [J]. 中國環(huán)境科學(xué), 2018,38(4):1473-1480.

Wang H L, Zang H L, Cheng Y, et al. Screening of a chlorimuron- ethyl-degrading strain and chlorimuron-ethyl-contaminated soil bioremediation [J]. China Environmental Science, 2018,38(4):1473- 1480.

[45] Semple K T, Reid B J, Fermor T R. Impact of composting strategies on the treatment of soils contaminated with organic pollutants [J]. Environ mental Pollution, 2001,112(2):269-283.

[46] Akbar S, Sultan S. Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement [J]. Brazilian Journal of Microbiology, 2016,47(3):563-570.

[47] Uqab B, Mudasir S, Nazir R. Review on bioremediation of pesticides [J]. Journal of Bioremediation and Biodegradation, 2016,7(3):343.

[48] Shishir T A, Mahbub N, Kamal N E. Review on bioremediation: a tool to resurrect the polluted rivers [J]. Pollution, 2019,5(3):555-568.

Biodegradation of 3,5,6-trichloro-2-pyridinol bysp. DT-1 in liquid and soil environments.

LU Peng*, ZHOU Hui, YUAN Meng

(Anhui Key Laboratory of Molecular Enzymology and Major Disease Mechanism research, College of Life Sciences, Anhui Normal University, Wuhu 241000, China)., 2021,41(6)：2780～2787

sp. DT-1 was a 3,5,6-trichloro-2-pyridinol (TCP)-degrading strain which could transform TCP to 2-hydroxypyridine (2-HP). Liquid-mass spectrometry (HPLC-MS) was used to detect the degradation products of 2-HP, And the methods of triparetal conjugation, quantitative real-time PCR (q-PCR) were used to evaluate the remediation effect of TCP-contaminated soils by the degrading-bacterium. Results showed that strain DT-1 was able to further degrade 2-HP, and sequentially produced nicotine blue, maleamic acid and fumaric acid, until it was transformed into the carbon source that could support the growth of strain DT-1. Pilot experiment showed that inoculation of strain DT-1 remarkably accelerated the elimination of TCP in soils. The degradation rates of TCP in inoculated soils were 94.4% and 86.7%, while those in uninoculated soils were 20.4% and 28.4%, respectively. Green fluorescent protein encoding gene gfp harbored strain DT-1-gfp could survive in soils for more than 35d. The results of q-PCR showed that inoculation of strain DT-1-gfp significantly improved the recovery of bacterial community abundance in the TCP-contaminated soils.

sp. DT-1；3,5,6-trichloro-2-pyridinol (TCP)；2-hydroxypyridine (2-HP)；bioremediation

X172

A

1000-6923(2021)06-2780-08

2020-10-28

安徽省高校自然科學(xué)研究項(xiàng)目(KJ2020A0084)

* 責(zé)任作者, 講師, lupeng_2007@126.com

陸 鵬(1986-),男,江蘇徐州人,講師,博士,主要研究方向?yàn)榄h(huán)境微生物學(xué).發(fā)表論文8篇.