一株耐酸產(chǎn)氫突變株P(guān)antoea agglomerans的篩選與產(chǎn)氫特性

劉洪艷,朱大玲,王文磊 (天津科技大學(xué)海洋科學(xué)與工程學(xué)院,天津市海洋資源與化學(xué)重點(diǎn)實(shí)驗(yàn)室,天津 300457)

一株耐酸產(chǎn)氫突變株P(guān)antoea agglomerans的篩選與產(chǎn)氫特性

劉洪艷*,朱大玲,王文磊 (天津科技大學(xué)海洋科學(xué)與工程學(xué)院,天津市海洋資源與化學(xué)重點(diǎn)實(shí)驗(yàn)室,天津 300457)

以紅樹林污泥中分離的厭氧發(fā)酵產(chǎn)氫細(xì)菌Pantoea agglomerans BH18為出發(fā)菌株,利用轉(zhuǎn)座子Tn7隨機(jī)插入菌株基因組DNA,通過卡那霉素篩選與 PCR擴(kuò)增驗(yàn)證,獲得一批轉(zhuǎn)座子插入突變菌株.起始 pH4.0培養(yǎng)條件下,以產(chǎn)氫量為指標(biāo)分離獲得一株耐酸產(chǎn)氫突變菌株TB220.多次傳代結(jié)果表明,突變菌株TB220具有穩(wěn)定的產(chǎn)氫遺傳特性.起始pH3.5～7.0范圍內(nèi),突變菌株TB220最適產(chǎn)氫pH值為6.0,產(chǎn)氫量為(2.39±0.08)mol H2/mol葡萄糖.起始pH4.0和葡萄糖濃度10g/L的海水培養(yǎng)條件下,突變菌株TB220產(chǎn)氫量為(0.47 ± 0.02)mol H2/mol葡萄糖,比野生菌株高70%,表現(xiàn)出較強(qiáng)耐酸性.

成團(tuán)泛菌；產(chǎn)氫量；轉(zhuǎn)座子耐酸

生物制氫是目前化石能源燃料的替代途徑之一,主要包括藻類、光合細(xì)菌和厭氧發(fā)酵細(xì)菌制氫3種方式,其中厭氧發(fā)酵細(xì)菌產(chǎn)氫效率要高于藻類和光合細(xì)菌[1-2].近年來,研究人員將有機(jī)廢水或廢渣等處理與厭氧發(fā)酵細(xì)菌制氫技術(shù)結(jié)合起來,使得厭氧發(fā)酵細(xì)菌制氫技術(shù)具有更廣闊的應(yīng)用前景[3].目前,已報(bào)道高效厭氧發(fā)酵產(chǎn)氫細(xì)菌,例如: Enterobacter cloacae IIT-BT 08、Enterobacter aerogenes 、 Clostridium acetobutylicum ATCC 824、 Ethanoligenens harbinense YUAN-3、Pantoea agglomerans等[4-8].這些菌株產(chǎn)氫量一般在 2mol H2/mol 葡萄糖左右,而厭氧發(fā)酵細(xì)菌產(chǎn)氫量理論值為 4mol H2/mol葡萄糖.厭氧發(fā)酵細(xì)菌在產(chǎn)氫過程中會(huì)同時(shí)產(chǎn)生有機(jī)酸,有機(jī)酸的累積將導(dǎo)致發(fā)酵液 pH值降低,發(fā)酵細(xì)菌的生理代謝活動(dòng)逐漸受到抑制,甚至停止,即產(chǎn)生有機(jī)酸反饋抑制[9-10],因而造成菌株實(shí)際產(chǎn)氫量與理論產(chǎn)氫量相差比較大,酸累積也成為了細(xì)菌發(fā)酵制氫工業(yè)化生產(chǎn)的制約因素.從目前研究來看,解決發(fā)酵細(xì)菌的有機(jī)酸反饋抑制主要有2種途徑:一是采用厭氧發(fā)酵細(xì)菌與光合細(xì)菌(或酵母菌)耦聯(lián)發(fā)酵產(chǎn)氫,通過后者對(duì)小分子有機(jī)酸的利用與消除,重新提高發(fā)酵液pH 值[9-14].然而,無(wú)論是光合細(xì)菌還是酵母菌,其自身產(chǎn)氫速率很低,耦聯(lián)發(fā)酵產(chǎn)氫的整個(gè)過程在經(jīng)濟(jì)可行性上仍是個(gè)問題[15].二是在實(shí)際生產(chǎn)中采用化學(xué)中和法維持產(chǎn)氫體系中的 pH值,但該法既不經(jīng)濟(jì),又容易導(dǎo)致反應(yīng)器生態(tài)系統(tǒng)受到破壞[16].因此,引入高酸條件下仍具有較強(qiáng)代謝活力的耐酸型產(chǎn)氫細(xì)菌,將有利于提高產(chǎn)氫效率.

在野生型菌株基礎(chǔ)上,通過誘變手段獲得產(chǎn)氫突變菌株的相關(guān)研究已經(jīng)開展[16-21].這些誘變手段主要集中于物理或化學(xué)手段,然而關(guān)于分子生物學(xué)手段誘變,比如轉(zhuǎn)座子插入突變技術(shù),應(yīng)用于厭氧發(fā)酵細(xì)菌的報(bào)道還比較少.本研究利用分離自海洋環(huán)境的產(chǎn)氫細(xì)菌 Pantoea agglomerans為出發(fā)菌株,采用Tn7轉(zhuǎn)座子構(gòu)建菌株的轉(zhuǎn)座子突變文庫(kù),在此基礎(chǔ)上篩選產(chǎn)氫突變菌株并進(jìn)行耐酸性能研究,為進(jìn)一步構(gòu)建耐酸性產(chǎn)氫工程菌株奠定基礎(chǔ).

1 材料與方法

1.1 菌種

厭氧發(fā)酵產(chǎn)氫細(xì)菌Pantoea agglomerans野生菌株分離自紅樹林污泥,由本實(shí)驗(yàn)室保存.

1.2 培養(yǎng)基

野生菌株培養(yǎng)采用LM培養(yǎng)基(g/L):葡萄糖20、胰蛋白胨 4,牛肉膏 2,酵母粉 1,NaCl 30, K2HPO41,MgCl20.1,FeSO4·7H2O 0.1,L-半胱氨酸 0.5.微量元素液(g/L,MnSO4·7H2O 0.01, ZnSO4·7H2O 0.05,H3BO30.01,CaCl2·2H2O 0.01, Na2MoO40.01,CoCl2·6H2O 0.2) 10mL,維生素溶液(g/L,L-抗壞血酸VC0.025,檸檬酸0.02,吡哆醛0.05,對(duì)氨基苯甲酸 0.01,生物素 0.01,維生素 B10.02,核黃素0.0250) 10mL.菌株活化采用LB培養(yǎng)基(g/L):胰蛋白胨 10,酵母提取物 5,NaCl 10.菌株感受態(tài)細(xì)胞制備采用2×YT培養(yǎng)基(g/L):胰蛋白胨 16,酵母提取物10,NaCl 4.復(fù)蘇電轉(zhuǎn)化產(chǎn)物采用SOC培養(yǎng)基(g/L):胰蛋白胨 2 0,酵母提取物5.0,NaCl 0.5,KCl 1.86,MgCl22.03,葡萄糖3.6.

1.3 轉(zhuǎn)座子突變菌株P(guān)antoea agglomerans

1.3.1 感受態(tài)細(xì)胞的制備 (1)活化 Pantoea agglomerans菌株接種于 50mL 2×YT培養(yǎng)基中,18℃ 120r/min培養(yǎng)使細(xì)菌分光光度值達(dá)到0.4～0.6. (2) 4℃,2500g離心15min,棄上清收集菌體,用預(yù)冷的 50mL雙蒸水輕輕懸浮菌體.(3) 重復(fù)步驟(2)3次,預(yù)冷雙蒸水的體積分別為 25,10, 5mL,最后于4℃,2500g離心15min收集菌體. (4)向沉淀中加140μL預(yù)冷雙蒸水和DMSO 2μL,懸浮的感受態(tài)細(xì)胞等量分裝(40μL)于無(wú)菌離心管中,立刻或-80℃保存用于電轉(zhuǎn)化.

1.3.2 轉(zhuǎn)座過程 首先,反應(yīng)體系中加入1μL轉(zhuǎn)座子質(zhì)粒 pGPS3、1μL DNA和 1μL轉(zhuǎn)座酶TnsABC,加水至總體積20μL,37 ℃溫育1h.然后,利用DNA聚合酶Ⅰ填補(bǔ)轉(zhuǎn)座插入反應(yīng)產(chǎn)生的缺口,利用酚/氯仿抽提后,加 1μL DNA聚合酶Ⅰ, 3μL 10×Buffer, 9μL dNTP,室溫放置 15min,加1μLT4連接酶(400U,ATP終濃度為 1mmol/L), 16℃溫育 4h.最后,利用稀有反應(yīng)酶 PISceⅠ ( VDE)酶切轉(zhuǎn)座產(chǎn)物,使轉(zhuǎn)座子重組 DNA能夠有效轉(zhuǎn)化到大腸桿菌細(xì)胞中.酚/氯仿抽提后,加入6μL PI-SceⅠ (6U),5μL 10×PI- SceⅠ緩沖液, 0.5μL牛血清蛋白,18.5μL水,37℃溫育1～2h后, -20℃中保存留用.

1.3.3 電轉(zhuǎn)化步驟 把0.2～0.5μg轉(zhuǎn)座產(chǎn)物加入到 40μL感受態(tài)細(xì)胞中,混合后于冰上放置.設(shè)置電轉(zhuǎn)化儀(BIO-RAD)參數(shù)為電壓 1.8kV.將冰上放置的混合物加入到 0.2cm預(yù)冷電轉(zhuǎn)杯中開始電擊.電擊產(chǎn)物快速?gòu)碾娹D(zhuǎn)化杯中轉(zhuǎn)移到裝有1mL SOC培養(yǎng)基的離心管內(nèi),37℃, 150r/min復(fù)蘇1h.復(fù)蘇產(chǎn)物于2000g離心1min,棄上清,剩余100μL用于懸浮細(xì)胞,涂布于含濃度卡那霉素的LB培養(yǎng)基中,37℃過夜培養(yǎng).

1.4 耐酸產(chǎn)氫突變菌株的篩選

1.4.1 轉(zhuǎn)座子突變菌株的鑒定 在 LB培養(yǎng)平板(卡那霉素濃度 40μg/mL)上生長(zhǎng)的菌落,即表示可能發(fā)生了轉(zhuǎn)座.隨機(jī)挑取單菌落340個(gè), 37℃過夜培養(yǎng), 2500g離心后,提取細(xì)菌基因組DNA,作為PCR反應(yīng)的模板.引物序列按轉(zhuǎn)座子突變?cè)噭┖刑峁?引物P1: 5'-TTAAGGATTATTTAGG-GAAG-3';P2: 5'-ACAATAAAGTCTTAAACTAG-3'.PCR反應(yīng)體系(50μL):buffer 5 μL(10×),引物P1和P2各1μL(10μmol/L),dNTP 1μL(10 mmol/ L),Taq DNA聚合酶1 μL(5U/L),模板lμL,超純水40μL.反應(yīng)程序?yàn)?94℃ 4min;94℃ 30s,49℃30s;72℃ 1min,30個(gè)循環(huán);72℃, 10min.

1.4.2 突變菌株耐酸性能測(cè)定 突變菌株接種于不同pH值(3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0)的發(fā)酵培養(yǎng)基,測(cè)定各突變菌株生長(zhǎng)及產(chǎn)氫指標(biāo)(細(xì)菌生物量、發(fā)酵液的pH值、氧化還原電位ORP、葡萄糖利用率和產(chǎn)氫量等,篩選耐酸產(chǎn)氫突變菌株.

1.5 分析方法

pH值和氧化還原電位測(cè)定采用 DLELTA–320型酸度計(jì)量.A600采用島津分光光度計(jì).氣體收集采用排水法.利用氣相色譜儀(型號(hào) 6820, Agilent)測(cè)定發(fā)酵氣體中氫氣的含量.氮?dú)庾鲚d氣,流量為 30mL/min,色譜柱填料為 5A分子篩(60/80目),柱長(zhǎng) 2m,檢測(cè)器為熱導(dǎo)檢測(cè)器(TCD),柱溫、進(jìn)樣器和檢測(cè)器分別為40,200,200℃.

2 結(jié)果與討論

2.1 轉(zhuǎn)座子插入突變菌株的鑒定

野生菌株P(guān)antoea agglomerans接種于不同起始pH值的培養(yǎng)液中,結(jié)果表明,菌株能夠在起始pH5.0的條件下產(chǎn)氫,產(chǎn)氫量為(0.6 ± 0.02) mol H2/mol葡萄糖.Chen等[22]報(bào)道 Clostridium butyricum CGS5在起始pH值為5.0時(shí),不能產(chǎn)氫.支曉鵬等[23]研究發(fā)現(xiàn) Enterobacter sp.C32在起始 pH值 5.0的產(chǎn)氫微弱,只有 3.3mL/L.菌株P(guān)antoea agglomerans能夠在起始5.0的條件下進(jìn)行產(chǎn)氫,表明該菌株具有一定的耐酸性,作為本實(shí)驗(yàn)轉(zhuǎn)座子Tn7插入突變的出發(fā)菌株.

利用轉(zhuǎn)座子對(duì)成團(tuán)泛菌進(jìn)行隨機(jī)突變,構(gòu)建轉(zhuǎn)座突變體文庫(kù),用以篩選耐酸型產(chǎn)氫突變株.在含卡那霉素濃度40μg/mL分離瓊脂平板上長(zhǎng)出的單菌落即為插入轉(zhuǎn)座子的突變體,陰性對(duì)照無(wú)菌落生長(zhǎng).提取突變菌株基因組DNA,經(jīng)PCR擴(kuò)增驗(yàn)證,突變菌株的PCR擴(kuò)增產(chǎn)物在1699bp處具有特異性片段(圖1),該片段大小與轉(zhuǎn)座質(zhì)粒陽(yáng)性對(duì)照的擴(kuò)增片段大小一致,證實(shí)突變菌株含有Tn7轉(zhuǎn)座子插入片段.以野生菌株 DNA為模板,PCR產(chǎn)物無(wú)擴(kuò)增片段.在隨機(jī)挑選340個(gè)菌落中,通過 PCR檢測(cè)發(fā)現(xiàn)有 331個(gè)突變菌株含有Tn7轉(zhuǎn)座子插入片段.

圖1 Tn7的PCR檢測(cè)Fig.1 PCR amplification of Tn7 from the genome of mutants

2.2 耐酸產(chǎn)氫突變菌株的篩選

轉(zhuǎn)座子插入突變菌株接種于 LM液體培養(yǎng)基中,在起始 pH4.0條件下進(jìn)行厭氧發(fā)酵產(chǎn)氫和耐酸馴化篩選.以Pantoea agglomerans野生菌株產(chǎn)氫量為對(duì)照,隨機(jī)挑取的331株突變株中,篩選出9個(gè)突變株的產(chǎn)氫量有明顯提高,其他突變菌株的產(chǎn)氫量與野生菌株相當(dāng)或降低.在起始pH4.0培養(yǎng)條件下,以單位葡萄糖產(chǎn)氫氣的摩爾數(shù)為標(biāo)準(zhǔn)進(jìn)行比較,突變菌株TB220的產(chǎn)氫量最高,為(0.47 ± 0.02 )mol H2/mol 葡萄糖,比野生菌株提高70%.

2.3 突變菌株TB220產(chǎn)氫特性

2.3.1 穩(wěn)定性 突變菌株TB220接種到厭氧平板培養(yǎng)基上作為第1代,以后每隔36h傳代1次,分別于血清瓶中厭氧培養(yǎng),測(cè)定其產(chǎn)氣量和氫氣含量,如此連續(xù)傳代,共傳代5次.取子代菌株提取DNA,進(jìn)行PCR檢測(cè),在5代菌株中都能擴(kuò)增出轉(zhuǎn)座子特異性序列(1699bp).以上結(jié)果表明,轉(zhuǎn)座子Tn7的插入具有穩(wěn)定遺傳性,這與Paul等[24]驗(yàn)證Tn5轉(zhuǎn)座子在Burkeholderia glumae中穩(wěn)定性的實(shí)驗(yàn)結(jié)果相一致.突變菌株TB220的產(chǎn)氫穩(wěn)定性是通過5代菌株之間產(chǎn)氫量的差異來確定.由圖2可以看出,突變菌株TB220連續(xù)傳代5次,產(chǎn)氫量和氫氣含量分別維持在(0.46±0.02)mol H2/mol葡萄糖和(25.93±2.39)%.

圖2 突變菌株TB220的產(chǎn)氫穩(wěn)定性Fig.2 Hydrogen production stability of mutantTB220

2.3.2 產(chǎn)氫過程 圖3可以看出,起始pH4.0時(shí),突變菌株TB220在發(fā)酵12h進(jìn)人對(duì)數(shù)生長(zhǎng)期,生物量最大值為 1.284,較原始菌株明顯提高(原始菌株為0.646).突變菌株TB220發(fā)酵液pH值在發(fā)酵12h后開始明顯下降,在發(fā)酵時(shí)間24h時(shí)發(fā)酵液 pH值基本不再變化,表明產(chǎn)氫主要發(fā)生在發(fā)酵時(shí)間 12～24h,產(chǎn)氫過程中發(fā)酵液 pH值在3.79～3.44之間.氧化還原電位(ORP)能夠作為產(chǎn)氫過程的一個(gè)實(shí)時(shí)監(jiān)測(cè)指標(biāo),高產(chǎn)氫率對(duì)應(yīng)于發(fā)酵液的低ORP值[8].在產(chǎn)氫過程中發(fā)酵液ORP始終處于一個(gè)比較低的值(-33mV左右).

圖3 起始pH 4.0培養(yǎng)條件下突變菌株TB220pH值, ORP和OD600變化Fig.3 Variation of pH,ORP and OD600 of mutantTB220 at the initial pH value of 4.0

2.3.3 耐酸性 由圖4可見,在起始pH3.5～10.0的范圍內(nèi)突變菌株都能生長(zhǎng),最適生長(zhǎng) pH7.0.突變菌株TB220在pH3.5時(shí)不能產(chǎn)氫,在pH4.0～7.0測(cè)定其產(chǎn)氫特性.由圖 4可以看出,突變菌株TB220在起始 pH4.0～7.0的范圍內(nèi)都能夠產(chǎn)氫,最適產(chǎn)氫pH6.0,產(chǎn)氫量為(2.39±0.08)mol H2/mol葡萄糖.Yokoi等[16]分離到的耐酸產(chǎn)氣腸桿菌HO-39最適發(fā)酵產(chǎn)氫pH值為6～7,在pH4.0下也能夠生長(zhǎng)與產(chǎn)氫,這與突變菌株TB220相當(dāng).

圖4 起始pH值對(duì)突變菌株TB220產(chǎn)氫量的影響Fig.4 Effect of initial pH on hydrogen production of mutantTB220

起始pH4.0的培養(yǎng)條件下,成團(tuán)泛菌野生菌株產(chǎn)氫能力明顯下降,產(chǎn)氫量?jī)H為(0.21±0.04) mol H2/mol葡萄糖,而突變菌株產(chǎn)氫量比野生菌株提高70%,顯示出較明顯耐酸產(chǎn)氫特性.與物理或化學(xué)誘變相比,轉(zhuǎn)座子插入突變可以利用轉(zhuǎn)座子的已知序列作為標(biāo)簽,在獲得耐酸產(chǎn)氫突變菌株TB220的基礎(chǔ)上,通過測(cè)定突變基因序列研究突變相關(guān)基因,為在分子水平上解釋相關(guān)耐酸機(jī)理提供前提.

3 結(jié)論

3.1 利用Tn7轉(zhuǎn)座子隨機(jī)插入成團(tuán)泛菌基因組DNA,獲得一批產(chǎn)氫突變菌株,其中突變菌株TB220在起始pH4.0條件下具有較高產(chǎn)氫能力.

3.2 突變菌株TB220連續(xù)傳代5次,通過卡那霉素選擇性篩選和PCR驗(yàn)證,表明該突變菌株具有穩(wěn)定的產(chǎn)氫遺傳特性.

3.3 與野生菌株相比,突變菌株 TB220的耐酸性和產(chǎn)氫能力明顯提高.在起始pH值4.0的海水培養(yǎng)條件下,突變菌株 TB220產(chǎn)氫量為(0.47 ± 0.02)mol H2/mol 葡萄糖,比野生菌株提高 70%,表現(xiàn)出較強(qiáng)耐酸性.

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Screening of an aciduric hydrogen producing mutant Pantoea agglomerans and characterization of hydrogenproduction.

LIU Hong-yan*, ZHU Da-ling, WANG Wen-lei (Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering,Tianjin University of Science and Technology, Tianjin 300457, China) . China Environmental Science, 2012,32(1)：125～129

A Tn7-based transposon was randomly inserted into genomic DNA of Pantoea agglomerans BH18, isolated from mangrove sludge. Mutants were screened by Kanrand amplification of the inserted sequences. At the initial pH 4.0, an aciduric and highly effective hydrogen producing mutant TB220 was screened using hydrogen production as screening index. The aciduric mutant TB220 was tested to have steady heredity and hydrogen-producing capability in several passages. The mutant TB220 was able to produce hydrogen over a wide rang of initial pH from 3.5 to 7.0, with an optimum initial pH of 6.0, and hydrogen production was (2.39 ± 0.08)mol H2/mol glucose. Under the marine conditions with the initial pH of 4.0 and glucose concentration of 10 g/L, hydrogen production of the mutant TB220 was (0.47 ± 0.02) mol H2/mol glucose, increasing by 70% compared with wild type. This indicated that the mutant showed high acid resistance capability.

Pantoea agglomerans；hydrogen production；transposon aciduric

2011-04-04

國(guó)家自然科學(xué)基金資助項(xiàng)目(40906074);天津市海洋資源與化學(xué)重點(diǎn)實(shí)驗(yàn)室開放基金(200912)

* 責(zé)任作者, 講師, hongyanliu1214@163.com

X172

A

1000-6923(2012)01-0125-05

劉洪艷(1977-),女,吉林通化人,講師,博士,主要從事海洋微生物研究.發(fā)表論文10余篇.