郭偉成+李作揚(yáng)+王斌+周文君+張凱+常安妮

摘要：用柴油為唯一碳源的培養(yǎng)基選擇分離海帶表面具有降解柴油性能的細(xì)菌；對(duì)分離菌株進(jìn)行了形態(tài)學(xué)、生理生化特性以及16SrDNA序列分析并測(cè)定其生長(zhǎng)特性；測(cè)定了接菌不同濃度（7×107 cfu/mL、7×108 cfu/mL、7×109 cfu/mL）分離菌對(duì)柴油的降解率；另外，測(cè)定了加入不同濃度葡萄糖（0.5 g/L、1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L）作用7 d后分離菌對(duì)柴油的降解率。結(jié)果顯示：從海帶表面分離到2株降油細(xì)菌，編號(hào)為：HD-4和HD-6。HD-4菌株菌落形態(tài)圓形、直徑1.5 mm、黃色、不透明、邊緣整齊，HD-6菌株菌落圓形，直徑1.0 mm，淺黃色、透明、邊緣整齊。兩株菌均為革蘭氏陰性短桿菌。生理生化特征和16SrDNA序列分析確定HD-4菌株為假交替單胞菌（Pseudoalteromonas sp.），其16SrDNA同源性為99%；HD-6菌株為交替單胞菌（Alteromonas_sp.），其16SrDNA同源性為98%。2株菌最適生長(zhǎng)溫度均為15 ℃。HD-4 和HD-6最適生長(zhǎng)pH分別為9和8。適宜NaCl濃度分別為 2%和4%；在25 ℃振蕩培養(yǎng)（150 r/min）7 d，接菌量為7×107 cfu/mL、7×108 cfu/mL、7×109 cfu/mL 時(shí)，HD-4對(duì)柴油的初始降解率分別為80%，22.1%和27.6%；HD-6初始降解率分別為23.7%、38.8%和432%。加入葡萄糖后2株菌的降油率均有所增高，加入4 g/L葡萄糖時(shí)達(dá)最高值，3個(gè)接種濃度下HD-4菌株分別為85.4%、82.3%和80.4%，HD-6菌株分別為86.8%、93.7%和89.3%。當(dāng)接菌量為7×107 cfu/mL，HD-4在葡萄糖含量為4 g/L 時(shí)作用7 d，降油率可達(dá)到最大值86.72%，HD-6菌株在葡萄糖濃度6 g/L達(dá)到最大值67.64%。葡萄糖濃度超過(guò)4 g/L和 6 g/L時(shí)HD-4和HD-6菌株的降油性能有所下降。

關(guān)鍵詞：石油降解菌；海帶；細(xì)菌鑒定；降油性

近年來(lái)，經(jīng)濟(jì)的高速發(fā)展使得對(duì)能源需求日益增加，而以化石能源為主的能源結(jié)構(gòu)帶來(lái)了海洋石油開(kāi)采和海上石油運(yùn)輸行業(yè)的大規(guī)模發(fā)展，而在石油開(kāi)采、運(yùn)輸過(guò)程中導(dǎo)致的海洋石油類(lèi)污染隨之成為世界性的問(wèn)題。根據(jù)國(guó)際海事組織的統(tǒng)計(jì)，全球每年流入海洋的石油數(shù)以千萬(wàn)噸計(jì)［1］，特別是在一些海灣，由于其獨(dú)特的半封閉水域特征，溢油事故一旦發(fā)生就將會(huì)導(dǎo)致可怕的生態(tài)危機(jī)［2］。據(jù)聯(lián)合國(guó)有關(guān)組織統(tǒng)計(jì)，每年由于海上油井井噴事故和油輪事故造成的溢油高達(dá)220萬(wàn)t［3］。如何治理和修復(fù)被石油污染的海洋環(huán)境及被其破壞的生態(tài)系統(tǒng)已經(jīng)成為當(dāng)下研究的熱點(diǎn)。微生物修復(fù)是目前研究最多、應(yīng)用也最為廣泛的一種生物修復(fù)方法。國(guó)內(nèi)外眾多研究者對(duì)清除石油污染微生物的篩選鑒定、生長(zhǎng)特性、菌株選育、降解原理、降解性能、影響因素等方面進(jìn)行了大量研究［4-8］。目前有報(bào)道從耐受石油污染的小型藻類(lèi)如藍(lán)藻和大型藻類(lèi)如江籬上篩選降解石油微生物進(jìn)行研究。Radwan［9］等發(fā)現(xiàn)，大量的石油分解菌附著于多種大型海藻上, 這種藻菌共同作用對(duì)石油類(lèi)污染物的降解有明顯效果。本研究通過(guò)分離海帶表面的附著微生物，并從中篩選出具有降油性能的菌株，為制備用于海洋石油污染修復(fù)的復(fù)合菌劑提供菌種，同時(shí)也為探索以海洋大型藻類(lèi)為載體強(qiáng)化定植降油細(xì)菌用于海洋環(huán)境的石油污染修復(fù)奠定基礎(chǔ)。

1實(shí)驗(yàn)材料與方法

1.1樣品來(lái)源和培養(yǎng)基

海帶樣品采自大連黑石礁潮間帶，菌株初篩選用2216E固體培養(yǎng)基，復(fù)篩選用柴油平板培養(yǎng)基，測(cè)量菌種柴油降解率選用基礎(chǔ)無(wú)機(jī)鹽培養(yǎng)基［4］。

柴油平板培養(yǎng)基：基礎(chǔ)無(wú)機(jī)鹽培養(yǎng)基 1000 mL、吐溫80 1 mL、瓊脂粉15 g、0號(hào)柴油10 mL (市售0號(hào)柴油，121 ℃高壓滅菌后待用)，pH 72，121 ℃高壓滅菌20 min。

基礎(chǔ)無(wú)機(jī)鹽培養(yǎng)基：氯化銨 0.5 g、氯化鈉 20 g、磷酸氫二鉀 1 g、磷酸二氫鉀 0.5 g、硫酸鎂 0.5 g、氯化鉀 0.1 g、硫酸鐵 0.01 g、氯化鈣 0.002 g、純水 1 000 mL，pH 7.2，121 ℃高壓滅菌20 min。

1.2降油細(xì)菌的分離和純化

首先將刮取的海帶表皮組織樣品置于5 mL滅菌離心管中，加入2 mL無(wú)菌生理鹽水并用勻漿器勻漿，對(duì)勻漿進(jìn)行梯度稀釋?zhuān)?0-1、10-2、10-3、10-4、10-5），各取稀釋度為0.1 mL分別涂布于2216E平板上，置于25 ℃恒溫培養(yǎng)箱培養(yǎng)24 h。根據(jù)菌落形態(tài)分別挑取單菌落于2216E平板進(jìn)行純化，每株菌分別純化三代后將其保種于2216E斜面用于初篩降油菌株，同時(shí)接種2216E液體25 ℃培養(yǎng)24 h，加入50%（V/V）滅菌甘油置于-80 ℃保存。將分離純化的菌株分別吸取10 μl點(diǎn)種到柴油平板中，25 ℃恒溫培養(yǎng)箱培養(yǎng)48～72 h。待平板上長(zhǎng)出較清晰菌苔，挑取生長(zhǎng)狀況良好的菌株作為目標(biāo)菌株進(jìn)行后續(xù)實(shí)驗(yàn)。

1.3菌落及菌體的形態(tài)學(xué)及生理生化鑒定

將純化后并在柴油平板上生長(zhǎng)良好的目標(biāo)菌株分別在2216E平板上進(jìn)行平板劃線，置于25℃培養(yǎng)24 h后，觀察菌落形態(tài)并挑取單菌落進(jìn)行革蘭氏染色鏡檢。按照《魚(yú)類(lèi)及其他水生動(dòng)物細(xì)菌實(shí)用鑒定指南》 ［10］和《常見(jiàn)細(xì)菌鑒定手冊(cè)》［11］鑒定其生理生化特性。

1.416S rDNA 基因測(cè)序及序列的擴(kuò)增

將待測(cè)菌株送至大連寶生物工程有限公司進(jìn)行16SrDNA基因測(cè)序及擴(kuò)增，將測(cè)序結(jié)果輸入 GenBank數(shù)據(jù)庫(kù)中，進(jìn)行Blast(Basic local Alignment Search Tool)同源性比對(duì)。

1.5系統(tǒng)發(fā)育樹(shù)的構(gòu)建

利用Mega5.05軟件，將所得16SrDNA序列，進(jìn)行多序列比對(duì)同時(shí)計(jì)算序列間的系統(tǒng)進(jìn)化距離，用鄰接法(Neighbor-Joining method)構(gòu)建系統(tǒng)發(fā)育樹(shù)后，以自舉數(shù)為1 000，通過(guò)自引導(dǎo)法(Bootstrap)進(jìn)行置信度的檢測(cè)。

1.6石油降解細(xì)菌的生長(zhǎng)特性

1.6.1溫度將目標(biāo)菌株用2216E液體培養(yǎng)基活化24 h后，接種到5 mL 2216E液體培養(yǎng)基中，以不同的溫度梯度（ 5 ℃、10 ℃、15 ℃、20 ℃、25 ℃、30 ℃、35 ℃、40 ℃）培養(yǎng)24 h，每三個(gè)平行為一組。對(duì)照組為未接菌的培養(yǎng)基，以波長(zhǎng)為600 nm，采用分光光度法測(cè)量實(shí)驗(yàn)組的吸光值(OD600nm)。以培養(yǎng)溫度為橫坐標(biāo)，吸光值為縱坐標(biāo)作圖，得到不同溫度條件下石油降解菌的生長(zhǎng)特性。

1.6.2pH將目標(biāo)菌株用2216E液體培養(yǎng)基活化24 h后，接種到5 mL 2%NaCl牛肉膏蛋白胨液體培養(yǎng)基中，以不同的pH梯度(pH=5、6、7、8、9、10，NaCl濃度2%)25 ℃培養(yǎng)24 h，每三個(gè)平行為一組。對(duì)照組為未接菌的培養(yǎng)基，以波長(zhǎng)為600 nm，采用分光光度法測(cè)量實(shí)驗(yàn)組的吸光值(OD600nm)。以培養(yǎng)pH為橫坐標(biāo)，吸光值為縱坐標(biāo)作圖，得到不同pH條件下石油降解菌的生長(zhǎng)特性。

1.6.3鹽度將目標(biāo)菌株用2216E液體培養(yǎng)基活化24 h后，接種到5 mL牛肉膏蛋白胨液體培養(yǎng)基中，以不同鹽度(NaCl濃度分別為1%、2%、3%、4%、5%)25 ℃培養(yǎng)24 h，每組三個(gè)平行。每三個(gè)平行為一組。對(duì)照組為未接菌的培養(yǎng)基，以波長(zhǎng)為600 nm，采用分光光度法測(cè)量實(shí)驗(yàn)組的吸光值(OD600nm)。以培養(yǎng)NaCl濃度為橫坐標(biāo)，吸光值為縱坐標(biāo)作圖，得到不同NaCl濃度條件下石油降解菌的生長(zhǎng)特性。

1.7細(xì)菌降油性能測(cè)定

配置 MMC液體培養(yǎng)基，高壓蒸汽滅菌鍋中121 ℃，0.1MPa滅菌20 min。將在2216E斜面培養(yǎng)24 h處于對(duì)數(shù)生長(zhǎng)期的菌斜面用無(wú)菌生理鹽水洗脫，梯度稀釋并用血球計(jì)數(shù)板計(jì)數(shù)，選擇細(xì)菌數(shù)為7×107 cfu/mL、7×108 cfu/mL、7×109 cfu/mL的梯度。

接種：分別將0.1 mL無(wú)菌柴油和1 mL濃度為7×107 cfu/mL、7×108 cfu/mL、7×109 cfu/mL的菌懸液依次加入到 MMC培養(yǎng)基中，每種接菌量設(shè)置三組平行實(shí)驗(yàn)，以不接種菌液的培養(yǎng)基作為對(duì)照組。將接種好的三角燒瓶置于25 ℃，150 r/min恒溫?fù)u床培養(yǎng)7 d。

柴油降解率的測(cè)定：將搖好的錐形瓶水平平穩(wěn)取出，分別加入適量無(wú)水硫酸鈉，蓋上棉塞，輕輕搖動(dòng)使無(wú)水硫酸鈉盡量溶解。吸取20 mL石油醚（透光率＞90%，沸程60～90 ℃）加入三角瓶中，放回?fù)u床搖10 min后取出錐形瓶，用移液槍吸取500 μL上層石油醚，加入到25 mL的比色管中，用石油醚定容至25 mL，充分混勻。在221 nm波長(zhǎng)處通過(guò)紫外分光光度計(jì)測(cè)定各組吸光值。根據(jù)以下公式計(jì)算出降解率。降解率(D) =（A0-Ai）/A0×100%，其中D為柴油降解率，A0為空白對(duì)照組柴油吸光值，Ai為實(shí)驗(yàn)組剩余柴油吸光值。

1.8測(cè)定葡萄糖對(duì)石油降解菌降油率的影響

以葡萄糖終濃度為4 g/L的 MMC培養(yǎng)基，分別按3個(gè)濃度接種（7×107 cfu/mL、7×108 cfu/mL、7×109 cfu/mL）兩株實(shí)驗(yàn)菌，每種接菌量設(shè)置三組平行實(shí)驗(yàn)，不接種菌液的培養(yǎng)基作為對(duì)照組，將接種好的三角燒瓶置于25 ℃，150 r/min恒溫?fù)u床培養(yǎng)7 d。同法測(cè)定柴油降解率，比較不同接種量對(duì)柴油降解率的影響。

1.9不同葡萄糖濃度對(duì)石油降解菌降油率的影響

配制不同葡萄糖濃度（0.5 g/L、1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L）的柴油培養(yǎng)基，分別接種兩株實(shí)驗(yàn)菌（107 cfu/mL），25 ℃、150 r/min恒溫?fù)u床分別培養(yǎng)7 d，測(cè)定柴油降解率。

2結(jié)果

2.1石油降解菌的初篩

經(jīng)柴油平板初篩得到2株不同形態(tài)的細(xì)菌，分別編號(hào)為：HD-4、HD-6。兩株菌可在以柴油為唯一碳源的培養(yǎng)基上生長(zhǎng)。在2216E平板上25 ℃ 24 h后，HD-4菌落形態(tài)特征為圓形、偏黃、不透明、邊緣整齊、=1.5 mm，有運(yùn)動(dòng)能力。HD-6菌落形態(tài)特征為圓形、微黃，邊緣整齊、無(wú)圓心、=1.0 mm、有運(yùn)動(dòng)能力，培養(yǎng)24 h時(shí)菌落透明，48 h后菌落變得不透明。兩株菌均為革蘭氏陰性桿菌，見(jiàn)圖1和圖2。

圖1HD-4菌株的鏡下和菌落形態(tài)

圖2HD-6菌株的鏡下和菌落形態(tài)

2.2HD-4 和HD-6菌株生理生化特征

HD-4是假單胞菌屬，革蘭氏染色陰性無(wú)芽孢桿菌，呈桿狀或略彎，菌體大小(0.5～1)×(1.5～4)μm。具端鞭毛，能運(yùn)動(dòng)，不發(fā)酵糖類(lèi)。HD-6是交替單胞菌屬：直或彎的桿狀，（0.7～1.5） μm×（1.8～3.0） μm，不積累聚-β-羥基丁酸鹽顆粒(PHB)作為胞內(nèi)貯存物，不形成微胞囊和芽孢，細(xì)胞染色革蘭氏陰性。大多數(shù)以單個(gè)無(wú)鞘和極生鞭毛運(yùn)動(dòng)，有帶鞘的鞭毛。化能異養(yǎng)菌，能呼吸而不發(fā)酵的代謝型。精氨酸雙水解酶陰性，所有的都耐鹽生長(zhǎng)，具體生理生化特征見(jiàn)表1。

表1HD-4, HD-6生理生化特征

鑒定指標(biāo)

HD-4

HD-6

運(yùn)動(dòng)性

+

+

脲酶

+

-

氧化酶

+

+

接觸酶

+

+

明膠水解

-

+

精氨酸雙水解

-

-

葡萄糖氧化

+

+

葡萄糖發(fā)酵

-

-

V-P試驗(yàn)

-

-

吲哚試驗(yàn)

+

-

甲基紅試驗(yàn)

-

-

檸檬酸鹽試驗(yàn)

-

-

硝酸鹽還原

-

-

O/129藥敏紙片敏感性

-

-

注：-反應(yīng)陰性；+ 反應(yīng)陽(yáng)性；d 不確定

2.316S rDNA序列及系統(tǒng)發(fā)育樹(shù)

2.3.1基因序列同源性比對(duì)將純化后得到的HD-4、HD-6菌株的單菌落移送至大連寶生物公司進(jìn)行16S rDNA片段的擴(kuò)增，對(duì)擴(kuò)增得到的16S rDNA序列進(jìn)行測(cè)序，將HD-4、HD-6菌株的16S rDNA序列測(cè)序結(jié)果與國(guó)際互聯(lián)網(wǎng)NCBI上GenBank中數(shù)據(jù)進(jìn)行BLAST比對(duì)，發(fā)現(xiàn)HD-4與Pseudoalteromonas sp. 同源性達(dá)到99%；HD-6菌株16SrDNA序列與Alteromonas sp.同源性98%。

2.3.2構(gòu)建系統(tǒng)發(fā)育樹(shù)序列的比對(duì)結(jié)果，利用Mega5.05軟件進(jìn)行多序列比對(duì)同時(shí)計(jì)算序列間的系統(tǒng)進(jìn)化距離，構(gòu)建的發(fā)育樹(shù)得出：HD-4與Pseudoalteromonas sp. CF4-10聚為一支；HD-6與Alteromonas sp. HB1聚為一支，如圖3。

圖3HD-4,HD-6系統(tǒng)發(fā)育樹(shù)

2.4細(xì)菌生長(zhǎng)特性的測(cè)定

圖4-圖6表明，HD-4和HD-6菌株最適生長(zhǎng)溫度分別為15 ℃和20 ℃，兩菌株在10～30 ℃范圍內(nèi)能較好生長(zhǎng)，說(shuō)明兩菌株能適應(yīng)較大的溫度范圍且具有一定的耐低溫能力。兩株菌最適生長(zhǎng)NaCl濃度分別為2%和4%；最適生長(zhǎng)pH分別為9和8；且兩菌株在NaCl濃度1%～5%、pH=5～9范圍內(nèi)，生長(zhǎng)情況良好，說(shuō)明兩菌株生長(zhǎng)能力較強(qiáng)，對(duì)于不同鹽度、不同pH有一定的耐受性。

圖4兩株菌的最適生長(zhǎng)溫度

圖5兩株菌的最適生長(zhǎng)NaCl濃度

圖6兩株菌的最適生長(zhǎng)pH

2.5HD-4和HD-6菌株原始降解率的測(cè)定

HD-4和HD-6菌株初始柴油降解率實(shí)驗(yàn)結(jié)果顯示：隨著接菌數(shù)量的增加，兩株菌的降解率均呈現(xiàn)上升趨勢(shì)，HD-4菌株接菌量為107 cfu/mL降解率只有8.0%；當(dāng)接菌量達(dá)到108 cfu/mL和109 cfu/mL時(shí)降解率有所提升，分別達(dá)到221%和27.6%。HD-6菌株接菌量為107 cfu/mL降解率為23.7%，而接菌量為108 cfu/mL和109 cfu/mL時(shí)，其降解率分別達(dá)到了388%和43.2%，兩株菌對(duì)柴油的降解率都與菌濃度正相關(guān)，且兩株菌差異性計(jì)算結(jié)果均顯示當(dāng)接菌量為107 cfu/mL時(shí)，柴油降解率與108 cfu/mL、109 cfu/mL降解率差異顯著（P＜0.05），接菌量在108 cfu/mL 和109 cfu/mL時(shí)降解率差異不顯著（P＞0.05），詳見(jiàn)圖7。

圖7HD-4和HD-6菌株初始柴油降解率

2.6加入葡萄糖對(duì)柴油降解率的影響

圖8添加葡萄糖對(duì)HD-4柴油降解率額影響

圖9添加葡萄糖對(duì)HD-6柴油降解率額影響

在以柴油為唯一碳源的 MMC培養(yǎng)基中加入終濃度為4 g/L的葡萄糖后，不同接菌量實(shí)驗(yàn)組對(duì)柴油降解率都有大幅度提升，均達(dá)到80%以上，且接菌量為107cells/mL時(shí)HD-4菌株和HD-6菌株的柴油降解率分別可達(dá)到85.4%和86.8%，不同接菌量?jī)芍昃鷮?duì)柴油的降解率差異不顯著（P＞0.05）。而在終濃度為4 g/L的葡萄糖的條件下，相同接菌量?jī)删甑牟裼徒到饴蕰?huì)因葡萄糖的增加而顯著增大，差異極顯著（P＜001）,且在菌濃度為107cells/mL時(shí)即可達(dá)到很高的降解率。詳見(jiàn)圖8-圖9。

2.7不同濃度葡萄糖對(duì)菌株降油性能的影響

在接菌量為107 cfu/mL的條件下，添加不同濃度葡萄糖，HD-4和HD-6兩株菌對(duì)柴油的降解率隨著葡萄糖濃度的增加都呈現(xiàn)先增大后減小的趨勢(shì)，HD-4在葡萄糖濃度為1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L時(shí)能保持較高的降解率，且在葡萄糖濃度為4 g/L時(shí)達(dá)到最大值86.72%；而HD-6在葡萄糖濃度為4 g/L、6 g/L、8 g/L、10 g/L濃度時(shí)對(duì)柴油的降解率才達(dá)到比較高的水平，在葡萄糖濃度為6 g/L時(shí)達(dá)到最大值67.64%，見(jiàn)圖10。

圖10不同濃度葡萄糖對(duì)兩株菌降油性能影響

3討論

通過(guò)以柴油為唯一碳源的柴油平板培養(yǎng)基，初篩得到兩株能利用柴油生長(zhǎng)的菌株，編號(hào)為HD-4、 HD-6，經(jīng)鑒定分別屬于假交替單胞菌屬和交替單胞菌屬。兩株菌均可耐鹽生長(zhǎng)，對(duì)柴油具有降解能力，其原始降解率與菌種接入量呈正相關(guān)。有關(guān)藻類(lèi)附著微生物石油污染物的利用性研究目前有關(guān)報(bào)道較少，S.S. Radwan等2002年報(bào)道在阿拉伯灣海域的幾種大型藻類(lèi)（滸苔Enteromorpha、馬尾藻Sargassum、石花菜Gelidium、江蘺Gracilaria等）表面附著多種可利用石油類(lèi)物質(zhì)的微生物，以放線菌(Actinomycetes)和不動(dòng)桿菌(Acinetobacter)為主，其中約64%～98%可利用烷烴，約 38%～56%可利用芳香烴（菲）［12］。并且這些微生物是定植在藻類(lèi)的表面，不易脫落，很可能與藻類(lèi)屬于共生關(guān)系。本研究分離的兩株降油細(xì)菌是否為海帶附著微生物中的優(yōu)勢(shì)種類(lèi)，以及與海帶的生態(tài)關(guān)系還需進(jìn)一步深入研究。

HD-4和HD-6兩株菌在添加葡萄糖后對(duì)柴油的降解率大大提高，在7 d內(nèi)可達(dá)到80%以上，并且菌種的接入量對(duì)降解率影響不明顯。葡萄糖為微生物代謝的初級(jí)能源，添加少量的初級(jí)能源物質(zhì)，促進(jìn)微生物對(duì)某些特殊物質(zhì)的代謝稱(chēng)為“共代謝” ［13］，它具有縮短生物處理系統(tǒng)適應(yīng)和繁殖期的優(yōu)勢(shì)［14］。微生物共代謝是時(shí)下研究較多的領(lǐng)域并具有較大的應(yīng)用空間，在很多污染地方，如廢污水治理、土壤修復(fù)等領(lǐng)域有著廣泛應(yīng)用，且對(duì)一些難降解的有機(jī)污染物，生物降解有其特殊優(yōu)勢(shì)［15-17］。有學(xué)者實(shí)驗(yàn)結(jié)果表明，當(dāng)葡萄糖作為外加碳源加入土壤后，可以提高原土壤中有機(jī)碳的礦化速率［18］，推測(cè)其機(jī)理可能正是由這種微生物共生作用增強(qiáng)了微生物的活性，增強(qiáng)了微生物的數(shù)量并且提高了其酶活力［19-20］。但是在測(cè)定添加不同濃度葡萄糖后兩株菌的降油率實(shí)驗(yàn)中HD-6菌株的降解率較之前出現(xiàn)了下降的情況，分析原因考慮是否為轉(zhuǎn)接過(guò)程中代謝能力受到一定影響。鑒于實(shí)驗(yàn)菌株的降油機(jī)理還不太清楚，其降解酶類(lèi)的表達(dá)情況、影響因素等還需進(jìn)行深入的研究。另外，本研究采用的兩株菌在柴油利用過(guò)程中是否具有“共代謝”作用也是今后需要深入研究的問(wèn)題，這對(duì)利用這兩株菌進(jìn)行石油污染海域或其他環(huán)境的生物修復(fù)具有重要的意義。

參考文獻(xiàn)：

［1］ 支振鋒.油輪泄漏與海洋生物災(zāi)難［J］.新安全，2004（6）：60-64

［2］ 陳鋒，陳偉琪，王萱.溢油事故造成的海灣生態(tài)系統(tǒng)服務(wù)損失的貨幣化評(píng)估［J］.環(huán)境科學(xué)與管理.2009，34（11）：1-5

［3］ United Nations Environment Programme.Keeping Track of Our Changing Environment: From Rio to Rio+20 (1992-2012)［R］.Nairobi：United Nations Environment Programme，2011：49

［4］ 鄭金秀，張甲耀，趙晴，等.高效石油降解菌的選育及其降解特性研究［J］.環(huán)境科學(xué)與技術(shù)，2006，29（3）：1-2，40

［5］ 吳業(yè)輝，邵宗澤.海洋烷烴降解菌Alcanivorax sp.A-11-3的分離鑒定及其降解酶基因研究［J］.臺(tái)灣海峽，2008，27（4）：427-434

［6］ 譚田豐，邵宗澤.海洋石油烴降解菌群構(gòu)建及其在降解過(guò)程中的動(dòng)態(tài)分析［J］.廈門(mén)大學(xué)學(xué)報(bào)（自然科學(xué)版），2006，45（增刊）：427-434

［7］ 賈燕，尹華，彭輝，等.石油降解菌株的篩選、初步鑒定及其特性［J］.暨南大學(xué)學(xué)報(bào)（自然科學(xué)版），2007，28（3）：296-301

［8］ 徐馮楠，馮貴穎，馬雯，等.高效石油降解菌的篩選及其降解性能研究［J］.生物技術(shù)通報(bào)，2010（7）：221-226

［9］ RadwanSS，Hasan R H，Alamah S S，et al.Bioremediation of oily sea water by bacteria immobilized in biofilmscoating macroalgae［J］.International Biodeterioration & Biodegradation，2002，50（1）：55-59

［10］ Nicky B. Buller. Bacteria from Fish and Other Aquatic Animals: A Practical Identification Manual ［M］.

［11］ 東秀珠, 蔡妙英，等. 常見(jiàn)細(xì)菌系統(tǒng)鑒定手冊(cè)［M］.北京：科學(xué)出版社，2001

［12］ RadwanSS, Al-Hasan R H，Khanafer M，Eliyas M，et al.Hydrocarbon accumulation by picocyanobacteria from the Arabian Gulf［J］.Journal of Applied Microbiology，2001，191：533-540

［13］ Jensen, H. L. Carbon nutrition of some microganisms decomposting halogen-substituted aliphatic acid. Acta. Agr. Scand., 1963, 13: 404-412

［14］ ANGELA V, GUIDO D M, SIMON A R, et al. Enhanced bioremediation of methyl tert-butyl ether (MTBE) by microbial consortia obtained from conta minated aquifer material［J］. Chemosphere, 2009, 75:149–155

［15］ YIN Z, TIANGANG L, XIAOWEI W, et al. Influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons by Sphingomonas sp. strain PheB4 ［J］. Applied Microbiology and Biotechnology, 2007, 75:175–186

［16］ 鞏宗強(qiáng), 李培軍, 王新，等. 芘在土壤中的共代謝降解研究［J］. 應(yīng)用生態(tài)學(xué)報(bào), 2001,12(3): 447–450

［17］ 李萍, 劉俊新. 廢水中難降解性有機(jī)污染物的共代謝降解［J］. 環(huán)境污染治理技術(shù)與設(shè)備, 2002,11(3):43–46

［18］ SHEN J, BARTHA R. The pri ming effect of substrate addition in soil–based biodegradation tests［J］. Applied and Environmental Microbiology, 1996, 62: 1428–1430.

［19］ 陳春梅, 謝祖彬, 朱建國(guó). 土壤有機(jī)碳激發(fā)效應(yīng)研究進(jìn)展［J］.土壤(Soils), 2006, 38(4):359-36

［20］ Bingemann C W, Varner J E, Martin W P. The effect of the addition of organic materials on the decomposition of an organic soil［J］. Soil Science Society of American Proceedings, 1953, 17:34–38

Isolation and Degradation Characteristic of Oil Degrading Bacteria from kelp

GUO Weicheng,LI Zuoyang,WANG Bin,ZHOU Wenjun,ZHANG Kai,CHANG Anni

(Key Laboratory of Marine Bio-resources Restoration ad Habitat Reparation in Liaoning Province, Dalian Ocean University, Dalian 116023, China)

Abstract：The experiment isolated strains from the surface of the kelp that grew in intertidal of dalian HeiShiJiao, And screening two strains named HD-4 and HD-6 which could grow in medium that diesel oil was the sole carbon source. Cellular morphological observations, biochemical reactions and molecular identifications of 16S rDNA were used to identify strain HD-4、HD-6，and the growth characteristics of HD-4、HD-6 were also studied.Deter mination the initial degradation rate of diesel with different inoculation quantity, meanwhile the oil-degrading efficiency was also studied when different concentrations of glucose was added .The result shows: HD-4 was yellow, opaque, edge neat, the diameter is 1.5 mm; HD-6 was light yellow, transparent, regular edge and its diameter is 1.0 mm, the above two strains of bacteria were gram-negative bacteria. The sequences of 16SrDNA indicate that HD-4 was related to Pseudoalteromonas sp. E407-2, and the homology was 99%;HD-6 was closely related to Alteromonas sp. HB1. , the homology was 98%.The growing characteristics of these two strains showed that the optimum growth temperature、pH and the adaptive of NaCl concentration for HD-4 was 15 ℃、pH 9 and 2%,while HD-6 was 15 ℃、pH 8 and 4% respectively.Ultraviolet spectrophotometermeasured HD-4 shows that the initial degradation rate of diesel oil were 8.0%, 22.1% and 27.6%,in the inoculation quantity of 7 x 107 cfu/mL, and 7 x 108 cfu/mL, 7 x 109 cfu/mL,cultured 7 days in constant temperature,the degradationrate of diesel oilincreased significantly after adding 4 g/L glucose,reach to 85.4%, 82.3% and 80.4%,respectively; In the same situation,the initial degradation rate of diesel oil of HD-6 were23.7%, 38.8% and 43.2%, after joining the 4 g/L glucose, reach to 86.8%, 93.7% and 89.3%, respectively. Meanwhlie, in the inoculation quantity of 7 x 107 cell/mL, adding different quantity of glucose for 0.5 g/L、1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L, cultured 3 days in constant temperature ,the degrading efficiency of strain HD-4 reach the best when adding glucose to a specific concentration of 4 g/L ,about 86.72%;and the most efficient concentration of HD-6 is 6 g/L ,reach to 67.64%. As more glucose was added,the degradations of these two bacterium were efficiency dropped.The experiments indicated that adding glucose in a appropriate level, both of them can obviously promote the degradation rate of diesel oil ,but as more glucose was added, the degradation efficiency dropped.In this paper, use bacterium isolated from kelp, provides a theoretical basis and experimental basis by using bacterium to remediate petroleum conta mination.

Key words：petroleum-degrading bacteria; kelp; degradation ;glucose.

［15］ YIN Z, TIANGANG L, XIAOWEI W, et al. Influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons by Sphingomonas sp. strain PheB4 ［J］. Applied Microbiology and Biotechnology, 2007, 75:175–186

［16］ 鞏宗強(qiáng), 李培軍, 王新，等. 芘在土壤中的共代謝降解研究［J］. 應(yīng)用生態(tài)學(xué)報(bào), 2001,12(3): 447–450

［17］ 李萍, 劉俊新. 廢水中難降解性有機(jī)污染物的共代謝降解［J］. 環(huán)境污染治理技術(shù)與設(shè)備, 2002,11(3):43–46

［18］ SHEN J, BARTHA R. The pri ming effect of substrate addition in soil–based biodegradation tests［J］. Applied and Environmental Microbiology, 1996, 62: 1428–1430.

［19］ 陳春梅, 謝祖彬, 朱建國(guó). 土壤有機(jī)碳激發(fā)效應(yīng)研究進(jìn)展［J］.土壤(Soils), 2006, 38(4):359-36

［20］ Bingemann C W, Varner J E, Martin W P. The effect of the addition of organic materials on the decomposition of an organic soil［J］. Soil Science Society of American Proceedings, 1953, 17:34–38

Isolation and Degradation Characteristic of Oil Degrading Bacteria from kelp

GUO Weicheng,LI Zuoyang,WANG Bin,ZHOU Wenjun,ZHANG Kai,CHANG Anni

(Key Laboratory of Marine Bio-resources Restoration ad Habitat Reparation in Liaoning Province, Dalian Ocean University, Dalian 116023, China)

Abstract：The experiment isolated strains from the surface of the kelp that grew in intertidal of dalian HeiShiJiao, And screening two strains named HD-4 and HD-6 which could grow in medium that diesel oil was the sole carbon source. Cellular morphological observations, biochemical reactions and molecular identifications of 16S rDNA were used to identify strain HD-4、HD-6，and the growth characteristics of HD-4、HD-6 were also studied.Deter mination the initial degradation rate of diesel with different inoculation quantity, meanwhile the oil-degrading efficiency was also studied when different concentrations of glucose was added .The result shows: HD-4 was yellow, opaque, edge neat, the diameter is 1.5 mm; HD-6 was light yellow, transparent, regular edge and its diameter is 1.0 mm, the above two strains of bacteria were gram-negative bacteria. The sequences of 16SrDNA indicate that HD-4 was related to Pseudoalteromonas sp. E407-2, and the homology was 99%;HD-6 was closely related to Alteromonas sp. HB1. , the homology was 98%.The growing characteristics of these two strains showed that the optimum growth temperature、pH and the adaptive of NaCl concentration for HD-4 was 15 ℃、pH 9 and 2%,while HD-6 was 15 ℃、pH 8 and 4% respectively.Ultraviolet spectrophotometermeasured HD-4 shows that the initial degradation rate of diesel oil were 8.0%, 22.1% and 27.6%,in the inoculation quantity of 7 x 107 cfu/mL, and 7 x 108 cfu/mL, 7 x 109 cfu/mL,cultured 7 days in constant temperature,the degradationrate of diesel oilincreased significantly after adding 4 g/L glucose,reach to 85.4%, 82.3% and 80.4%,respectively; In the same situation,the initial degradation rate of diesel oil of HD-6 were23.7%, 38.8% and 43.2%, after joining the 4 g/L glucose, reach to 86.8%, 93.7% and 89.3%, respectively. Meanwhlie, in the inoculation quantity of 7 x 107 cell/mL, adding different quantity of glucose for 0.5 g/L、1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L, cultured 3 days in constant temperature ,the degrading efficiency of strain HD-4 reach the best when adding glucose to a specific concentration of 4 g/L ,about 86.72%;and the most efficient concentration of HD-6 is 6 g/L ,reach to 67.64%. As more glucose was added,the degradations of these two bacterium were efficiency dropped.The experiments indicated that adding glucose in a appropriate level, both of them can obviously promote the degradation rate of diesel oil ,but as more glucose was added, the degradation efficiency dropped.In this paper, use bacterium isolated from kelp, provides a theoretical basis and experimental basis by using bacterium to remediate petroleum conta mination.

Key words：petroleum-degrading bacteria; kelp; degradation ;glucose.

［15］ YIN Z, TIANGANG L, XIAOWEI W, et al. Influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons by Sphingomonas sp. strain PheB4 ［J］. Applied Microbiology and Biotechnology, 2007, 75:175–186

［16］ 鞏宗強(qiáng), 李培軍, 王新，等. 芘在土壤中的共代謝降解研究［J］. 應(yīng)用生態(tài)學(xué)報(bào), 2001,12(3): 447–450

［17］ 李萍, 劉俊新. 廢水中難降解性有機(jī)污染物的共代謝降解［J］. 環(huán)境污染治理技術(shù)與設(shè)備, 2002,11(3):43–46

［18］ SHEN J, BARTHA R. The pri ming effect of substrate addition in soil–based biodegradation tests［J］. Applied and Environmental Microbiology, 1996, 62: 1428–1430.

［19］ 陳春梅, 謝祖彬, 朱建國(guó). 土壤有機(jī)碳激發(fā)效應(yīng)研究進(jìn)展［J］.土壤(Soils), 2006, 38(4):359-36

［20］ Bingemann C W, Varner J E, Martin W P. The effect of the addition of organic materials on the decomposition of an organic soil［J］. Soil Science Society of American Proceedings, 1953, 17:34–38

Isolation and Degradation Characteristic of Oil Degrading Bacteria from kelp

GUO Weicheng,LI Zuoyang,WANG Bin,ZHOU Wenjun,ZHANG Kai,CHANG Anni

(Key Laboratory of Marine Bio-resources Restoration ad Habitat Reparation in Liaoning Province, Dalian Ocean University, Dalian 116023, China)

Abstract：The experiment isolated strains from the surface of the kelp that grew in intertidal of dalian HeiShiJiao, And screening two strains named HD-4 and HD-6 which could grow in medium that diesel oil was the sole carbon source. Cellular morphological observations, biochemical reactions and molecular identifications of 16S rDNA were used to identify strain HD-4、HD-6，and the growth characteristics of HD-4、HD-6 were also studied.Deter mination the initial degradation rate of diesel with different inoculation quantity, meanwhile the oil-degrading efficiency was also studied when different concentrations of glucose was added .The result shows: HD-4 was yellow, opaque, edge neat, the diameter is 1.5 mm; HD-6 was light yellow, transparent, regular edge and its diameter is 1.0 mm, the above two strains of bacteria were gram-negative bacteria. The sequences of 16SrDNA indicate that HD-4 was related to Pseudoalteromonas sp. E407-2, and the homology was 99%;HD-6 was closely related to Alteromonas sp. HB1. , the homology was 98%.The growing characteristics of these two strains showed that the optimum growth temperature、pH and the adaptive of NaCl concentration for HD-4 was 15 ℃、pH 9 and 2%,while HD-6 was 15 ℃、pH 8 and 4% respectively.Ultraviolet spectrophotometermeasured HD-4 shows that the initial degradation rate of diesel oil were 8.0%, 22.1% and 27.6%,in the inoculation quantity of 7 x 107 cfu/mL, and 7 x 108 cfu/mL, 7 x 109 cfu/mL,cultured 7 days in constant temperature,the degradationrate of diesel oilincreased significantly after adding 4 g/L glucose,reach to 85.4%, 82.3% and 80.4%,respectively; In the same situation,the initial degradation rate of diesel oil of HD-6 were23.7%, 38.8% and 43.2%, after joining the 4 g/L glucose, reach to 86.8%, 93.7% and 89.3%, respectively. Meanwhlie, in the inoculation quantity of 7 x 107 cell/mL, adding different quantity of glucose for 0.5 g/L、1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L, cultured 3 days in constant temperature ,the degrading efficiency of strain HD-4 reach the best when adding glucose to a specific concentration of 4 g/L ,about 86.72%;and the most efficient concentration of HD-6 is 6 g/L ,reach to 67.64%. As more glucose was added,the degradations of these two bacterium were efficiency dropped.The experiments indicated that adding glucose in a appropriate level, both of them can obviously promote the degradation rate of diesel oil ,but as more glucose was added, the degradation efficiency dropped.In this paper, use bacterium isolated from kelp, provides a theoretical basis and experimental basis by using bacterium to remediate petroleum conta mination.

Key words：petroleum-degrading bacteria; kelp; degradation ;glucose.