印度洋表层海水石油降解菌多样性分析及生物表面活性剂发酵工艺优化
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摘要
海洋微生物在海洋石油污染的自然消除及生物修复中发挥着重要的作用。本文利用2007年“大洋一号”第19次科考期间从德班到珀斯走航过程中采集的印度洋表层海水,通过石油富集,对降解菌多样性进行了分析。此外,本文还对石油降解菌Alcanivorax dieselolei B-5进行了产表面活性剂的发酵培养基优化。
主要研究内容及结论如下：
(1)为了研究印度洋石油降解菌多样性,并获得新的石油降解菌。本研究通过印度洋表层海水样品采集、以柴油与原油1：1混合物作为碳源,从中富集、分离筛选石油降解菌,并通过PCR-DGGE对13个站点富集菌群的菌群结构进行分析。通过形态观察、生理生化反应和16SrRNA分析,共得到29个属的51株不同的细菌,它们主要是属于α亚群和γ亚群。其中,Alcanivorax属(占18%),Novosphingobium属(占10%),Marinobacter(占6%)和Thalassospira(占6%)为主要的优势菌。通过生态多样性分析表明,Shannon-Winner指数(H)为4.57968,说明其具有较高的多样性;均匀度指数(E)为0.8664771,表示其分布比较均匀。单菌实验表明,49株具有石油降解能力,其中,Sinomonas, Knoellia, Mesoflavibacter等属的细菌为首次发现有降解能力。DGGE分析表明Alcanivorax属的细菌是印度洋表层海水中的重要石油降解菌。本研究首次揭示了印度洋表层海水中石油降解菌的多样性,并获取了若干在海洋石油污染生物修复中具有应用前景的降解菌。
(2)由于缺乏快速有效准确的方法,生物表面活性剂产量的定量一直是一个耗时且困难的工作。本研究旨在利用表面活性剂在低pH时会形成沉淀而浑浊的原理,将浑浊度与标准曲线结合,用于定量表面活性剂的产量。由不同碳源诱导菌株A. dieselolei B-5所产的表面活性剂经配成标准浓度后沉淀,实验表明浑浊度与标准浓度制成的曲线具有良好的线性相关性。经MSM、MMSM和DMSM培养基验证后显示,该方法可适用于多种培养基中生物表面活性剂产量的定量。
利用以上实验的表面活性剂产量定量方法为检测手段。本研究对菌株A.dieselolei B-5产生的生物表面活性剂进行了发酵培养基的优化。采用Plackett-Burman设计进行优化实验,结果分析表明,影响表面活性剂产量的3个重要的培养基成分为：液体石蜡、TWEEN-80、SrCl2。进一步用Design Expert软件的响应面回归方法对培养基进行了优化。结果表明,其最适浓度分别为33.18ml/L、4.15 ml/L和0.22 g/L。此时表面活性剂的产量为最大(4.31 g/L),比优化前提高了95.91%。
Marine microorganisms play an important role in elimination and bioremediation of marine oil pollution. Biosurfactant play a role of promoting dissolution and degradation in hydrophobic substrate. In this study, we have analyzed the diversity of oil-degrading bacteria acrossing the Indian Ocean surface seawater during the 19th scientific expedition of "DAYANG YIHAO" in 2007. In addition, we optimized the production of a biosurfactant from the oil-degrading bacteria Alcanivorax dieselolei B-5.
The main contents and findings are as following:
(1) In order to investigate the diversity of the oil-degrading bacteria in the surface seawater across the India Ocean, and obtain new oil-degrading bacteria. Potential oil-degrading bacteria were selected out via 1:1 mixture of diesel and crude oil as sole carbon source. Meanwhile, the community structure of 13 enrichments was analyzed by polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE). We obtained 51 unique strains of 29 genera after screening via morphological, physiological, biochemical and 16S rRNA analyses. They mainly belonged to a andγ-Proteobacteria. The four genera Alcanivorax (accounting for 18%), Novosphingobium (10%), Marinobacter (6%) and Thalassospira (6%) were the most predominant bacteria. Ecological analyses showed that the bacteria had high diversity with Shannon-Winner index (H) of 4.57968, and distributed even with Evenness index (E) as 0.8664771. Then Further experiments revealed oil-degrading capability of 49 strains. In addition, our investigation revealed oil-degrading ability of genera Sinomonas, Knoellia and Mesoflavibacter for the first time. DGGE fingerprint patterns indicated that the genus Alcanivorax was an important oil-degrading bacteria in the surface seawater across the India Ocean. This study demonstrated a high diversity of the oil-degradation bacteria in the surface seawater of Indian Ocean, these bacteria are of potential in bioremediation of marine oil pollution.
(2) Quantification of the biosurfactants produced by a variety of microorganisms is a time taking and dif fi cult task due to the lack of rapid, ef fi cient and accurate methods. This work presents a simple turbidometric method forquanti fi cation of crude biosurfactants based on their property to become insoluble at low pH values. Biosurfactants obtained from A. dieselolei B-5 using different carbon substrates showed a good linear correlation(R2>0.95) between biosurfactant concentrations and turbidity in the range of 2 to 10 g/L of crude biosurfactants. The MSM, MMSM, DMSM developed in this work effectively predicted the amount of crude biosurfactant produced in different sets of fermentation experiments validating the method.
The biosurfactant produced in the fermentation medium was quantified directly by cell-free solution turbidity corresponding standard curve. The nutritional medium requirement for biosurfactant production by A. dieselolei B-5 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were liquid paraffin, TWEEN-80 and SrCl2. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were:liquid paraffin, 33.18 ml/L; TWEEN-80,4.15 ml/L; SrCl2,0.22 g/L respectively. Using this statistical optimization method, the biosurfactant yield was increased from 2.2 g/L to 4.31 g/L, which is 95.91 percent higher than the non-optimized rich medium.
主要研究内容及结论如下：
(1)为了研究印度洋石油降解菌多样性,并获得新的石油降解菌。本研究通过印度洋表层海水样品采集、以柴油与原油1：1混合物作为碳源,从中富集、分离筛选石油降解菌,并通过PCR-DGGE对13个站点富集菌群的菌群结构进行分析。通过形态观察、生理生化反应和16SrRNA分析,共得到29个属的51株不同的细菌,它们主要是属于α亚群和γ亚群。其中,Alcanivorax属(占18%),Novosphingobium属(占10%),Marinobacter(占6%)和Thalassospira(占6%)为主要的优势菌。通过生态多样性分析表明,Shannon-Winner指数(H)为4.57968,说明其具有较高的多样性;均匀度指数(E)为0.8664771,表示其分布比较均匀。单菌实验表明,49株具有石油降解能力,其中,Sinomonas, Knoellia, Mesoflavibacter等属的细菌为首次发现有降解能力。DGGE分析表明Alcanivorax属的细菌是印度洋表层海水中的重要石油降解菌。本研究首次揭示了印度洋表层海水中石油降解菌的多样性,并获取了若干在海洋石油污染生物修复中具有应用前景的降解菌。
(2)由于缺乏快速有效准确的方法,生物表面活性剂产量的定量一直是一个耗时且困难的工作。本研究旨在利用表面活性剂在低pH时会形成沉淀而浑浊的原理,将浑浊度与标准曲线结合,用于定量表面活性剂的产量。由不同碳源诱导菌株A. dieselolei B-5所产的表面活性剂经配成标准浓度后沉淀,实验表明浑浊度与标准浓度制成的曲线具有良好的线性相关性。经MSM、MMSM和DMSM培养基验证后显示,该方法可适用于多种培养基中生物表面活性剂产量的定量。
利用以上实验的表面活性剂产量定量方法为检测手段。本研究对菌株A.dieselolei B-5产生的生物表面活性剂进行了发酵培养基的优化。采用Plackett-Burman设计进行优化实验,结果分析表明,影响表面活性剂产量的3个重要的培养基成分为：液体石蜡、TWEEN-80、SrCl2。进一步用Design Expert软件的响应面回归方法对培养基进行了优化。结果表明,其最适浓度分别为33.18ml/L、4.15 ml/L和0.22 g/L。此时表面活性剂的产量为最大(4.31 g/L),比优化前提高了95.91%。
Marine microorganisms play an important role in elimination and bioremediation of marine oil pollution. Biosurfactant play a role of promoting dissolution and degradation in hydrophobic substrate. In this study, we have analyzed the diversity of oil-degrading bacteria acrossing the Indian Ocean surface seawater during the 19th scientific expedition of "DAYANG YIHAO" in 2007. In addition, we optimized the production of a biosurfactant from the oil-degrading bacteria Alcanivorax dieselolei B-5.
The main contents and findings are as following:
(1) In order to investigate the diversity of the oil-degrading bacteria in the surface seawater across the India Ocean, and obtain new oil-degrading bacteria. Potential oil-degrading bacteria were selected out via 1:1 mixture of diesel and crude oil as sole carbon source. Meanwhile, the community structure of 13 enrichments was analyzed by polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE). We obtained 51 unique strains of 29 genera after screening via morphological, physiological, biochemical and 16S rRNA analyses. They mainly belonged to a andγ-Proteobacteria. The four genera Alcanivorax (accounting for 18%), Novosphingobium (10%), Marinobacter (6%) and Thalassospira (6%) were the most predominant bacteria. Ecological analyses showed that the bacteria had high diversity with Shannon-Winner index (H) of 4.57968, and distributed even with Evenness index (E) as 0.8664771. Then Further experiments revealed oil-degrading capability of 49 strains. In addition, our investigation revealed oil-degrading ability of genera Sinomonas, Knoellia and Mesoflavibacter for the first time. DGGE fingerprint patterns indicated that the genus Alcanivorax was an important oil-degrading bacteria in the surface seawater across the India Ocean. This study demonstrated a high diversity of the oil-degradation bacteria in the surface seawater of Indian Ocean, these bacteria are of potential in bioremediation of marine oil pollution.
(2) Quantification of the biosurfactants produced by a variety of microorganisms is a time taking and dif fi cult task due to the lack of rapid, ef fi cient and accurate methods. This work presents a simple turbidometric method forquanti fi cation of crude biosurfactants based on their property to become insoluble at low pH values. Biosurfactants obtained from A. dieselolei B-5 using different carbon substrates showed a good linear correlation(R2>0.95) between biosurfactant concentrations and turbidity in the range of 2 to 10 g/L of crude biosurfactants. The MSM, MMSM, DMSM developed in this work effectively predicted the amount of crude biosurfactant produced in different sets of fermentation experiments validating the method.
The biosurfactant produced in the fermentation medium was quantified directly by cell-free solution turbidity corresponding standard curve. The nutritional medium requirement for biosurfactant production by A. dieselolei B-5 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were liquid paraffin, TWEEN-80 and SrCl2. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were:liquid paraffin, 33.18 ml/L; TWEEN-80,4.15 ml/L; SrCl2,0.22 g/L respectively. Using this statistical optimization method, the biosurfactant yield was increased from 2.2 g/L to 4.31 g/L, which is 95.91 percent higher than the non-optimized rich medium.
引文
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