Article pubs.acs.org/est
Carbon Source Dependence of Cell Surface Composition and Demulsifying Capability of Alcaligenes sp. S‑XJ‑1 Xiangfeng Huang, Kaiming Peng, Lijun Lu, Ruofei Wang, and Jia Liu* College of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Ministry of Education Key Laboratory of Yangtze River Water Environment, Tongji University, Shanghai 200092, People’s Republic of China S Supporting Information *
ABSTRACT: Biodemulsifiers are environmentally friendly agents used in recycling oil or purifying water from emulsion, yet the demulsifying feature of cell-surface composition remains unclear. In this study, potentiometric titration, attenuated total reflectanceFourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and matrix-assisted laser desorption/ionization-timeof-flight mass spectrometry were combined to characterize cellsurface chemical composition of demulsifying strain Alcaligenes sp. SXJ-1 cultivated with different carbon sources. Cells cultivated with alkane contained abundant elemental nitrogen and basic functional groups, indicating that their surface was rich in proteins or peptides, which contributed to their highest demulsifying efficiency. For cells cultivated with fatty acid ester, the relatively abundant surface lipid contributed to a 50% demulsification ratio owing to the presence of more acidic functional group. The cells cultivated with glucose exhibited a high oxygen concentration (O/C ∼0.28), which indicated the presence of more polysaccharides on the cell surface. This induced the lowest demulsification ratio of 30%. It can be concluded that cell surface-associated proteins or lipids other than the polysaccharide of the demulsifying strain played a positive role in the demulsification activity. In addition, the cell-surface oligoglutamate compounds identified in situ were crucial to the demulsifying capability.
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INTRODUCTION Biodemulsifiers have a variety of potential applications in diverse industries such as mining, food, nuclear, fuel reprocessing, cosmetics, and pharmaceuticals.1 Due to their environmental-friendly feature and resistance to some chemical reagents, pH changes, high salinity, and extreme temperature, biodemulsifiers have become a research hotspot in the field of crude oil production and oil field pollution control.2 Biodemulsifiers can be classified into cell-bound biodemulsifiers, such as Nocardia,2 Corynebacterium,3 Micrococcus,4 Alcaligenes,5 Ochrobactrum anthropi,6 and hybrid strains;7 and extracellular metabolic biodemulsifiers, such as fengycin,8 proteins, or lipopeptides.9 The demulsification capability exists in the cultivated cells for the former and in culture supernatant for the latter. Previous research has revealed intact cells as excellent biodemulsifiers.5,6 Compared with extracellular demulsifiers, cellular demulsifiers are more suitable for the demulsification of complex emulsion systems, owing to their more complicated demulsification mechanism. It was found that the abundant cell wall-bound compounds and functional groups, structure and morphology of cells, hydrophobicity, and charge of cell-surface are all crucial to the demulsifying capability.10,11 Carbon sources have a noticeable impact on the demulsifying capability of the demulsifying strain,12 probably because they change the chemical composition of the cell surface. The most © 2014 American Chemical Society
widely used carbon sources belong to three types of chemical compounds: alkanes, fatty acid esters, and carbohydrates. Hydrophobic carbon sources, such as the petroleum hydrocarbons,5−7 pure alkanes,2,4 fatty acid esters of vegetable oils, and waste oil,13 were used to cultivate demulsifying strains. Meanwhile, some hydrophilic (ethanol,6 sucrose,14 glucose,3 etc.) or mixed hydrophilic and hydrophobic compounds (such as paraffin and glucose)9 were also used as carbon sources. Hydrophobic carbon sources were more favorable to the synthesis of cell-bound biodemulsifiers with higher demulsifying efficiency than hydrophilic carbon sources.12,15 And some research indicated the demulsification performance of the biomass cultivated with alkane seemed to be better than that cultivated with fatty acid ester.16,17 However, few studies have reported the effect of carbon sources on the chemical composition of cell bound demulsifying substances and the demulsifying ability. This is probably because the complexity of the cell surface makes it difficult to identify the active component. In our previous study, 17 strains were screened as potential demulsifying bacteria from oilfields, oil-contaminated soil, and Received: Revised: Accepted: Published: 3056
October 16, 2013 January 22, 2014 January 29, 2014 January 29, 2014 dx.doi.org/10.1021/es404636j | Environ. Sci. Technol. 2014, 48, 3056−3064
Environmental Science & Technology
Article
activated sludge, in which strain Alcaligenes sp. S-XJ-1 showed the highest demulsifying capability.5 In addition, the S-XJ-1 belongs to a typical cell-bound biodemulsifier, which could be cultivated in different carbon sources, high pH conditions, or high salinity conditions.11,15 It showed that demulsifying activity of the S-XJ-1 was closely correlated with protein and fatty acids on the cell surface.11,13 The active demulsifying substance extracted from the cell of Alcaligenes sp. was identified as a carbohydrate−protein−lipid mixture. But it exhibited lower demulsifying capability than the original cells,10 which suggests that the extraction did not fully obtain all the active components. So in situ identification of cell-surface chemical composition will be more appropriate to reveal how it affects demulsifying capability. Recently, many analytical techniques appeared for characterizing cell-surface functional groups and composition, including potentiometric titration, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS), and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF/MS). The potentiometric titration is a macroscopic methodology to characterize protonation behavior of bacterial cell-surface, and determine the concentrations and acidity of proton-active surface function groups.18 To further understanding of cell surface composition, spectroscopic techniques (IR and XPS) and mass spectra (MALDI) may be useful tools to characterize the cell surfaces. The FTIR spectra of microorganisms analyzed with chemometrics is widely used for to rapid and accurate differentiation and classification of microorganisms, which is based mainly on the difference of outer membrane cell components.19 XPS provides information for element and functional groups on a 2−10 nm thick surface layer of dried microorganism in many researches.20,21 In addition, MALDI-TOF-MS has been used for classification and identification of intact microorganism according to their cell wall.22 These powerful analytical tools can be used to reveal different cell-surface composition of demulsifying strains obtained with different carbon sources. Some of these methods have been combined together to characterize microorganism surface.23 With the above-mentioned analytical tools, a more comprehensive for in situ characterization of the cell-surface chemical composition of the demulsifying strain Alcaligenes sp. S-XJ-1 was carried out in this study. The relationship between the composition of the cell surface and the demulsifying capability was also elucidated.
bacteria were harvested from the top oil layer by centrifugation at 13 000g for 10 min. To remove residual oil, the harvested bacteria were rinsed three times with n-hexane, followed by centrifugal separation at 13 000g . The bacteria cell cultivated with glucose was obtained by direct centrifugation of fermented culture at 13 000g directly, followed by rinsing with distilled water. The washed bacteria cells were dried in a freeze drier (Scientz-10N; Ningbo Scientz Biotechnology, Zhejiang, China) at −50 °C for 24 h. The dried cells were used to characterize biomass, demulsifying capability, and cell surface chemical composition. Demulsification Test. A water−in−oil(W/O) model emulsion was prepared according to the following protocol. Aviation kerosene (80 mL containing 1.526 g Tween 80 and 0.074 g Span 80) and distilled water (40 mL) were mixed at 10 000 rpm by a high-speed emulsifying machine (WL-500CY; Shanghai Wei Yu Mechanical and Electrical Manufacture, Shanghai, China) for 3.5 min. Distilled water (80 mL) was slowly added at ∼2 mL/s when the mixing started. In the demulsification test, the dried demulsifying cells were resuspended in distilled water to make 10 000 mg/L of cell suspension. Then 2 mL of cell suspension was added in a 20mL graduated test tube containing 18-mL model emulsion. The test tubes were vigorously inverted 120 times to achieve complete mixing and then left undisturbed in water baths at 35 °C. The demulsification activity at a set time interval was calculated by the following eq 1. demulsification ratio = (oil volume + water volume) × (original emulsion volume added biodemulsifier volume)−1 × 100%
(1)
A blank was conducted with 2 mL distilled water, and it had an emulsion breaking ratio of
Carbon Source Dependence of Cell Surface Composition and Demulsifying Capability of Alcaligenes sp. S‑XJ‑1 Xiangfeng Huang, Kaiming Peng, Lijun Lu, Ruofei Wang, and Jia Liu* College of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Ministry of Education Key Laboratory of Yangtze River Water Environment, Tongji University, Shanghai 200092, People’s Republic of China S Supporting Information *
ABSTRACT: Biodemulsifiers are environmentally friendly agents used in recycling oil or purifying water from emulsion, yet the demulsifying feature of cell-surface composition remains unclear. In this study, potentiometric titration, attenuated total reflectanceFourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and matrix-assisted laser desorption/ionization-timeof-flight mass spectrometry were combined to characterize cellsurface chemical composition of demulsifying strain Alcaligenes sp. SXJ-1 cultivated with different carbon sources. Cells cultivated with alkane contained abundant elemental nitrogen and basic functional groups, indicating that their surface was rich in proteins or peptides, which contributed to their highest demulsifying efficiency. For cells cultivated with fatty acid ester, the relatively abundant surface lipid contributed to a 50% demulsification ratio owing to the presence of more acidic functional group. The cells cultivated with glucose exhibited a high oxygen concentration (O/C ∼0.28), which indicated the presence of more polysaccharides on the cell surface. This induced the lowest demulsification ratio of 30%. It can be concluded that cell surface-associated proteins or lipids other than the polysaccharide of the demulsifying strain played a positive role in the demulsification activity. In addition, the cell-surface oligoglutamate compounds identified in situ were crucial to the demulsifying capability.
■
INTRODUCTION Biodemulsifiers have a variety of potential applications in diverse industries such as mining, food, nuclear, fuel reprocessing, cosmetics, and pharmaceuticals.1 Due to their environmental-friendly feature and resistance to some chemical reagents, pH changes, high salinity, and extreme temperature, biodemulsifiers have become a research hotspot in the field of crude oil production and oil field pollution control.2 Biodemulsifiers can be classified into cell-bound biodemulsifiers, such as Nocardia,2 Corynebacterium,3 Micrococcus,4 Alcaligenes,5 Ochrobactrum anthropi,6 and hybrid strains;7 and extracellular metabolic biodemulsifiers, such as fengycin,8 proteins, or lipopeptides.9 The demulsification capability exists in the cultivated cells for the former and in culture supernatant for the latter. Previous research has revealed intact cells as excellent biodemulsifiers.5,6 Compared with extracellular demulsifiers, cellular demulsifiers are more suitable for the demulsification of complex emulsion systems, owing to their more complicated demulsification mechanism. It was found that the abundant cell wall-bound compounds and functional groups, structure and morphology of cells, hydrophobicity, and charge of cell-surface are all crucial to the demulsifying capability.10,11 Carbon sources have a noticeable impact on the demulsifying capability of the demulsifying strain,12 probably because they change the chemical composition of the cell surface. The most © 2014 American Chemical Society
widely used carbon sources belong to three types of chemical compounds: alkanes, fatty acid esters, and carbohydrates. Hydrophobic carbon sources, such as the petroleum hydrocarbons,5−7 pure alkanes,2,4 fatty acid esters of vegetable oils, and waste oil,13 were used to cultivate demulsifying strains. Meanwhile, some hydrophilic (ethanol,6 sucrose,14 glucose,3 etc.) or mixed hydrophilic and hydrophobic compounds (such as paraffin and glucose)9 were also used as carbon sources. Hydrophobic carbon sources were more favorable to the synthesis of cell-bound biodemulsifiers with higher demulsifying efficiency than hydrophilic carbon sources.12,15 And some research indicated the demulsification performance of the biomass cultivated with alkane seemed to be better than that cultivated with fatty acid ester.16,17 However, few studies have reported the effect of carbon sources on the chemical composition of cell bound demulsifying substances and the demulsifying ability. This is probably because the complexity of the cell surface makes it difficult to identify the active component. In our previous study, 17 strains were screened as potential demulsifying bacteria from oilfields, oil-contaminated soil, and Received: Revised: Accepted: Published: 3056
October 16, 2013 January 22, 2014 January 29, 2014 January 29, 2014 dx.doi.org/10.1021/es404636j | Environ. Sci. Technol. 2014, 48, 3056−3064
Environmental Science & Technology
Article
activated sludge, in which strain Alcaligenes sp. S-XJ-1 showed the highest demulsifying capability.5 In addition, the S-XJ-1 belongs to a typical cell-bound biodemulsifier, which could be cultivated in different carbon sources, high pH conditions, or high salinity conditions.11,15 It showed that demulsifying activity of the S-XJ-1 was closely correlated with protein and fatty acids on the cell surface.11,13 The active demulsifying substance extracted from the cell of Alcaligenes sp. was identified as a carbohydrate−protein−lipid mixture. But it exhibited lower demulsifying capability than the original cells,10 which suggests that the extraction did not fully obtain all the active components. So in situ identification of cell-surface chemical composition will be more appropriate to reveal how it affects demulsifying capability. Recently, many analytical techniques appeared for characterizing cell-surface functional groups and composition, including potentiometric titration, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS), and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF/MS). The potentiometric titration is a macroscopic methodology to characterize protonation behavior of bacterial cell-surface, and determine the concentrations and acidity of proton-active surface function groups.18 To further understanding of cell surface composition, spectroscopic techniques (IR and XPS) and mass spectra (MALDI) may be useful tools to characterize the cell surfaces. The FTIR spectra of microorganisms analyzed with chemometrics is widely used for to rapid and accurate differentiation and classification of microorganisms, which is based mainly on the difference of outer membrane cell components.19 XPS provides information for element and functional groups on a 2−10 nm thick surface layer of dried microorganism in many researches.20,21 In addition, MALDI-TOF-MS has been used for classification and identification of intact microorganism according to their cell wall.22 These powerful analytical tools can be used to reveal different cell-surface composition of demulsifying strains obtained with different carbon sources. Some of these methods have been combined together to characterize microorganism surface.23 With the above-mentioned analytical tools, a more comprehensive for in situ characterization of the cell-surface chemical composition of the demulsifying strain Alcaligenes sp. S-XJ-1 was carried out in this study. The relationship between the composition of the cell surface and the demulsifying capability was also elucidated.
bacteria were harvested from the top oil layer by centrifugation at 13 000g for 10 min. To remove residual oil, the harvested bacteria were rinsed three times with n-hexane, followed by centrifugal separation at 13 000g . The bacteria cell cultivated with glucose was obtained by direct centrifugation of fermented culture at 13 000g directly, followed by rinsing with distilled water. The washed bacteria cells were dried in a freeze drier (Scientz-10N; Ningbo Scientz Biotechnology, Zhejiang, China) at −50 °C for 24 h. The dried cells were used to characterize biomass, demulsifying capability, and cell surface chemical composition. Demulsification Test. A water−in−oil(W/O) model emulsion was prepared according to the following protocol. Aviation kerosene (80 mL containing 1.526 g Tween 80 and 0.074 g Span 80) and distilled water (40 mL) were mixed at 10 000 rpm by a high-speed emulsifying machine (WL-500CY; Shanghai Wei Yu Mechanical and Electrical Manufacture, Shanghai, China) for 3.5 min. Distilled water (80 mL) was slowly added at ∼2 mL/s when the mixing started. In the demulsification test, the dried demulsifying cells were resuspended in distilled water to make 10 000 mg/L of cell suspension. Then 2 mL of cell suspension was added in a 20mL graduated test tube containing 18-mL model emulsion. The test tubes were vigorously inverted 120 times to achieve complete mixing and then left undisturbed in water baths at 35 °C. The demulsification activity at a set time interval was calculated by the following eq 1. demulsification ratio = (oil volume + water volume) × (original emulsion volume added biodemulsifier volume)−1 × 100%
(1)
A blank was conducted with 2 mL distilled water, and it had an emulsion breaking ratio of
