Article pubs.acs.org/EF
Manipulation of Surface Hydrophobicity and Charge of Demulsifying Bacteria Using Functional Magnetic Nanoparticles: A Mechanistic Study of Demulsification Performance Xiangfeng Huang,† Yongjiao Xiong,† Liju Lu,† Jia Liu,† and Kaiming Peng*,‡ †
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 ‡ Post-Doctoral Research Station, Tongji University, Shanghai 200092, People’s Republic of China S Supporting Information *
ABSTRACT: Hydrophobicity and an electric charge are thought to be key factors that influence demulsification performance of demulsifying bacteria. Nonetheless, the exact mechanism of action of these two factors are not clear. Two series of functional magnetic nanoparticles with gradually varied hydrophobicity or surface charge were synthesized and combined with demulsifying bacteria, for manipulation of surface hydrophobicity or a charge of these bacteria. Demulsification results indicated that stronger cell surface hydrophobicity resulted in better demulsification performance of magnetically responsive bacterial demulsifiers (MRBDs), whereas a higher or lower surface charge (in the range from −30 mV to −20 mV) can inhibit it. Hydrophobicity accelerated droplet coalescence by 1.0 h in relation to its stimulatory effect on cell translocation to the interface. A proper surface charge made sure that bacteria aggregate to a certain extent at the interface. These results should be useful for broader applications of bacterial demulsifiers in the future.
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INTRODUCTION Demulsifying bacteria have a variety of potential applications in diverse industries, such as mining, food, cosmetics, and pharmaceutical industries. Because of their environmentally friendly features and resistance to some chemical reagents, pH changes, high salinity, and extreme temperatures, demulsifying bacteria have become an important component in the fields of oil production and oil field pollution control.1 Surface hydrophobicity and a charge are thought to be key factors that influence the demulsification performance of demulsifying bacteria.2,3 In this field, more attention is given to cell surface hydrophobicity, whereas the effects of the surface charge are rarely investigated probably, because of difficulties with manipulation of these bacteria. Cell surface hydrophobicity has been measured by several methods, such as polystyrene microsphere attachment, hydrophobic interaction chromatography, microbial adhesion to hydrocarbons (MATH), and water contact angle (WCA).4 MATH and WCA are two of the most widely utilized techniques. The relationship between surface hydrophobicity and demulsification performance of demulsifying bacteria has attracted broad attention in recent years. Liu et al. found that the cell surface hydrophobicity (WCA = 50°−114.6° and MATH = 50%−80%) shows a positive correlation with the demulsifying ability of the Alcaligenes sp. S-XJ-1 that was cultivated at various pH levels of the culture medium.5 Huang et al. reported a linear relation between hydrophobicity (WCA = 40°−90° and MATH = 74%− 90%) and demulsification performance, namely, a more hydrophobic strain shows better demulsification performance after they adjusted the carbon source of this strain.1 Ma et al. drew the same conclusion as well, where the Rhodococcus sp. PR-1 with 87% MATH showed better demulsification performance.6 © XXXX American Chemical Society
There are also researchers who think that the surface hydrophobicity is irrelevant to demulsification performance. Another research group reported that the correlation between cell surface hydrophobicity and the demulsifying capability is absent, negative, or positive, depending on the microbial strain.7 The above-mentioned relationships between demulsification performance and cell surface hydrophobicity were mostly observed when cultivation conditions of bacteria were adjusted. In these studies, the researchers focused on determining the cultivation conditions and growth characteristics of strains, rather than focusing on adjusting the surface hydrophobicity of the cells. Along with cell surface hydrophobicity, other surface properties are also altered as a function of cultivation conditions, such as cell morphology and surface substance constitution, among others. Moreover, the variation range has been limited, and located either in the relatively hydrophilic part (in the WCA range of 40°−114.6°) or hydrophobic part (within the MATH range of 50%−87%). Although a MATH assay and WCA measurements have been commonly used in environmental microbiology applications, these tests are often reported to yield poor correlations.8 The MATH assay may be affected by other solution-phase interactions. For WCA measurements, there appears to be no consensus on the threshold contact angle for a bacterial surface that defines hydrophilicity and hydrophobicity.9 All in all, a more precise assessment method for cell surface hydrophobicity would be useful, and a target-specific method for adjusting the surface properties of demulsifying bacteria independently in a wider range is needed. Received: October 14, 2016 Revised: December 27, 2016 Published: February 7, 2017 A
DOI: 10.1021/acs.energyfuels.6b02674 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels
Figure 1. Synthesis scheme of the functional magnetic nanoparticles (MNPs).
determined here. This research may facilitate the future development of industrial applications of bacterial demulsifiers.
As the correlation between demulsification performance and cell surface hydrophobicity attracts more and more attention, the specific function of hydrophobicity during the demulsification process is being studied. It was hypothesized that the bacterial cell surface hydrophobicity mainly governs the bacterial attachment to the interface in emulsions.10 Some researchers reported that higher hydrophobicity improves dispersion and adhesion abilities of the strains, accelerating the transfer rate of the bacteria in oil.11 In contrast, other researchers found that too strong hydrophobicity can make particles remain in the oil for a long period, whereas medium hydrophobicity can force particles to remain at the interface and interact with droplets effectively.12 Nonetheless, these studies revealed qualitatively the mechanism of surface properties’ action on demulsification performance. The concrete effects of the surface hydrophobicity and the charge on demulsification performance, and which stage of the demulsification process is affected by them, have yet to be elucidated. A combination of magnetic nanoparticles (MNPs) and bacteria has been widely used in pathogen detection, strengthening of microbial desulfurization, and pollutant degradation.13−15 The binding of MNPs and bacteria is mostly driven by an electrostatic force, hydrophobic interaction, or specific binding between them. These interactions are rather strong and make the complex especially firm.16 Some researchers found that the cell surface charge17 and cell adhesion characteristics18 are altered after cell surface adsorption of MNPs. That is to say, the complex may change surface properties of the demulsifying bacteria, and it is possible to use MNPs for manipulating surface properties of demulsifying bacteria. In our previous study,19 MNPs were used to enhance demulsification performance of demulsifying bacteria. We found that the combination of hydrophilic MNPs with hydrophobic bacteria decreases hydrophobicity of the demulsifying bacteria. In the present study, functional MNPs with gradually varied hydrophobicity or surface charge were designed and synthesized. The two series of MNPs were used to manipulate the surface hydrophobicity and charge of a bacterial strain in a wide range. Surface properties of the bacteria and their demulsification performance were analyzed using various methods. Finally, the mechanism of action of surface properties of demulsifying bacteria on a demulsification process was
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MATERIALS AND METHODS
Synthesis and Characterization of Functional MNPs. Functional MNPs with gradually varied hydrophobicity were prepared by a series of modification procedures for MNPs. First, Fe3O4 MNPs were prepared by chemical coprecipitation, as reported by Liang et al.20 Then, Fe3O4@SiO2 was synthesized by the method of hydrolysis with silicon alkylation reagents.16 Functional MNPs with gradually varied hydrophobicity and charge were prepared on the basis of Fe3O4@SiO2 modification, which is shown in Figure 1. MNPs with gradually varied hydrophobicity were obtained by grafting alkyl trimethoxysilane onto Fe3O4@SiO2, denoted as M@Cn. “Cn” represents alkyl trimethoxysilane with different length of the carbon chain, and “n” denotes the carbon number of alkylation reagents during preparation of hydrophobic MNPs. Briefly, 2.0 g of a Fe3O4@ SiO2 powder was dispersed in an alkyl trimethoxysilane solution for 6 h. The alkyl trimethoxysilane solution was prepared by mixing toluene with an alkyl trimethoxysilane solution in a volume ratio of 10:1. Methyl trimethoxysilane (n = 1), trimethoxyoctylsilane (n = 3), dodecyltrimethoxysilane (n = 12), and hexadecyltrimethoxysilane (n = 16) were selected as modification substances. The as-prepared powder collected by a magnet was washed with ethanol and dried for subsequent use. MNPs with a gradually varied surface charge (M@NH2) were prepared by grafting triethylene tetramine onto Fe3O4@SiO2. That is, 2.0 g of the Fe3O4@SiO2 powder was dispersed in 100 mL of a toluene solution, which contained 2 mL of a silane coupling agent (KH-560) solution, for 6 h. The as-prepared powder collected by a magnet was washed by ultrasonic treatment in toluene three times, and then redispersed in 100 mL of toluene for another 6 h. Various volumes of triethylene tetramine were added to the mixture for modifying the MNPs. The as-prepared powder collected by a magnet was washed with ethanol and dried for subsequent use. The zeta potentials (ZPs) of functional MNPs were measured using a Zetasizer NanoZ analyzer (Malvern Instruments, Malvern, U.K.). To prepare samples for the ZP test, the MNPs were resuspended in distilled water to initial optical density of 0.8−1.0 (at a wavelength of 600 nm). The suspensions then were diluted with deionized (DI) water until there was no obvious sedimentation of the particles. After that, pH of the samples was consistent with the pH of DI water (∼7). The WCA value of the functional MNPs was used to characterize their surface hydrophobicity. The newly prepared MNPs were pressed to form a slice (by means of a vacuum compressor). Contact angles were then measured with water on a Model SL200B instrument (Shanghai Solon Technology Science Company, Shanghai, China). To confirm that alkyl B
DOI: 10.1021/acs.energyfuels.6b02674 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels trimethoxysilane and triethylene tetramine were successfully grafted onto the MNPs, the composition of the two types of functional MNPs was determined by thermogravimetric analysis (Discovery TGA system, TA Instruments, New Castle, DE, USA) in a N2 atmosphere at a heating rate of 10 °C/min up to 800 °C. Weight loss could be calculated from the resulting curve. Modification of Surface Properties of Demulsifying Bacteria. Alcaligenes sp. S-XJ-1 was cultivated in a fermentation tank, as reported in our early study.3 A magnetically responsive bacterial demulsifier with hydrophobicity modification (MRBD-Hs) and a magnetically responsive bacterial demulsifier with positive charge modification (MRBD-Ps) were prepared by grafting a silylating reagent-modified MNPs (named M@Cn) and triethylene tetramine-modified MNPs (named M@NH2) onto the surface of the demulsifying bacteria, respectively. A certain amount of dry demulsifying bacteria powder was resuspended in DI water and sonicated for 20 min with stirring by a glass rod at the same time to achieve the suspension concentration of 16 g/L.19 Two milliliters (2 mL) of a demulsifying-bacteria suspension (16 g/L) were taken immediately and blended with 2 mL of a functional MNP suspension (4.8 mg/L for M@Cn, 3.2 mg/L for M@NH2) in a container within a vortex rotator for 90 s at 2000 rpm.21 Thus, magnetically responsive demulsifying bacteria with different surface properties were obtained. The ZPs of MRBDs and the demulsifying cells were measured as described in the previous section (“Synthesis and Characterization of Functional MNPs”). The WCA value of the MRBDs and the free energy of the interfacial interaction between two MRBDs immersed in water (ΔGbwb) were used to characterize their surface hydrophobicity.19 For a contact angle test, 10 mL of the prepared MRBDs or a demulsifying cell suspension (8 g/L) was filtered through a 0.45 μm (pore size) cellulose acetate membrane (50 mm diameter) to deposit a uniform lawn. The membranes were transferred to Petri plates containing 1% agar and 10% glycerol and were maintained at 35 °C for more than 2 h to standardize the humidity of the membranes. The membranes were cut into strips, mounted on glass slides with double-sided tape, and allowed to dry for 20−40 min at 35 °C. The contact angles between the droplet and the demulsifier lawn were recorded by a contact angle meter SL200B (Shanghai Solon Technology Science Company, Ltd., Shanghai, China) and were calculated by a circular fitting analysis, using the CAST3.0 software (Solon Tech Co. Ltd., Shanghai, China). ΔGbwb can be calculated through the surface free energy of MRBDs and water, according to the method reported in our study.21 Briefly, contact angles of the MRBDs were measured using formamide and diiodomethane as an apolar liquid, and the surface energy parameters of each demulsifying bacterium was calculated from the measured contact angle based on the acid−base theory proposed by van Oss et al.22 Demulsification Tests. A water/oil (W/O) model emulsion (containing 1.526 g of Tween 80, 0.074 g of Span 80, 80 mL of aviation kerosene, and 120 mL of distilled water) was prepared as described in another study.19 The pH of the thus-prepared emulsion was 6.4 and it had an emulsion breaking ratio of
