Saponin-enhanced biomass accumulation and demulsification capability of the demulsifying bacteria Alcaligenes sp. S-XJ-1

Abstract

Surfactants have been widely applied to the bioremediation of hydrophobic organic compound (HOC) contamination. In this study, the biosurfactant saponin was used for improving rape oil utilization to increase the biomass and demulsification capability of the demulsifying bacterial strain Alcaligenes sp. S-XJ-1. A saponin concentration of 0.05%, near its critical micelle concentration, was optimal to enhance biodemulsifier synthesis; the biomass and demulsification ratio increased by 2.4-fold and 18%, respectively. Biomass promotion was revealed to be attributed to the saponin-enhanced utilization of rape oil resulting from improved oil solubilization, cell membrane permeability, and cell activity. The demulsification boost was found to correlate to saponin-strengthened hydrophobicity via an increased protein content and decreased polysaccharide content of the cell-surface. This study advances our understanding of surfactant-enhanced biodemulsifier synthesis with respect to HOC utilization and cell-surface composition responses.

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1. Introduction

Biodemulsifiers synthesized by microorganisms are used for separation of the oil and water phases from emulsions.^1,2 They have great potential applications in petroleum oil recovery and oily wastewater treatment, owing to their resistance to extreme environmental conditions (such as pH variation, high salinity, and high temperature), wide applicability to different types of emulsions, and minimal secondary pollution risk, which are superior to those of traditional chemical demulsifiers.^1,3 Concerns about biodemulsifiers have persisted for 30 years. To date, the applications of biodemulsifiers are constrained by low yield. To address the bottleneck, previous studies have primarily focused on screening high-yield strains,^4,5 and fermentation optimizations of medium compositions and culture conditions, such as carbon source, nitrogen source, and pH, etc.^3,6 One of the major factors limiting biodemulsifier production is the poor aqueous solubility and bioavailability of hydrophobic organic compounds (HOCs), which are the preferred carbon and energy source for biodemulsifier-producing strains.^3,7 Therefore, the development of targeted and effective strategies to purposefully improve HOC utilization is important to increase the yields of biodemulsifiers.

A promising strategy to intensify the bioavailability of HOCs is the introduction of surfactants. Presently, this strategy is not common in the production of biological agents using an HOC–surfactant system, but is widely applied to the bioremediation of HOC contamination.^8,9 Common chemical surfactants include Triton X-100,¹⁰ Tweens,¹¹ Brijs,⁹ and sodium dodecyl benzenesulfonate (SDBS).¹² Li et al.¹² demonstrated that 30 mg L⁻¹ SDBS was optimal for the promotion of phenanthrene degradation, resulting in 28.2% greater degradation than the surfactant-free control. In recent years, biosurfactants are more popular, such as rhamnolipids⁸ and saponins,¹³ owing to their high activity, ecological compatibility, and especially minimal or no toxicity.¹⁴ Kaczorek et al.¹⁵ reported that the diesel oil biodegradation rate increased from 28% without rhamnolipids to 90% with 120 mg L⁻¹ rhamnolipids. Among the biosurfactants, plant-derived saponins are cheaper and more available than rhamnolipids. Furthermore, Kaczorek et al.¹³ showed that saponins were more effective than rhamnolipids and Triton X-100 during diesel oil biodegradation. The abovementioned features suggest that saponin is advantageous for HOC degradation.

Surfactant-enhanced HOC degradation may be mediated by positive interactions between the surfactant and either HOCs or microbes.¹⁶ Numerous studies just threw light on the impacts of surfactant on HOCs.^9,17 In the HOC–bacteria–surfactant system, the surfactant chiefly regulates the distribution of HOC substrates. Solubilization is a widely accepted mechanism for bioavailability-enhancement of HOCs by surfactants.^9,17 The action of surfactants on microbes is also considered to be crucial for the HOC biodegradation process. That is because the cell surface and cytoplasmic membrane act as a sensitive and selective barrier between the cell and its environment, and intracellular metabolism is the biochemical reaction core. Concerning the positive effects of surfactants on the cell surface, most studies focused on modifications of cell surface hydrophobicity (CSH).^11,18 However, the cell-surface molecular basis for the CSH response is unclear. Taken altogether, the complexity of HOC–bacteria–surfactant system makes it hard to discriminate the roles of surfactant in HOC degradation. The influences of surfactants should be comprehensively explored with respect to both HOC substrates and bacteria.

Our previous study found that a highly efficient demulsifying strain Alcaligenes sp. S-XJ-1 preferred alkane or fatty acid ester as a carbon source, and the biomass cultivated with rape oil was better than that cultivated with paraffin (4.95 g L⁻¹ vs. 1.53 g L⁻¹).¹⁹ In this study, we preferentially combined rape oil substrate with saponin additives to increase the biomass and demulsification ability of Alcaligenes sp. S-XJ-1. HOC solubilization, cell membrane permeabilization, and metabolic activity were determined to illuminate changes in biomass in response to saponin. Furthermore, the cell-surface composition and properties were characterized to clarify the demulsification shifts affected by saponin. This is the first report on the enhanced production of biodemulsifiers via surfactant medium additives.

2. Materials and methods

2.1 Strain and cultivation

Alcaligenes sp. S-XJ-1 was enriched in 100 mL of nutrient broth for 3 days. Ten milliliters of fermented broth was transferred to 100 mL of modified mineral salts medium (MMSM) containing 4% (v/v) paraffin as a sole carbon source for another 7 days of cultivation to obtain the seed culture. MMSM (L⁻¹) contained NH₄NO₃ 4.0 g, K₂HPO₄ 4.0 g, KH₂PO₄ 6.0 g, MgSO₄·7H₂O 0.2 g, and 1 mL of a trace element solution (pH 9). The trace element solution (L⁻¹) contained 1.0 g of CaCl₂·2H₂O, 1.0 g of FeSO₄·7H₂O, and 1.4 g of EDTA.²⁰ For biodemulsifier production, MMSM containing rape oil (4%, v/v) and a saponin additive (0%, 0.01%, 0.03%, 0.05%, 0.1%, 0.5%, and 1%, w/v) were inoculated with the seed culture (10%, v/v). Cultivation was carried out at 30 °C with a rotation speed of 130 rpm for 7 days.

2.2 Biomass preparation and rape oil utilization

After cultivation, the cell pellets and the free oil phase were collected by centrifugation (19 800 × g, 10 min) from the fermented broth. The harvested cells were rinsed several times with n-hexane to obtain the hexane phase. The rinsed cells were dried in a vacuum freezing dryer (Scientz-10N; Ningbo Scientz Biotechnology, Zhejiang, China) at −50 °C for 24 h, and then used to characterize biomass, demulsifying capability, and cell-surface composition and properties. The original free oil and the hexane phase containing oil were volatilized at 60 °C in a rotary evaporator and then weighed as residual rape oil. The determinations of biomass and the utilization ratio of rape oil were based on triplicate samples.

2.3 Demulsification test

A water-in-kerosene model emulsion was prepared following previously published procedures.¹⁹ Two milliliters of cell suspension (10 g L⁻¹) was added to a 20 mL graduated test tube containing 18 mL of the model emulsion. The test tube was vigorously inverted 120 times and placed in a water bath at 35 °C. The demulsification ratio was calculated by eqn (1). The demulsification ratio of the blank, which contained 2 mL of distilled water instead of the cell suspension, was less than 10% within 24 h. The values were based on triplicate samples.

2.4 Solubilization of rape oil by saponin

Rape oil (4%, v/v) and the saponin additives (0%, 0.01%, 0.03%, 0.05%, 0.1%, 0.5%, and 1%, w/v) were added to a flask containing 100 mL of MMSM without any bacteria. The flask was mixed on a reciprocal shaker at 30 °C and 130 rpm. After 7 days of shaking, the contents of the flask were transferred to a funnel, which was allowed to settle for 24 h, during which two separate phases formed: an oil-rich phase and an aqueous phase. The oil-rich phase was collected from the top of the funnel, and the oil content was weighed after n-hexane extraction, and rotary evaporation. The molar solubility ratio (MSR, defined as the increased molar quantity of solubilized rape oil per mole increase of saponin concentration) was calculated. The solubilization ratios of rape oil were based on triplicate samples.

2.5 Preparation of transmission electron microscopy

The sample for transmission electron microscopy (TEM) was prepared using the methods described by Zhang et al.¹¹ The active cells cultivated without and with saponin (0%, 0.01%, 0.05%, and 0.5%) for 7 days were washed three times with phosphate buffered saline (PBS, 0.1 M, pH 7.0), and then fixed with 2.5% glutaraldehyde and 1% OsO₄ in PBS. After gradient dehydration and infiltration, the specimen was dried, embedded in epoxy resin, sliced, stained, and observed at an accelerating voltage of 120 kV (Tecnai G2 Spirit Biotwin; FEI, Hillsboro, OR, USA).

2.6 Cellular metabolic activity determination

Cellular metabolic activity was examined by measuring the fluorescence intensity of cells labeled with fluorescein diacetate (FDA) on a Varian Cary Eclipse Fluorescence Spectrophotometer (HORIBA MAX, Kyoto, Japan). The active cells (cultivated as described in Section 2.1) were washed three times with PBS and suspended in distilled water with an optical density at 600 nm (OD₆₀₀) of 0.5. Seven milliliters of cell suspension was mixed with 875 μL of FDA acetone solution (2 mg mL⁻¹) and shook (500 rpm, 5 min) before fluorescence intensity was detected. The maximal excitation wavelength for FDA was 492 nm, and the emission wavelength was 516 nm; the slit widths of EX and EM were 1.5 and 5 nm, respectively. The values were estimated based on triplicate samples.

2.7 Cell-surface property analysis

Cell surface hydrophobicity was assessed based on the water contact angle (θ_W) and the interfacial interaction energy between bacterial cells immersed in water (ΔG_bwb). Contact angle measurements were performed following previously described procedures.²¹ To prepare the bacterial lawns, 8 mL of bacterial suspension (10 g L⁻¹) was collected on a 0.45 μm nitrocellulose filter, which was then transferred to an agar plate containing 10% glycerol to prevent complete dehydration. The contact angle between the droplet (distilled water, formamide, and diiodomethane were used as probe liquids) and the air-dried bacterial lawn was recorded using the contact angle meter SL200B (Shanghai Solon Technology Science Company, Ltd, Shanghai, China). Each measurement was repeated on three different sites of the bacterial lawn for each of the three liquids. Details of ΔG_bwb calculation were provided in the ESI† (surface free energy calculation).^22,23 Components and parameters of the surface free energy of probe liquids were shown in Table S1.†

Cell-surface charge was characterized by measuring Zeta Potential (ZP) using a Zetasizer NanoZ (Malvern Instrument Ltd., Malvern, Worcestershire, UK). The freeze-dried bacterial powder was resuspended in distilled water to turbidity at an OD₆₀₀ of 1.0. One milliliter of bacterial suspension (the original pH state of approximately 6.0) was injected into an electrophoresis cell using a syringe for measurement. All samples were run in duplicate.

2.8 Cell-surface composition analysis

The bacterial lawn (made in Section 2.7) was thoroughly dried and analyzed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) (Nicolet5700; Thermo Electron Corporation, Beverly, MA, USA). The FTIR spectra were collected with wavenumbers ranging from 4000 to 800 cm⁻¹, and processed using OMNIC software. To further interpret the variation in functional groups among all samples, spectra in the region between 1800 and 900 cm⁻¹, which contained signatures of nearly all cellular components, were examined in a principal component analysis (PCA) implemented in SPSS.²⁴ The transmittances, as variables, were normalized to calculate the correlation matrix, eigenvalue, eigenvector, and the contribution rate. The principal components (F) explaining over 80% of cumulative variances were chosen. Moreover, the wavenumbers, corresponding to more than 0.9 of principal component scores, were assigned to some substances.²⁵ Experiments were performed in triplicate.

X-ray photoelectron spectroscopy (XPS) was performed according to Ojeda et al.²⁶ The freeze-dried bacterial powder was mounted on standard sample studs using double sided adhesive tape. The measurements were made on a KRATOS AXIS 165 Ultra Photoelectron spectrometer at 10 kV and 20 mA. Each analysis consisted of a wide survey scan (pass energy 160 eV, 1.0 eV step size) and high-resolution scan (pass energy 20 eV, 0.1 eV step size) for component speciation. All experiments were conducted in three replicates. The translation of the elemental differences from XPS into the three major classes of proteins, lipids, and polysaccharides was provided in the ESI† (cell-surface substance analysis).^27,28

3. Results and discussion

3.1 Effects of various saponin concentrations on biodemulsifier synthesis

Whole Alcaligenes sp. S-XJ-1 cells have been used as demulsifiers;¹⁹ accordingly, it is important to evaluate biomass (or demulsifier yield) and the corresponding demulsifying activity for this strain. The impacts of saponin at various concentrations on biomass and demulsification are summarized in Fig. 1.

Fig. 1 Biomass and demulsification capability (within 24 h) of the demulsifying strain S-XJ-1 cultivated with saponin additives at various concentrations.

Biomass increased markedly as the saponin concentration increased from 0% to 0.05%, and stabilized when the saponin concentration exceeded 0.05%. For 0.05% saponin, biomass reached 8.11 g L⁻¹, which was 2.4 times higher than that observed in samples lacking saponin. Demulsification ability was clearly higher for cells cultivated with saponin at concentrations of between 0.01% and 0.05% than in cells obtained without saponin, but lower for concentrations ranging from 0.1% to 1%. The demulsification ratio for 0.05% saponin rose from 53.5% to 71.5%. Interestingly, the notable changes in biomass and demulsification depended on the dose with respect to the critical micelle concentration (CMC = 0.094%, Fig. S1; its measurement procedure was shown in the ESI†) of saponin as an inflection point. This might be explained by striking shifts in many physicochemical properties of surfactant solution at its CMC, such as oil solubilization,²⁹ interfacial adsorption,³⁰ and cell membrane modification,³¹ etc.

In a previous study of Alcaligenes sp. S-XJ-1 using rape oil as the sole carbon source, a maximum biomass of 5.99 g L⁻¹ was achieved in a flask culture at pH 7, with a poor demulsification ratio of 26.0%.²⁰ The highest demulsification ratio (41.8%) reported to date was obtained in a fermenter culture at pH 9, with 4.95 g L⁻¹ biomass.¹⁹ In this study (using a flask culture at pH 9), there was a more substantial growth in the biomass (8.11 g L⁻¹) and demulsifying capability (71.5%) of Alcaligenes sp. S-XJ-1 incubated with low-concentration (especially 0.05%) saponin additives. Overall, the saponin supplement is an effective measure to improve biomass and demulsification, which has the potential to reduce a synthetic cost.

3.2 Role of saponin in biomass growth

Biodemulsifier-producing strains typically prefer HOCs as a carbon source;^3,7 however, biomass growth is limited by the bioavailability of insoluble HOC substrates. In general, HOC utilization involves initial contact with the cell, crossing the cell membrane, and intracellular catabolism.¹¹ Surfactants, which exhibit high surface activity, may play an active role in the solubilization, the permeabilization, and the stimulation of enzyme activity, and these properties may promote the use of HOCs and biomass synthesis. Hence, for the saponin–bacteria–rape oil culture system, it is necessary to examine the roles of saponins in biomass growth with respect to the use of rape oil by cells.

3.2.1 Effect of saponin on the solubilization of rape oil. Effective contact between bacteria and HOCs is necessary. Solubilization is a widely accepted mechanism for bioavailability-enhancement of HOCs by surfactants,^9,17 but little is known about the solubilization properties of saponins.

The detailed behaviors of rape oil solubilization by saponins at various concentrations are presented in Fig. 2. As the saponin dosage increased, the solubilization ratio of rape oil increased from 0.3% (without saponin) to 37.2% (with 1% saponin) (Fig. 2a). Additionally, the amount of solubilized rape oil was linearly correlated with the saponin concentration, with different slopes for values greater than or less than the CMC (Fig. 2b). The MSR obtained from the slope was significantly higher below the CMC than above the CMC (i.e., 9.529 and 0.430, respectively, Fig. 2b), indicating saponins at sub-CMC concentrations exhibited a noticeable solubilization capacity for rape oil. These results indicate that sub-CMC solubilization occurred, which differed from the conceptualized micellar solubilization starting at surfactant concentrations above the CMC. Similar phenomena have been observed for rhamnolipids, SDBS, and Triton X-100 in the solubilization of long chain alkanes.^32–34 Zhang et al.³⁴ inferred that the formation of octadecane/monorhamnolipids aggregates increased specific surface area of solubilization below the CMC, while self-aggregation of monorhamnolipids reduced the solubilization efficiency above the CMC, leading to sub-CMC solubilization.

Fig. 2 Solubilization and utilization of rape oil in the presence of saponin additives at various concentrations (a), and molar solubility ratio (MSR) of rape oil with added saponin (b).

There are three major HOC uptake modes: access to dissolved HOCs in water, direct adhesion to mass oil, and contact with pseudosolubilized oil mediated by surfactants.³³ In the saponin–bacteria–rape oil culture system, the solubility of rape oil itself was minimal (0.3%, Fig. 2a); accordingly, rape oil in the form of either mass oil or pseudosolubilized oil was used as the substrate. When the saponin concentrations were below the CMC, the utilization ratios were higher than the solubilization ratios of rape oil (Fig. 2a). This implied that, in addition to pseudosolubilized oil uptake, cells were also allowed to directly adhere to mass oil to sustain growth. In the presence of saponin at concentrations above the CMC, the utilization ratios were lower than the solubilization ratios of rape oil (Fig. 2a), which indicated that partial pseudosolubilized oil was sufficient to support the growth of the strain. It was plausible that rape oil solubilization induced by saponin favored the increased bioavailability.

3.2.2 Effect of saponin on cell membrane permeability. The limited permeability of the cell envelope is a general problem in whole-cell bioprocesses.³⁵ Transmembrane transport is often believed to be a rate-limiting step in HOC utilization.¹¹ Surfactants, as permeabilizing agents, can speed up the transmembrane diffusion rate of HOCs by improving the fluidity and permeability of cell membranes. The cell membrane structure of S-XJ-1 was characterized by TEM with the embedding and ultrathin sectioning, to evaluate the effects of saponin on the permeability of cell membranes (Fig. 3).

Fig. 3 TEM images showing cell morphological changes of Alcaligenes sp. S-XJ-1 incubated with saponins at various concentrations ((a) 0%, (b) 0.01%, (c) 0.05%, (d) 0.5%).

Cells obtained without the saponin additive had evenly distributed cytochylema and intracellular electron densities as well as intact, clear cell envelopes and thick cell walls (Fig. 3a). Nonetheless, the exposure of bacteria to increased saponin caused remarkable changes in cell surface morphology, including blurred and incomplete envelopes, thin walls, and more irregular cells (Fig. 3b–d). This illustrated that saponins promote membrane permeability to varying extents. However, saponins at the tested concentrations were non-lethal to cells, which was confirmed by the rapid multiplication in biomass. In the present study, the saponin supplement overcame the permeability barrier of the cell envelope and accelerated the transport of rape oil into cells, providing benefits for biomass synthesis.

3.2.3 Effect of saponin on metabolic activity of cells. The metabolic state of a bacterial cell can be characterized based on esterase activity, which is typically detected by FDA.³⁶ This method was applied to evaluate the metabolic activity of Alcaligenes sp. S-XJ-1 cultivated in the presence of a saponin additive at various concentrations (Fig. 4). As the saponin amount increased, the relative fluorescence intensities initially increased and then dropped, but were consistently higher than that in the absence of saponin. Also, this indicated that a similar trend was presented for the changes of esterase activity (or cell activity). Cell activity was strongest for the bacteria cultivated with 0.05% saponin. In brief, saponin addition promotes intracellular metabolism to varying degrees. These observations are consistent with previous studies showing that enzyme activities are indeed affected by surfactant additives.^12,16

Fig. 4 Fluorescent intensity of cells labeled with FDA cultivated with saponin additives at various concentrations.

At the tested concentrations, saponin had positive influences on the utilization of rape oil. Saponin at concentrations below its CMC promoted the solubilization of rape oil, cell membrane permeability, and cell activity, and thus rapidly increased biomass. When the saponin concentrations were above the CMC, improvements in permeability may be offset by the inhibition of metabolic activities; thus, biomass growth was directly related to the solubilization of rape oil with similar changes. Therefore, the uptake of pseudosolubilized oil is important for maintaining high-yield biomass.

3.3 Role of saponin in changes in demulsifying capability

Saponins had a marked impact on the demulsification ability of Alcaligenes sp. S-XJ-1, according to the results presented in Section 3.1. Changes in demulsifying capability can be attributed to differences in cell-surface composition and corresponding macroscopic properties.^19,23 Consequently, the mechanisms by which saponin influences the demulsifying activities of S-XJ-1 were firstly analyzed with respect to cell surface properties.

3.3.1 Effect of saponin on cell-surface properties. Cell surface hydrophobicity and cell-surface charge are two important factors affecting the demulsification of W/O emulsions by demulsifying cells.³⁷ Table 1 presents the effects of various concentrations of saponin on cell-surface properties. When ΔG_bwb < 0 mJ m⁻², bacteria are considered hydrophobic; otherwise, bacteria are considered hydrophilic.³¹

Table 1 Hydrophobicity and surface charge of Alcaligenes sp. S-XJ-1 cultivated with saponin additives at various concentrations

Index	Addition amount of saponin (%)
	0	0.01	0.03	0.05	0.1	0.5	1
θW (°)	53.46 ± 0.99	94.80 ± 2.61	104.51 ± 2.48	69.73 ± 0.68	35.31 ± 2.28	21.29 ± 3.75	23.57 ± 1.82
θF (°)	38.78 ± 1.10	80.92 ± 2.87	90.92 ± 3.24	64.95 ± 1.86	47.64 ± 3.85	13.59 ± 3.26	19.43 ± 2.39
θD (°)	58.58 ± 0.78	68.69 ± 5.12	67.72 ± 4.41	68.35 ± 0.42	75.61 ± 2.75	83.05 ± 4.00	84.36 ± 3.08
ΔGbwb (mJ m^−2)	−4.19 ± 2.20	−59.49 ± 2.90	−70.20 ± 3.54	−8.51 ± 2.59	34.24 ± 4.19	9.38 ± 3.50	9.69 ± 2.68
ZP (mV)	−40.1 ± 0.35	−40.6 ± 1.12	−43.0 ± 2.31	−42.1 ± 0.67	−52.0 ± 2.51	−50.6 ± 1.20	−49.9 ± 1.85

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As shown in Table 1, the θ_W of 53.46° and negative ΔG_bwb value indicate that cells obtained from culture medium without saponin were hydrophobic. In contrast, for bacteria cultivated with 0.01–0.05% saponin, θ_W was greater (above 69°) and ΔG_bwb was lower, suggesting an increase in the CSH of cells. For bacteria cultivated with 0.1–1% saponin, the θ_W was smaller (below 36°) and ΔG_bwb > 0 mJ m⁻², so cells were considered hydrophilic with a reduced CSH. In the case of ZPs, the ZPs of all tested samples were negative at the initial pH (approximately 6.0). For bacteria cultivated with 0.01–0.05% saponin, the ZPs were approximately −42 mV, similar to that of the cells obtained without saponin. Bacteria cultivated with 0.1–1% saponin exhibited more negative ZPs (approximately −50 mV). The results of ZPs indicated that saponin at concentrations above CMC had a great influence on the cell-surface charge. Overall, distinct effects on the CSH and surface charge were observed below and above the CMC of saponin.

In this study, for the bacteria cultivated with saponin at various concentrations, the demulsification ratio initially increased and then almost stabilized with increasing θ_W (Fig. S2†). Many studies have shown that the CSH of demulsifying bacteria has a positive correlation with emulsion breaking performance.^4,23 One possible explanation for this observation was that the cells with high CSH and interfacial activity were allowed to adsorb onto the oil-water interface and replace some emulsifier molecules, resulting in the drainage of the interfacial film and the demulsification of the W/O emulsion.³⁸

3.3.2 Effect of saponin on cell-surface composition. Cell-surface properties result from the unique chemical structure of the cell surface. The effects of saponin on the functional groups and elements present on the cell-surface were examined by ATR-FTIR and XPS, respectively.

Cell-surface differentiation of demulsifying bacteria cultivated with saponin was analyzed by FTIR spectra (Fig. S3†) and PCA (Fig. 5a). There were striking differences in the functional groups between cells cultivated without and with (0.01% and 0.03%) the saponin additive. This indicated that a small amount of saponin can cause large changes in the cell-surface. However, a relatively minor difference was observed among cells cultivated with higher saponin concentration (0.05–1% saponin, Fig. 5a). To further characterize the specific differences in cell-surface composition, elements (such as C, N, O, and P, detailed in Table S2†) detected by XPS were further modeled with respect to the surface composition for three classes:²⁷ proteins, polysaccharides, and lipids (Fig. 5b). As summarized in Fig. 5b, as saponin increased, the protein content of the cell surface tended to increase initially and then drop, which was contrary to the changes in polysaccharide content. The cells cultivated with 0.01% and 0.03% saponin showed an excess of proteins (30.6–35.9%) and fewer polysaccharides, and were quite different from those that were not exposed to saponin, consistent with the results of the PCA analysis of the FTIR spectra. The protein or polysaccharide contents of bacteria with 0.05% saponin were similar to those of bacteria without the addition of saponin. These results indicated that differences among the tested samples reflected differences in the protein and polysaccharide contents of the cell-surfaces. Saponin may modify cells via simple physicochemical adsorption^39,40 and via physiological stimulation.¹⁰ When the saponin concentrations were below the CMC, the cells exhibited almost no adsorption of saponin (Fig. S4; its measurement procedure was shown in the ESI†). Thus, it was inferred that saponin adjusts the relative amounts of cell-surface substances (i.e., increases in the proteins and decreases in the polysaccharides) by stimulating cellular metabolism. When the saponin concentrations were above the CMC, several saponin molecules were adsorbed onto the cell-surface (Fig. S4†), which likely increased polysaccharides (perhaps primarily in the form of saponin) and reduced the protein content of the cell-surface. Alternatively, changes in the cell-surface composition might be explained by the inhibited anabolism of demulsifying active substances due to the high concentration of saponin.

Fig. 5 Discrimination between bacterial cells cultivated with saponins at various concentrations. (a) PCA score plots from ATR-FTIR spectra (1800–900 cm⁻¹), (b) surface compositions in terms of model compounds, as deduced from XPS.

A positive relationship was observed between protein concentration and θ_W for all tested samples (Fig. S5†). This suggests that intrinsic CSH was conveyed to the cell surface proteins. Mukherjee et al.⁴¹ found that an increase in surface-exposed proteins on Escherichia coli cells during biofilm formation is accompanied by an increase in CSH. Huang et al.¹⁹ and Peng et al.²³ characterized Alcaligenes sp. S-XJ-1 cells cultivated with diverse carbon sources in situ, and found that the excess of cell surface proteins or lipids increased hydrophobicity, and surface proteins were particularly important. Our results are consistent with these previous studies.

In summary, saponin at concentrations below the CMC, as a medium additive, increased the protein content and deceased the polysaccharide content, and thus enhanced the CSH, leading to the improved demulsification, contrary to concentrations above the CMC. In the culture system of using both oil rape and saponin, flexible responses in the properties and composition of cell-surfaces with respect to saponin provide an adaptive mechanism for the growth of Alcaligenes sp. S-XJ-1.

4. Conclusions

Saponin at concentrations of 0.01–0.05% enhanced both the biomass and demulsification capability of Alcaligenes sp. S-XJ-1. Biomass growth was attributed to the saponin-enhanced utilization of rape oil resulting from oil solubilization, improved membrane permeability, and increased cellular metabolism. The improved demulsification was attributed to saponin-strengthened CSH via an increased protein content and reduced polysaccharide content of the cell-surface. The obvious concentration-dependent effects of saponin, with the CMC as a boundary, were reflected in the interactions between saponin and both rape oil and S-XJ-1 cells. This study provides a targeted and effective strategy for increasing the yield and reducing the cost of biodemulsifiers.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (51478325).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02237e

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