Strategy to improve crude oil biodegradation in oligotrophic aquatic environments: W/O/W fertilized emulsions and hydrocarbonoclastic bacteria

Abstract

We studied petroleum biodegradation by biostimulation by using water in oil in water (W/O/W) double emulsions. These emulsions were developed using seawater, canola oil, surfactants, and mineral salts as sources of NPK. The emulsions were used in the simulation of hydrocarbon bioremediation in oligotrophic sea water. Hydrocarbon biodegradation was evaluated by CO₂ emissions from microcosms. We also evaluated the release of inorganic nutrients and the stability of the emulsion’s droplets. The double emulsions improved CO₂ emission from the microcosms, suggesting the increase in the hydrocarbon biodegradation. Mineral nutrients were gradually released from the emulsions supporting the hydrocarbon biodegradation. This was attributed to the formation of different diameters of droplets and therefore, varying stabilities of the droplets. Addition of the selected hydrocarbonoclastic isolates simulating bioaugmentation improved the hydrocarbon biodegradation. We conclude that the nutrient-rich W/O/W emulsion developed in this study is an effective biostimulation agent for bioremediation in oligotrophic aquatic environments.

Introduction

Seawater and other aquatic environments are subject to contamination by various chemicals used by modern societies. Among them, one of the most prominent and dangerous contaminants is petroleum hydrocarbons [1–3]. Accidents involving oil spillage in aquatic environments result in both immediate and chronic disturbances to the local biota, due to toxicity and recalcitrance of many hydrocarbons, along with their interaction with all lifeforms [3–5].

Different technologies have been developed to remediate aquatic environments affected by oil spillage, aiming to minimize environmental damages and economic losses caused by the presence of hydrocarbons. However, despite all efforts to remove the contaminant plumes that concentrate at the water surface, at least 25% of the oil from an oil spill remains in the environment [6]. The remediation alternatives include physical, chemical, and biological methods. Physical and chemical treatments typically cause dispersion of the pollutants and do not remove them completely from the environment [7]. However, biological treatments using microorganisms (i.e., bioremediation) can eliminate the pollutants by converting them into non-toxic waste although can also convert pollutants into more toxic compounds. These treatments have low cost, high efficiency, and avoid secondary pollution [7, 8]. When used in combination with other remediation technologies, bioremediation can lead to a successful cleanup due to complementary effects [9–11]. In situ bioremediation can rely on different strategies, including natural attenuation, biostimulation, and bioaugmentation [12]. Although bioremediation technologies are effective for treating hydrocarbon-contaminated soils [13], there is still a lack of effective technologies to accelerate the biodegradation of hydrocarbons in aquatic environments, especially in oligotrophic water (e.g., pelagic zones of marine ecosystems) [14].

Hydrocarbonoclastic bacteria are ubiquitous, although they usually occur in low abundance in pristine environments [14–16]. This fact is not surprising, since hydrocarbons have been present on Earth for millions of years as a result of natural leakage of oil from natural reservoirs and their synthesis by plants and animals [17–19]. However, when a site receives a large input of hydrocarbons (e.g., from an oil spillage), hydrocarbonoclastic populations become dominant and may represent 100% of the local microbial community [15].

The direct contact between the contaminants and the bacteria’s cells is essential, due to the low water solubility of hydrocarbons and the involvement of membrane-bound oxygenases in the initial steps of the catabolism of hydrocarbon biodegradation [14]. Cell adhesion to hydrocarbons [20, 21] and the emulsification of such compounds by biosurfactants [22–24] have been proposed as mechanisms used by hydrocarbonoclastic bacteria to gain access to these organic substrates.

Biodegradation in marine pelagic zones and other oligotrophic aquatic environments is limited by inorganic nutrients, especially nitrogen and phosphorus. The biostimulation of hydrocarbonoclastic bacteria in such environments by fertilizer application is not effective, due to the dispersion of the nutrients in the water body [25–28], and the development of a strategy to retain or minimize the loss of nutrients is desirable.

Double emulsions of water in oil in water (W/O/W) are complex liquid dispersions, in which oil droplets containing an internal aqueous phase are dispersed in a continuous aqueous phase [29]. Among the characteristics of such emulsions, it is noteworthy the high capacity of retention of hydrophilic compounds in the inner aqueous phase, the ability to introduce incompatible substances into the same system, and the protection and sustained release of chemicals originally entrapped in the internal water phase.

The double emulsions are used for encapsulation and controlled release of active compounds employed in food systems [30], and in pharmaceutical [31] and biomedical applications [32]. However, this emulsion system has not been tested with the purpose of environmental bioremediation.

In this way, this study describes the development of double W/O/W emulsions as a biostimulation agent to treat oil spills in pelagic or intertidal oligotrophic areas. These emulsions are characterized by a nutrient-rich aqueous internal phase, which is involved by a biodegradable oily droplet dispersed in a continuous aqueous external phase. We also propose the use of two hydrocarbonoclastic bacteria as bioaugmentation agents to improve the effectiveness of this new bioremediation strategy.

Material and methods

Materials

The aqueous phase was composed of seawater obtained near Trindade Island/Brazil, about 100 m from the Tartarugas beach. Other ingredients included gelatin (porcine skin, Type A) (Sigma-Aldrich); the inorganic salts NH₄NO₃ and K₂HPO₄ (Vetec Química Fina Ltda.); canola oil (Bunge Alimentos S.A.); polyglycerol polyricinoleate (PGPR) (Dhaymer’s Fine Chemicals); and coco glucoside (Biotek Fenchem LTD.).

Production of the W/O/W emulsions

The internal aqueous phase (W₁) was prepared by melting 7.5 g L⁻¹ gelatin for 30 min at 65 °C, followed by supplementation with the mineral salts (680.25 g L⁻¹ NH₄NO₃ and 133.75 g L⁻¹ K₂HPO₄). The oily phase (O) was prepared by dissolving 20 g L⁻¹ of PGPR (Dhaymer’s Fine Chemicals) in canola oil (Bunge Alimentos S.A.). The external aqueous phase (W₂) was prepared by dissolving 8 g L⁻¹ of coco glucoside (Biotek Fenchem LTD.) in seawater.

The double emulsion (W₁/O/W₂) was obtained in two steps. Water-in-oil emulsion (W₁/O) was prepared by mixing the aqueous internal phase (W₁) and the oily phase in a 2:3 ratio (v:v). The mixture was stirred in a high speed agitator (Yellowline ID25 basic) at 24,000 rpm for 4 min. The resulting emulsion was immediately cooled to 4 °C in a water bath to solidify the gelatin present in the internal aqueous phase (W₁), followed by incubation at room temperature for 1 h to improve the efficiency of emulsification [33]. The double emulsion (W₁/O/W₂) was prepared by gradual addition of W₁/O (1:5 v/v) to the external aqueous phase (W₂), followed by further stirring at 20,000 rpm for 2 min and subsequent cooling at 4 °C in a water bath. Control emulsions were produced omitting the inorganic salts from the internal aqueous phase. The W/O/W emulsions were characterized by images from a Q-Color 3 Olympus digital camera that was coupled in an optical microscope (Olympus BX50), with the QCapture Pro 6.0.0.412 software system.

Microbial inoculants

In a previous research, we isolated some strains of hydrocarbonoclastic bacteria from microbial biofilms formed on the surface of crude oil incubated in the coastal region of the Trindade Island/Brazil [34]. Two of those hydrocarbonoclastic marine bacterial strains previously isolated (Rhodococcus rhodochrous TRN7 and Nocardia farcinica TRH1) presented the ability of fast hydrocarbon biodegradation [10, 34], and both of them were selected as microbial inoculants in the present study.

Stability of the W/O/W emulsions

Photomicrographs were taken under optical microscope during 30 days to check the stability of the oil droplets present in the external aqueous phase. The emulsions were stored in screw-capped tubes and kept under an end-over-end agitation at 15 rpm, average temperature (21 ± 2) °C, and 45° tilt. Photomicrographs were analyzed using the ImageJ software (National Institutes of Health). The average size of the oil droplets was calculated by using approximately 40 droplets/image and 5 images of each treatment.

Stimulation of hydrocarbon biodegradation by fertilized W/O/W emulsions

Hydrocarbon biodegradation was analyzed in microcosms prepared in respirometric flasks containing 50 mL (92.6% of total microcosm volume) of water collected at approximately 100 m from the coast of the Trindade Island (storage for 5 days before the start of the experiment and kept at 4 °C), 0.5 mL (6.5% of total microcosm volume) of weathered oil (Marlim Field, Rio de Janeiro, Brazil, API = 20.3), and 3.5 mL (0.9% of total microcosm volume) of W/O/W emulsions. The bottles were coupled to a respirometer with intermittent air flow (Sable Systems, Las Vegas, USA). Abiotic factors, as temperature, air flow, nutrients added, and luminosity were controlled. All the treatments were conducted using three replicates. Before use, the oil was heated gradually to 210 °C for elimination of volatile compounds, simulating a natural weathering process in the water surface. The experiment was composed of 13 treatments (Table 1). We decided not to use other controls treatments such as “Water + oil + R. rhodochrous”, “Water + oil + N. farcinica”, and “Water + oil + N. farcinica + R. rhodochrous” due to previous studies conducted under the same conditions by Rodrigues et al. [10], which demonstrated that the hydrocarbon mineralization rate is low when the biostimulating agents are not added in the treatments. In addition, it would not be possible to perform the experiment with many controls because of the number of channels available in the used respirometer.

Table 1.

Description of the microcosms used in the oil biodegradation experiment using W/O/W emulsions

Treatment	Description
W	Water from the coast of the Trindade Island (control)
NFE	Water + non-fertilized emulsion
FE	Water + fertilized emulsion
FERr	Water + fertilized emulsion + R. rhodochrous
FENf	Water + fertilized emulsion + N. farcinica
FERrNf	Water + fertilized emulsion + R. rhodochrous + N. farcinica
CE	Water + emulsion components (canola oil, nutrients, coco glucoside, PGPR) + R. rhodochrous + N. farcinica
O	Water + oil
ONFE	Water + oil + non-fertilized emulsion
OFE	Water + oil + fertilized emulsion
OFERr	Water + oil + fertilized emulsion + R. rhodochrous
OFENf	Water + oil + fertilized emulsion + N. farcinica
OFERrNf	Water + oil + water + fertilized emulsion + R. rhodochrous + N. farcinica

Rhodococcus rhodochrous TRN7 and Nocardia farcinica TRH1 cells were grown for 48 h at 30 °C and 150 rpm in nutrient broth (Merck), centrifuged and washed twice in sterile saline to remove components of the growth medium. Bacterial cells were added to the microcosms at a concentration of 1.0 × 10⁶ cells mL⁻¹ in each microcosm. F_ERrNf and OF_ERrNf microcosms received 5.0 × 10⁵ cells mL⁻¹ of each bacterial species.

A P1000 pipette tip (Kasvi) was added to each microcosm after removing the tip end to give access for a Pasteur pipette to collect the bottom layer. To analyze the rate of hydrocarbon mineralization, the microcosms were incubated at 24 °C (average temperature of coastal water in Trindade) for 30 days under ambient pressure. Atmospheric air was injected at a 500 mL min⁻¹ flow during 5 min at a 5.9 h interval. The CO₂ accumulated during this period was analyzed in a CO₂ infrared detector. During the first 5 days of the experiment, approximately half the aqueous volume of the flasks (25 mL) was removed daily from the bottom layer and replaced with the same volume of water from the coast of Trindade Island stored at 4 °C. From the 6th until the 15th day, this same operation was done every 2 days, and from the 16th to the 30th day, the replacement was done every 3 days. This procedure was adopted to simulate the effect of dispersion of the mineral nutrients in the ocean, supposing they were applied as regular mineral fertilizers. In this way, it is expected that the nutrients contained in the inner aqueous phase of the emulsions would remain in the water/oil interface, instead of suffering dispersion into the water body, thus stimulating hydrocarbon biodegradation in the water/oil interface. The content removed from the flasks was used to quantification of total nitrogen and phosphorus in according to the “Analysis of micronutrients” section.

Analysis of macronutrients

Total organic carbon, Kjeldahl-nitrogen, and phosphorus were analyzed in the water used to prepare the microcosms and in the aqueous phase collected from the bottom layer of the F_E treatment (three replicates). The total phosphorus was quantified by the Mehlich-1 method [35]. Organic carbon was measured using a total organic carbon analyzer (TOC-L, Shimadzu Corporation). Kjeldahl-nitrogen was determined in a semimicro-Kjeldahl destillation apparatus [36, 37].

Statistical analysis

Statistical analyses were performed using the “R” software. The F and Shapiro-Wilk tests were used to test the variance and normality of the data, respectively. Means of nonparametric data were compared by the Wilcoxon test at 5% probability.

Results and discussion

Microscopic characterization of W/O/W emulsions

The optical microscopic analysis revealed the presence of oil droplets of varying sizes dispersed in the external aqueous phase (Fig. 1). The mean diameter of the droplets varied between 1.4 and 7.2 μm. The presence of oil droplets with different sizes can be advantageous for the bioremediation process, because they may differ also in terms of stability. This could assure a gradual release of nutrients from the internal aqueous phase, meeting the demand of the microbial hydrocarbonoclastic populations for these elements during a prolonged period, and thus stimulating a continuous biodegradation of the organic contaminants.

Fig. 1.

Fertilized W/O/W double emulsion produced for use in oil bioremediation in oligotrophic aquatic environments. The visual intensity of the dark color inside the droplets varies with the amount of inner aqueous phase

In the production of the emulsions, the use of canola oil, which serves as carbon source by many heterotrophic microorganisms, such as hydrocarbonoclastic populations, can potentially compete with the target hydrocarbons as substrate for the later populations. However, the canola oil is recognized as one of the most promptly degradable vegetable oils in marine environments [38]. Thus, this oil can stimulate a fast initial growth of hydrocarbonoclastic populations, with further biodegradation stimulation of hydrocarbons to which the oil droplets tend to adhere.

Coco glucoside was selected as one of the emulsifying agents due to its low toxicity and high degradability, in addition to be synthesized from renewable raw materials [39]. Similarly, PGPR is a synthetic surfactant used in the food and pharmaceutical industry, and it does not have known toxic effects towards living organisms [40]. Both surfactants are classified as “generally recognized as safe” (GRAS).

Over the past two decades, W/O/W emulsions proved to be suitable for the encapsulation of various active compounds, such as hormones [41, 42], steroids [43], analgesics [44], and antiseptics [45]. They are also used in the cosmetic [46] and food industries (e.g., for the encapsulation of vitamins and minerals) [47–50]. Apart from the many possible applications of double emulsions, we are not aware of any study that has reported the use of fertilized W/O/W emulsions as a strategy for gradual release of nutrients in open water bodies or in coastal areas contaminated with petroleum hydrocarbons.

Nutrient limitation in marine and other aquatic ecosystems can limit the biodegradation rates of spilled oil [51]. Our objective was to obtain a biostimulating agent that sticks to the oily layer in the water surface or that covers rocks in polluted shorelines, and gradually releases its mineral nutrients to meet the demand of the hydrocarbonoclastic populations feasting on the pollutants at these interfaces. This biostimulation methodology is quite different from applying non-encapsulated soluble nutrients directly into the affected site, which tend to disperse into the water body or be washed by the tidal movement when the contamination occurs in the shoreline. Even if the movements of the sea spread the oil layer over a large surface, the nutrient-enriched oil droplets would remain attached to the contaminants.

To confirm this hypothesis, we evaluated the kinetics of nitrogen and phosphorus loss from the emulsion to the aqueous phase of microcosms, which represents the water column of a pelagic zone (Fig. 2). The release of both nitrogen and phosphorus from the fertilized emulsions was initially fast. In the occasion of each sampling, half the volume of the aqueous phase was removed from the bottom of the microcosms for nutrient analysis and replaced with the same amount of water. After 5 days, the aqueous phase took 70% of the phosphorus and 45% of the nitrogen from the emulsion. The variation about the release of nitrogen and phosphorous is related to different diffusion coefficients between these compounds [52]. A gradual loss of nitrogen was then observed, while phosphorus release to the aqueous phase became negligible (Fig. 2). After the 7th day, the phosphorus concentration in the aqueous phase was not detected, and the release of the element from the emulsion to the aqueous phase was considered to have stopped. By the end of the experiment, the loss of nitrogen from the emulsion to the aqueous phase was close to that found for phosphorus in the first 5 days. Our results indicate that around 30% of the nutrients added to the microcosms as components of the inner aqueous phase of the double emulsion were retained in the water-oil interface after 30 days, confirming our expectations.

Fig. 2.

Total phosphorus and nitrogen released during 30 days after the application of the W/O/W emulsion to seawater from Trindade Island containing a layer of weathered crude oil on the surface. *No detection of phosphorous after the 7th day. The hypothetical loss of both elements was estimated based on the amount of the elements that would be removed with sample collection (i.e., 50% in the first sampling, 75% in the second sampling, 87.5% in the third sampling, and so on, which is equivalent to the equation of exponential saturation 100 % [1 − e^−t ln(2)], where t is the time in days), if the phosphorous and nitrogen were to suffer dispersion into the water column

In marine environments, especially in pelagic zones distant from the shoreline, microbial growth is limited by mineral nutrients, particularly when the environment receives a sudden input of organic substrates, as occurs during an oil spillage [53]. The attachment of the nutrients to the contaminant plume and their gradual release from the inner aqueous phase of the oil droplets allow microorganisms to use these nutrients to support the catabolism of hydrocarbons and thus accelerate the elimination of the contaminants.

Stability of the W/O/W emulsion

The average size distribution of the oil droplets in the W/O/W emulsion during the 30 days under gently rotation at room temperature is shown in Fig. 3a. The average size of the oil droplets increased from 1.5 μm to approximately 3 μm during the first 15 days, after, they increased sharply to approximately 7 μm. It is well known that emulsions are thermodynamically unstable, so even in the presence of emulsifiers agents, the system will eventually be destabilized by coalescence of droplets. This phenomenon may explain the increase in the droplet’s average size throughout the days. We discarded the Ostwald ripening as a growing mechanism of the droplets since the solubility of the oil in water is negligible. We also observed an increase in size variability among the oil droplets during the incubation period (Fig. 3b). As the smaller droplets coalesce, the higher variability resulted in the formation of oil droplets of higher dimension.

Fig. 3.

Stability of the oil droplet sizes (n = 200) in the W/O/W emulsions during incubation at gently rotation at room temperature. a Average droplet size. The error bars indicate the standard deviation. b Droplet size distribution. The boxes indicate where 50% of the data are contained; the line inside each box indicates the median; the outer bars correspond to extreme, but not disparate, data. The asterisk (*) represents outliers

Oil droplets dispersed in an aqueous continuous phase tend to coalesce [54], but this phenomenon may be reduced by the presence of the emulsifiers and NaCl in the continuous phase [55]. In our work, the use of seawater to prepare the W/O/W emulsion may have contributed to increase the stability of the fertilized water-containing oil droplets.

Effect of W/O/W emulsions on oil biodegradation

The water used both in the production of emulsions and in the microcosms for bioremediation studies initially contained a concentration of 1.12 mg L⁻¹ of total nitrogen and 29.98 mg L⁻¹ of organic carbon. Phosphorus concentration in the aqueous phase was not detected by the Mehlich-1 method (Mehlich, 1953).

There was a great difference in CO₂ emission between the treatments during the 30 days of the experiment incubation period (Fig. 4). The addition of only weathered crude oil to water (treatment O) did not affect CO₂ emission, when compared with the control (W). According to Harayama et al. [56], the amounts of nitrogen and phosphorous are insufficient to support the microbial growth in marine environments. Among the treatments involving the use of fertilized W/O/W emulsions, OF_ERrNf, which contained the fertilized emulsion and the bacteria R. rhodochrous TRN7 and N. farcinica TRH1, showed the highest amount of released CO₂ (4857 μmol), followed by treatments with these same bacteria added separately. CO₂ emission by NF_E and ONF_E treatments, which received non-fertilized double W/O/W emulsions, was significantly lower than the corresponding treatments receiving fertilized emulsions. This result was expected, when one considers the low availability of mineral nutrients in the seawater used in this study. Both bacterial species used in the microcosms seem to have mechanisms to modify the hydrophobicity of cell surface to possible adhesion to hydrocarbons and then facilitate the catabolism of these molecules [57, 58]. This fact can be related to the increase of CO₂ emission from inoculated treatments.

Fig. 4.

Carbon dioxide released after 30 days of incubation treatments simulating bioremediation strategies using W/O/W emulsions and hydrocarbonoclastic bacteria. The bars followed by the same letter do not differ significantly by the Wilcoxon test at 5% probability. Three replicates were used for error bars (representing the standard deviation). W, water; NF_E, water with double non-fertilized emulsion; F_E, water with double fertilized emulsion; F_ERr, water with fertilized double emulsion and R. rhodochrous; F_ENf, water with double fertilized emulsion and N. farcinica; F_ERrNf, water with double fertilized emulsion and R. rhodochrous and N. farcinica; C_E, water with canola oil, NH₄NO₃, and K₂HPO₄; O, oil with water; ONF_E, oil in water with double non-fertilized emulsion; OF_E, oil in water with double fertilized emulsion; OF_ERr, oil in water with double fertilized emulsion and R. rhodochrous; OF_ENf, oil in water with double fertilized emulsion and N. farcinica; OF_ERrNf, oil in water with double fertilized emulsion and R. rhodochrous and N. farcinica

There was no significant difference in CO₂ emission between the treatments containing non-contaminated water and fertilized emulsions (F_ERr, F_ENf, and F_ERrNf). When oil is present, CO₂ emission by these same treatments (OF_ERr, OF_ENf, and OF_ERrNf) differs significantly. The highest CO₂ emission by OF_ERrNf suggests that R. rhodochrous TRN7 and N. farcinica TRH1 do not exert antagonistic effects on each other and are effective in improving hydrocarbon biodegradation. Bioaugmentation with these two microbial strains, coupled to biostimulation by the nutrients present in the inner aqueous phase, was shown to be beneficial for crude oil bioremediation in oligotrophic seawater.

CO₂ emission by OF_ERrNf (4857 μmol CO₂) was around 40% greater than by F_ERrNf (3516 μmol CO₂). The only significant carbon source in the later treatment was the canola oil used to prepare the fertilized emulsion. These results indicate that additional CO₂ emission by OF_ERrNf occurred due to the mineralization of hydrocarbons from the crude oil added to it. CO₂ emission by OF_ERrNf was also significantly higher than by OF_E (3342 μmol CO₂), indicating that the two bacteria included in the OF_ERrNf improved the effect of the fertilized emulsion. The result is coherent with the potential of use of a variety of hydrocarbon molecules [34] by the Rhodococcus rhodochrous TRN7 and Nocardia farcinica TRH1 strains. Furthermore, the highest values for CO₂ emission using both bacteria allow us to infer that no antagonist compounds are produced during the hydrocarbon biodegradation. Using respirometric assays, McFarlin et al. [59] obtained an increase from 0 to 11% on the hydrocarbon mineralization in assays of biostimulation (with nitrogen and phosphorous addition) at − 1 °C. In our study, the increase was more accentuated (comparing ONF_E and OF_E) probably due to the temperature of 24 °C and autochthonous microbial community.

When a comparison is made between the treatments with and without fertilized W/O/W emulsions, there is an increase of almost 20% in the release of CO₂. The increase is even more significant when comparing F_ERrNf/OF_ERrNf treatments, which contain the two bacterial isolates used in this study. In this case, the difference was 28%. Therefore, although it is possible that the bacteria preferably metabolize canola oil from the emulsion, there is a significant increase in bacterial activity when crude oil is present as a contaminant. A potential technology for ocean water bioremediation was tested by Warr et al. [27], consisting in fertilized clay for applications on the contaminated sites. Quantifying the O₂ dissolved in the water, the authors showed that in presence of fertilized clay, the O₂ consumption is much faster comparing with the treatments without fertilized clay. According to the authors, the oxygen consumption by the microbial community is due to direct oxidation of the small quantities of mineral matter or hydrocarbon molecules. Although it seems interesting, this technology may have limitations for in situ application, due to the low surface/volume ratio. Comparing the fertilized clay with our W/O/W fertilized emulsion, our product has a higher surface/volume ration and therefore seems to be more efficient in the field applications.

The C_E treatment was initially designed as control treatment. However, due to the air injection into the aqueous medium to supply oxygen and collect the accumulated CO₂ for respiration measurement, macro water-in-oil emulsions were formed in the microcosm, thereby trapping nutrients within them and rendering the use of this treatment as a control. However, when comparing CO₂ emission by this treatment and by the treatments with fertilized emulsions (F_E, F_ERr, F_ENf, and F_ERrNf), we conclude that the micrometric emulsions in these later treatments were more effective in sustaining microbial catabolism in the oligotrophic water present in the microcosms (Fig. 4). It is prudent to emphasize that the proportions of crude oil, emulsions, and water in microcosms occur under experimental conditions. Therefore, in possible field applications, concentrations tend to be different.

Comparing ours results with findings of Rodrigues et al. [10] is possible to affirm that the W/O/W fertilized emulsions are promising to biostimulation. Using the same methodology and similar conditions (R. rhodochrous and N. farcinica as inoculants), Rodrigues et al. [10] obtained mean maximum value of 2415 μmol CO₂, in contrast, our mean maximum value was 4857 μmol CO₂ (OF_ERrNf treatment) using the emulsions. On the same way, without the bacterial inoculation, the maximum value reached by Rodrigues et al. [10] was 535 μmol CO₂, and 3.342 μmol CO₂ was found at this work.

The application of inorganic macronutrients as N, P, and K is widely used for biostimulation in environments contaminated with petroleum hydrocarbons [60–62]. This methodology has been successfully employed in soils contaminated with hydrocarbons [63–65], because the added nutrients remain in place unless when there is leaching by rainwater. The application of inorganic nutrients aiming at promoting biostimulation in oligotrophic aquatic environments is limited by the rapid dispersal of the compounds added to the water [27]. Gradual applications of nutrients using nautical infrastructure are unfeasible due to the high costs and problems with logistics and infrastructure. Therefore, the W/O/W double emulsions appear as a new possibility to provide interventions with the purpose of biostimulation. In studies conducted after the accident involving the oil spillage in the Gulf of Mexico that was caused by the Deepwater Horizon platform in 2010, it was concluded that the low concentration of phosphorus in oligotrophic water was a limiting factor to promote the removal of the oil by natural attenuation [28]. Some strategies have been proposed to support biostimulation in oligotrophic ocean water, including the use of fertilized mineral clay flakes [27], uric acid and soybean lecithin (compounds with low solubility in water) [66], and oleophilic fertilizers, such as EAP Inipol 22 [51, 67, 68]. The use of fertilized mineral clay flakes, proposed by Warr and collaborators [27], appears to be an alternative to biostimulate open waters. However, the contact area between fertilized clay flakes and the oil plume is small, which can result in low effectiveness in the field.

Regarding the application of fertilizers, such as EAP Inipol 22, there are studies disputing its effectiveness to remediate the contamination caused by the Exxon Valdez accident [69]. One of its components, 2-butoxyethanol, is potentially toxic to live organisms. Moreover, when in contact with water, the emulsion breaks almost instantaneously, releasing all the urea into the aqueous phase, which is quickly lost [14]. Among the alternatives mentioned for biostimulation of hydrocarbonoclastic microorganisms in oligotrophic water, the use of uric acid as a source of nitrogen and soybean lecithin as a phosphorus supply seems to be the best available option suggested until now [66]. In this sense, the fertilized double emulsion developed in this work is an alternative to the currently available products aimed at biostimulation in oligotrophic water. In this W/O/W emulsion, water-soluble nutrients are carried inside hydrophobic oil droplets, which have affinity for the contaminants, and they gradually release the nutrients around the target pollutants (Fig. 2).

The use of the previously characterized hydrocarbonoclastic bacteria, R. rhodochrous and N. farcinica [34], had a significant effect on petroleum hydrocarbon biodegradation. This behavior is expected to occur in the open sea under a real situation. Therefore, both bacteria have the potential to be used in bioaugmentation to stimulate biodegradation of petroleum hydrocarbons in aquatic environments.

The initial bacterial colonization of the oil pellicle by the selected hydrophobic and hydrocarbonoclastic bacteria (R. rhodochrous and N. farcinica) may have benefited further colonization by autochthonous populations, as a result of the initial hydrocarbon biodegradation [34, 70]. The microbial ecological succession in the water/oil interface within the affected regions increases the likeliness of colonization by new hydrocarbonoclastic microorganisms, thus increasing the efficiency of contaminant removal. As previously suggested by Rodrigues et al. [34], the movement of ocean currents promotes the dispersion of microbial cells, which when in contact with oil, they may use it as a substrate. Furthermore, horizontal transference of catabolic genes to autochthonous bacterial populations via mobile genetic elements is another mechanism [71] by which hydrocarbonoclastic bacteria used in bioaugmentation can stimulate hydrocarbon biodegradation [72].

Conclusions

Water in oil (W/O/W) emulsion was used as a viable alternative for biostimulation in oligotrophic marine environments contaminated with petroleum hydrocarbons. The gradual release of nutrients from the inner aqueous phase supports prolonged microbial growth in the water/oil interface and assists in the metabolism of hydrocarbons. The use of the versatile hydrocarbonoclastic bacteria R. rhodochrous TRN7 and N. farcinica TRH1 is also indicated for bioaugmentation to remediate oil contamination in marine environments.

Funding information

This work was supported by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq), which provided all approvals, permits (project grant number 405544/2012-0), and authorization access to genetic resources (process number 010645/2013-6).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.