Surfactants are amphiphilic molecules, consisting of hydrophilic and hydrophobic domains, which tend to partition preferentially at the interface between fluids of different degrees of polarity and hydrogen bonding. Due to their unique interfacial behavior, surfactants find applications in various industrial processes involving emulsification, foaming, detergency, wetting and phase dispersion or solubilization (Lin at al., 1998).

In bioremediation processes, they can promote the biodegradation of hydrophobic pollutants such as hydrocarbons by emulsification and solubilization (De¨ziel at al., 1999).

Crude oil hydrocarbons are highly hydrophobic materials that can hardly be degraded or decomposed due to their poor availability to microorganisms. The solubilization of hydrocarbons may be restrained by their existence in oil matrix and may be also dependent on the attachment of microorganisms to oil surface. Thus the direct contact of cells with the surface of oil is thought to be important in the bioremediation of contaminated area with crude oil (Choi at al., 1999). Biosynthesis and excretion of biosurfactants into the medium are considered to be another cell mechanism aiming at an adaptation of the microorganism for using external lipophilic compounds as carbon and energy sources.

Biosurfactants are produced mainly by aerobically growing microorganisms in aqueous media from a carbon source feedstock, e.g. carbohydrates, hydrocarbons, oils and fats or of mixtures thereof.  The emulsifiers are secreted into the culture medium during the growth of the microorganism and assist in the transport and translocation of the insoluble substrates across the membranes.

Several petroleum aliphatic and polycyclic aromatic hydrocarbons can act as source of carbon and energy for the growth of microorganisms. One main factor that influences the extent of their biodegradation is their bioavailability and this is a priority research objective in the bioremediation field (Bardi et al., 2000).

Crude oil contains mutagenic, carcinogenic, growth inhibitory compounds, which can cause severe damage to aquatic and terrestrial environment.  It is estimated that 0.08–0.46% of the total oil production is wasted to the environment, eventually causing pollution to waters and shores. Surface-active agents assist in the degradation of hydrocarbon pollutants by facilitating the desorption from the soil and/or by dispersing in small droplets that are more easily attacked by microorganisms (Bognolo, 1999).

‘Hydrocarbono-clastic’ microorganisms often make and secrete one or more surface-active agents (bio-surfactants and/or bioemulsifiers). Any new or unexamined hydrocarbonoclastic environmental isolate offers a potential for the discovery of a new biosurfactant structure (Esch et al., 1999).

In the oil field, the two basic types of emulsions are water-in-oil (w/o) and oil-in-water (o/w). More than 95% of the crude oil emulsion formed in the oil field are of the w/o type (Ali & Algam, 2000).

Petroleum fuel spills have resulted in accumulation of petroleum products at refineries, fuel storage areas, airports, military bases and gasoline service stations. If these compounds are introduced to the environment at high concentrations (e.g. spills), the problem with recalcitrance to microbial degradation is considerably enhanced. Currently, considerable effort is being spent to design cheap and feasible strategies for the clean up of contaminated sites. Bioremediation, in particular, shows promise as a relatively cheap clean-up strategy. In situ bioremediation by indigenous microbial population is an increasingly popular option for clean up of sites with readily degradable contaminants (Jansson et al., 2000).

The COD (Chemical Oxygen Demand) and BOD (Biochemical Oxygen Demand) are indirect parameters to determine the pollution of industrial effluents. Treatments that could contribute to low COD or BOD values are of interest to industries (Von Sperlins, 1996).

The purpose of this research was to analyze the use of bacteria and/or their biosurfactants to bioremediate oily industrial effluents, through the determination of biosurfactant activities and of COD reduction.

1. Material and Methods

Isolation of Microbial Strains: Samples from soil contaminated with diesel oil were collected near Paulinia's petroleum refinery in Campinas (SP, Brazil), and used for the isolation of microorganisms as follows: homogenization of soil samples, surface plating, incubation at 30 ⁰C and isolation of pure colonies.

Identification and Maintenance of the Bacterial Strains: For the tentative identification of the five bacteria, various biochemical tests were undertaken and observation was made of all morphological characteristics (Holt et al., 1994). The isolated bacterial strains were identified as Acinetobacter calcoaceticus (5C2), Flavobacterium sp (5B4), Moraxella nonliquefaciens (5E2), Pantoea agglomerans (Pa) and Planococcus citreus (Pc). All strains were maintained in Nutrient agar, with the exception for Planococcus citreus, with was in YPD agar.

Culture Conditions: The bacteria were cultivated in a medium containing either 3%, 5%, 15% or 30% (vol/vol) of petroleum derived compounds (kerosene, toluene or vaseline) obtained from FLUKA and diesel oil from TEXACO as the carbon source plus 0.5 g MgSO₄, 3.0 g NaNO₃, 1.0 g KH₂PO₄, 1.0 g yeast extract and 0,3 g peptone per liter of medium. Cultures were grown in 100 mL Erlenmeyer flasks with 50 mL of medium and incubated with shaking (150 rpm) at 30 ⁰C. Duplicate flasks were collected at 48, 72 and 120 hours of incubation and the emulsification activities were determined as described below. Non-inoculated flasks were also incubated under the same conditions and were used as control.

Emulsification Activities Measurements: Culture broth was made cell free by centrifugation. 3.5 ml of the cell free broth was vigorously shaken with 2.0 ml of toluene, xylene or diesel oil on a vortex shaker and left undisturbed. After one hour, optical density of the oil-in-water emulsion phase was measured at 610 nm. The O.D. was reported as emulsification activity (Johnson et al., 1992). After 24 hours the height of the emulsion layer (water-in-oil) was measured and emulsification activity was expressed in cm (Cooper & Goldenberg, 1987). The test tubes utilized for activities measurements have diameters of 1.0 cm, producing a 2.0 cm substrate layer, which corresponds to 2 ml of tested substrate.

Biosurfactants production: The selected bacteria Pantoea agglomerans and Planococcus citreus were cultivated in a medium containing 1.5% (vol/vol) of kerosene and olive oil, respectively, plus 0.5 g MgSO₄, 3.0 g NaNO₃, 1.0 g KH₂PO₄, 1.0 g yeast extract and 0.3 g peptone per liter of medium. Cultures were grown in 500 ml Erlenmeyer flasks with 300 ml of medium and incubated with shaking (150 rpm) at 30 ⁰C for 48 hours.

Biosurfactants isolation: Culture broth was made cell free by centrifugation at 9.000 rpm during 15 minutes. The biosurfactants were isolated (Rocha et al., 1992), lyophilized and maintained on dessecador. 

Bioremediation assays: Oily effluent was obtained from a truck garage near Campinas city, São Paulo - Brazil and the bioremediation assays were carried out using Pantoea agglomerans and Planococcus citreus.

Six experiments were developed: 1a) 10 ml of Pantoea agglomerans was added to 50 ml  effluent; 1b) 10 ml of Planococcus citreus was added to 50 ml  effluent; 2) 5 ml of Pantoea agglomerans and 5 ml of Planococcus citreus were added simultaneously to 50ml effluent; 3a) 0.25 g biosurfactant isolated produced by Pantoea agglomerans was added to 50 ml  effluent; 3b) 0.25 g biosurfactant isolated produced by Planococcus citreus was added to 50 ml  effluent; 4) 0.125 g of each biosurfactant was added to 50 ml  effluent. Samples were collected at 0, 24, 48, 72 and 120 hours of incubation at 150 rpm, 30° C.

COD reduction: COD determination was carried out utilizing potassium dichromate as oxidant agent (Greenberg et al., 1992) in a COD reactor Model 2000, Hach Company. The COD reduction percentage was established as the difference among samples value and samples value at zero time. The COD reduction was determined on the six-bioremediation assays at 24, 48, 72 and 120 hours.

Oily Effluent Biodegradation: The oily effluent biodegradation, were determined with a Shimadzu GC-14A capillary gas chromatograph equipped with a flame ionization detector (FID), a split/splitless injector and a Supelcowax-10 Supelco column (30m, 0.53mm ID, 1mm film thickness). 

Gas Chromatographic conditions: The analysis were realized with a 280ºC detector, 250ºC injector, split ratio on 50:1, carrier gas helium flow rate of 20cm/s and samples of 1mL injection. The column temperature was programmed as initial temperature of 40ºC held for 5 minutes, and then increased at a rate of 5ºC/min and final temperature 200ºC for 5 minutes.

2. Results

Figure 1 shows the best results obtained for the bacterial strains grown on 5%, 15% and 30% of the carbon sources, i.e., emulsification activities higher than 0.2 for oil-in-water emulsions.

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Figure 1: Oil-in-water emulsification activities in the cell-free culture fluids of the bacterial strains grown and tested on diesel oil. 5B4: Flavobacterium sp; 5C2: Acinetobacter calcoaceticus; 5E2: Moraxella nonliquefaciens; Pc: Planococcus citreus.

Figure 2 shows the best results obtained when Pantoea agglomerans was grown for 72 hours on 3% and 5% of the carbon sources, i.e., emulsification activities higher than 1 cm for water-in-oil emulsions.

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Figure 2: Water-in-oil emulsification activities in the cell-free culture fluids of the Pantoea agglomerans. K=kerosene, T=toluene, X=xylene.

Figure 3 shows the results obtained when the bacterial strains were grown on 5%, 15% and 30% of diesel oil, i.e., emulsification activities higher than 1 cm for water-in-oil emulsions.

Figure 3: Water-in-oil emulsification activities in the cell-free culture fluids of the bacterial strains. Legends: time of incubation. A: data from Acinetobacter calcoaceticus; B: data from Flavobacterium sp; C: data from Moraxella nonliquefaciens.

Figure 4 shows the biosurfactant production by Pantoea agglomerans and Planococcus citreus.

Figure 4: Biosurfactant production by Pantoea agglomerans and Planococcus citreus after 48 hours of incubation.

Figures 5 and 6 show the COD reduction obtained after bioremediation assays with Pantoea agglomerans and Planococcus citreus and their isolated lyophilized biosurfactants.

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Figure 5:  Oily effluent COD reduction (%) after bioremediation assays 1a (Planococcus citreus strain), 1b (Pantoea agglomerans strain) and 2 (bacterial strains consortium).

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Figure 6: Oily effluent COD reduction (%) after bioremediation assays 3a (Planococcus citreus biosurfactant), 3b (Pantoea agglomerans biosurfactant) and 2 (bacterial biosurfactant consortium).

The biodegradation of the oily effluent after the various bioremediation assays, which was expressed as the percentages of the peak reduction areas following the analyses by gas chromatography, are demonstrated in figure 7 (A and B).

Figure 7: Oily effluent biodegradation during 24 hours (A) and 120 hours (B) of bioremediation assays analyzed by gas chromatography. Bioremediation Assays: 1a) 10 ml of Pantoea agglomerans; 1b) 10 ml of Planococcus citreus; 2) 5 ml of Pantoea agglomerans and 5 ml of Planococcus citreus; 3a) 0.25 g biosurfactant isolated produced by Pantoea agglomerans; 3b) 0.25 g biosurfactant isolated produced by Planococcus citreus; 4) 0.125 g of each biosurfactant.

1.Discussion

Acinetobacter calcoaceticus, Flavobacterium sp and Moraxella nonliquefaciens were able to produce a significant amount of biosurfactants detected by water-in-oil and oil-in-water emulsions, during their growth on different percentages of diesel oil.  Acinetobacter calcoaceticus showed good results (halos higher than 2.0 cm), when grown for 120 hours in diesel oil concentrations up to 5%. Moraxella nonliquefaciens showed good emulsification activities detected by absorbance increased when grown on 3% and 5% of diesel oil for 120 hours and water-in-oil emulsions when grown on 5% and 15% of diesel for 120 hours of incubation.

Apparently higher diesel oil concentrations stimulated the biosurfactant production by Flavobacterium sp, Fig. 3 (B).

As could be noted in Fig. 1 (A), Planococcus citreus grew better on kerosene and vaseline that resulted in significant oil-in-water emulsification. Planococcus citreus emulsified all the diesel oil present in the growth medium, regardless of the concentration used and of the incubation period. Unexpectedly, low emulsification activities were obtained from this strain at the incubation time tested. Probably, the greater amount of biosurfactant produced by Planococcus citreus was released to culture medium during the first hours of incubation, being utilized to emulsify the diesel oil present in the culture medium.

Pantoea agglomerans showed best results on kerosene, toleuene and vaseline as the carbon sources for growth, in different concentrations (3% and 5%). Significant results were obtained at 48 hours of incubation, detected by higher halos superior than 1.5 cm.

Planococcus citreus and Pantoea agglomerans produced 2 g/L and 0.7 g/L biosurfactants, respectively, during 48 hours of incubation in a oily medium. These amounts of biosurfactant obtained are significant when compared to the production of the well-known Alasan (2.2 g/L in 2.5-liter fermentor), (Navon-Venezia et. al., 1995).

The higher COD reduction of the effluent occurred following growth of Planococcus citreus for 120 hours (Fig. 5). When the mixture of the bacteria biosurfactants was used, a significant COD reduction was detected after 48 hours of incubation (Fig. 6). As biosurfactants do not degrade the oily compounds but emulsify it into small oil drops (Àscon-Cabrera & Lebeault, 1995), these results of COD reduction could be explained by the utilization of immediately emulsified oily compounds by indigenous microorganisms present in the effluent, which was not autoclaved.

A significant reduction on most of the peaks areas of the CG chromatograms was observed following the bioremediation assays, indicating that these treatments were effective. During the first 24 hours of incubation, every bioremediation assay showed a tendency to reduce peaks areas, indicating that biodegradation was occurring, because of the total number of peaks were reduced in 70 to 100% of their original area, independently of the bioremediation assay.

The biosurfactants and emulsification produced following growth of these bacterial strains in petroleum-derived compounds demonstrate their potential to be used in bioremediation process.

The bioremediation assays showed that Planococcus citreus was able to degrade the oily effluent, which is rich in diesel oil, and capable to reduce the COD of this effluent.  The treatment using the isolated biosurfactants showed that the bacterial biosurfactants were able to emulsify the oily compounds present in the effluent and turned this substrate available to microbial metabolism.

2. Acknowledgments

We thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for financial support.  

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