Biosurfactant-mediated biodegradation of straight and methyl-branched alkanes by Pseudomonas aeruginosa ATCC 55925

Leaking from oil wells, tanks, pipes and transportation vehicles together with the inadequate waste disposal from the oil industry at large (oil exploration and recovery) have become important sources of environmental contamination (Leahy and Colwell 1990). Alkanes, particularly n-alkanes, are important components of crude oils and its derivatives, such as heating oil, jet fuel, gasoline and kerosene (Marin et al. 1995; Berekaa and Steinbüchel 2000). In nature, some microorganisms oxidize aerobically (Berekaa and Steinbüchel 2000; Solano-Serena et al.2000; Dutta and Harayama 2001) and anaerobically (Chayabutra and Ju 2000; Kniemeyer et al. 2003), co-metabolize (Whyte et al. 1997; Garnier et al. 2000) and detoxify most of the C4-C20 compounds from linear, branched and cyclic alkanes (Scott and Finnerty 1976; Leahy and Colwell 1990), including low-carbon hydrocarbons, which may affect cell membrane integrity (Marin et al. 1995). Particularly, alkanes that are metabolized via oxidation are used as a carbon source for cell growth. Generally, oxidation of alkanes occurs by terminal C-H oxidation followed by β-oxidation. Alternatively, bacteria use α, ω, and Finnerty oxidations as well as β-alkyl group removal by β-descarboxymethylation (Schaeffer et al. 1979). The fate of alkanes during the biodegradation process can be used as a practical tool for assessing bioremediation of oil-polluted sites, which involves some biological-based engineering techniques to improve the microorganisms' ability to biotransform the contaminant to a less or non-toxic state (mineralization), resulting in a more economic and environmentally friendly approach.

Besides the effects of environmental conditions on oil biodegradation, other factors intrinsic to oil, such as oil solubility, partition coefficient, dissolution rate, viscosity and physical state become rate-limiting in the cell-oil uptake and biodegradation by the cell. Consequently, only a small fraction of hydrocarbons will be present in the bulk water phase ready for oxidation, co-metabolism or detoxification (Zhang and Miller 1992), most of it being concentrated in the oil-water interface. In response to this, cell adaptations to growth on oily substrates are also depicted in nature. Particularly, biosurfactant production can occur, which would enhance oil dispersion into the aqueous phase and retard volatilization of low carbon atom-hydrocarbons (below C7), favoring biodegradation (Kretschmer et al. 1982; Neu 1996; Bruheim et al. 1999; Rocha et al. 1999; Rocha et al. 2000). However, this type of amphipathic molecules can also render inhibitory and neutral effects (Bruheim et al. 1999). Despite of that, surfactants, especially rhamnolipidic biosurfactants, have been reported to enhance the biodegradation of crude oil (Rocha and Infante 1997) and many other oil derivatives (Zhang and Miller 1992; Zhang and Miller 1994; Zhang and Miller 1995; Al-tahhan et al. 2000). This type of tensio-active glycolipids are produced by some strains of Pseudomonas aeruginosa, which also depict the ability to undertake the oxidation of a wide variety of oil components, including alkanes. In addition to biosurfactant production, cell-to-cell and cell-to-substrate interactions play an important role on alkane biodegradation. In relation to this, hydrophobic compounds can alter cell membranes (Heipieper and Bont 1994; Whyte et al. 1999), including cell surface hydrophobicity, which enhances adhesion of cells to hydrocarbons in the water-hydrocarbon interface (Scott and Finnerty 1976; Rosenberg 1991; Baldi et al. 1999) and transportation through the cell membrane. In response to all these factors, oil-biodegrading bacteria have shown different patterns of alkane oxidation.

In this study we investigated the patterns and kinetics of alkane degradation by a biosurfactant-producing Pseudomonas aeruginosa (ATCC 55925) grown on natural heating oil (mainly of 11-21 carbon atoms) and commercial n-alkanes (from C7 to C19 carbon atoms) in relation to biosurfactant. Cell growth and CO₂ production are commonly used as indirect indicators of hydrocarbon biodegradation; however, these techniques do not demonstrate the real changes that hydrocarbons suffer when they are used as carbon sources for cell growth, such as the degree of hydrocarbon depletion, the patterns of hydrocarbon biodegradation and other cell-hydrocarbon and hydrocarbon-hydrocarbon interactions. In this study we followed directly the hydrocarbon biodegradation by analyzing the substrate through the Gas chromatography technique. Also, some structural and morphological cell strategies for the uptake of hydrocarbons were elucidated by electron microcopy.

Pseudomonas aeruginosa ATCC 55925 was used in this study because of its ability to produce a potent tensio-active agent in a rich culture medium and because of its potential to biodegrade a wide variety of hydrocarbon compounds (Rocha and Infante 1997; Rocha et al. 1999; Rocha et al. 2000).

Hydrocarbon degradation has been usually reported as total saturate or aromatic loss. In this study we determined the fate of each hydrocarbon under different conditions in other to establish a pattern of biodegradation. P. aeruginosa ATCC 55925 showed different patterns of alkane biodegradation in the context of a single aliphatic compound present alone or as part of different hydrocarbon mixtures, such as a heating oil and an artificial mixture of alkanes. Also, the alkane chain length, alkane branching and the biosurfactant-mediated dispersion of alkanes into the aqueous medium were investigated.

Since no low molecular weight-hydrocarbon species were found in the heating oil (which has been reported to be either toxic to cells or volatile) all n-alkane species degraded and supported cell growth, while iso-alkanes showed some degree of recalcitrance. In this latter case, methylation of alkanes, as in iso-alkanes, could have decreased the solubility of the aliphatic compounds, which in turns would have rendered resistance to or discouraged biodegradation. This is especially true when methylation occurs at the saturate β-carbon, which is known to inhibit β-oxidation unless the bacterial population is able to β-descarboxymethylate (Schaeffer et al. 1979; Singer and Finnerty 1984; Berekaa and Steinbüchel. 2000). In addition, n-alkanes probably inhibited iso-alkane degradation as previously reported (Marin et al. 1995). However, we also showed in this study that n-alkanes could instead enhance the biodegradation of branched alkanes.

On the other hand, the use of the biosurfactant significantly enhanced degradation of all alkane species, including recalcitrant iso-alkanes. These results suggested that biosurfactant-mediated dispersion of hydrocarbons played a very important role in the degradation of saturated compounds (Neu 1996; Bruheim and Eimhjellen 2000; Noordman and Janssen 2002), regardless of the metabolic strategy used by the bacterial population. In the case of iso-alkanes, biosurfactant-induced emulsions probably compensated the reduction of hydrocarbon solubility caused by methyl branching, which would have lowered substrate availability to cells. Highly volatile alkanes showed the highest hydrocarbon loss in the abiotic systems, and hence, the lowest demonstrable degradation in the biotic systems. Contrary to what we expected, the biosurfactant did not seem to affect volatilization of low molecular weight hydrocarbons. According to these results we suggest that the biosurfactant increased the low solubility caused either by methyl branching or by the carboxylic derivative obtained at the initial oxidation steps of alkanes when they became slow-moving compounds.

Pristane, a low solubility multi-methyl branched alkane, usually remains recalcitrant in biodegradation systems, and it is even used as an internal marker to determine biotic hydrocarbon loss. In this study, the recalcitrance of pristane observed under some conditions suggested that low solubility and probably the substitution pattern after several cycles of β-oxidation would have inhibited oxidation. Particularly, the methyl substitutions at carbon 3 would have rendered pristane recalcitrant, unless they were bypassed by a β-decarboxymethylation event (Cantwell et al. 1978). Opposed to those results, our data suggested that the biosurfactant and the presence of some types of n-alkanes directly enhanced degradation of pristane by increasing its solubility and indirectly by allowing pristane to reach more easily the β-oxidation steps. This novel result contrasted with previous reports which indicated that n-alkanes inhibited the biodegradation of methyl branched alkanes (Leahy and Colwell 1990). It was therefore shown that in terms of net degradation value and pattern of degradation, alkanes behaved differently depending on whether they were a unique carbon source or part of a particular hydrocarbon mixture (natural or artificial), indicating that several types of hydrocarbon-hydrocarbon and hydrocarbon-cell interactions occurred.

In addition, it was demonstrated in this study that the different patterns of biodegradation became similar when hydrocarbons were dispersed by the biosurfactant. Even though it has been reported that biosurfactants usually enhance biodegradation of single hydrocarbons, it is also known that micellar solubilization can affect the biodegradation of hydrocarbon mixtures depending upon their ability to partition into the micellar core. In mixed systems, alkanes compete among themselves to partition into the micelle and a decreased rate of degradation may result due to exclusion, or very low levels of solubilization within the micelle (Kniemeyer et al. 2003). By the contrary, our results demonstrated that biosurfactant enhanced biodegradation of alkanes under all conditions.

Since no detectable bacterial growth was associated with low molecular weight alkanes in any biotic system (data not shown) and considering that such hydrocarbons were exhausted in the abiotic systems due to volatilization, we suggest that substrate unavailability was the main limiting factor that affected the time-course and fate of such hydrocarbons. Nevertheless, the toxicity of low molecular weight alkanes (Solano-Serena et al. 2000) or the lack of capability of P. aeruginosa to degrade these hydrocarbons (Scott and Finnerty 1976) cannot be ruled out with the data at hand.

These findings report neatly the different patterns of biodegradation and the fate of particular n-alkanes when they impact individually or as part of an alkane mixture, together with the effect of a biosurfactant under such conditions. These results would impact the expectations and interpretation of the alkane degradation under the context of bioremediation.

It was also shown in this study that P. aeruginosa ATCC 55925 depicted interesting cell strategies to degrade hydrocarbons, such as biosurfactant (Marin et al. 1995; Wolfaardt et al. 1998) and non biosurfactant-mediated cell surface changes as well as the formation of inclusions. SEM analysis revealed several types of extracellular bacterial structures when P. aeruginosa ATCC 55925 was grown on hydrocarbons, probably to increase the substrate surface area, and hence, to facilitate biodegradation. Based on our results we propose for the first time, to our best knowledge, that cell clusters and cell flocks were part of an initial phase in the formation of the final spherical structures surrounding the oily substrate. Wu and Ju (1997) and Whyte et al. (1998) have reported this type of cell clusters and cell flocks as unique cell adaptations while growing on hydrocarbons, suggesting a cross-linked polymeric structure. However, these authors failed to demonstrate the step-by-step formation of the final polymeric spheres. Based on our evidence, we have suggested that cells linked to each other by extracellular appendages could participate in the waxy particles formation and eventually in the formation of the final polymeric spheres through the addition of successive cell layers. All these results suggest that P. aeruginosa ATCC 55925 exhibited many structural changes at the cell surface level, some of them probably mediated by the biosurfactant, and certainly in combination with biosurfactant as strategies to adapt to oily substrates. It is worth noting that the production of extracelullar polymeric substances, changes in the fatty acid composition of membranes (Garnier et al. 2000) and cell appendages (Scott and Finnerty 1976; Kretschmer and Wagner 1982; Takeda et al. 1991; Whyte et al. 1999; Wolfaardt et al. 1994; Marin et al. 1996) have been reported previously as responses of bacterial growth on hydrocarbons. However, the multi-layer composition of the polymeric spheres observed in the study is, to our best knowledge, the first report of such structural pattern. Even though cell hydrophobicity was not investigated in this study, it has been reported to occur on P. aeruginosa strains growing on hydrocarbons by altering the LPS or shortening LPS O-antigen on the cell surface (Zhang and Miller 1994; Al-tahhan et al. 2000; Norman et al. 2002).

TEM observation also revealed intracellular transparent vesicles not depicted in cell cultures growing on non-hydrocarbon culture media. We were unable, with the data at hand, to determine the nature of these inclusions and their content. Nevertheless, it has been speculated that these structures could contain metabolic waste from the hydrocarbon catabolism or may function as reservoirs for untouched hydrocarbons (Marin and Laborda 1996). Other energy-dependent mechanisms (efflux-influx) and metabolic strategies such as reduction of low molecular weight aliphatic toxicity could also be involved in P. aeruginosa cultures growing on alkanes. None of these could be ruled out in this study.

In summary, different patterns of hydrocarbon degradation and cell strategies were shown by P. aeruginosa ATCC 55925 growing on aliphatic compounds as the sole carbon and energy source. Susceptibility of alkanes to degradation depended upon the presence of other readily available hydrocarbons, type of hydrocarbon, dispersion into aqueous phase, hydrocarbon volatilization, cell metabolic pathways and several structural changes from inclusions to complex extracellular polymeric spheres. This study investigated total hydrocarbon loss as well as individual alkane utilization in terms of specific patterns of microbial and biosurfactant mediated-biodegradation and cell adaptations to hydrocarbon growth. We believe that determining different degradation profiles for specific hydrocarbon families under different conditions will contribute to improving oil bioremediation techniques.