Results of bioremediation assays on supra-tidal rocks affected by the Prestige oil spill



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VERTIMAR-2005
Symposium on Marine Accidental Oil Spills

Results of bioremediation assays on supra-tidal rocks affected by the Prestige oil spill

M.A. MURADO, J. MIRÓN, M.P. GONZÁLEZ, J.A. VÁZQUEZ, M.L. CABO and J. PINTADO


Instituto de Investigacións Mariñas (CSIC). r/ Eduardo Cabello, 6. Vigo-36208. Galicia, Spain

ABSTRACT This work presents the comparative results of different biorremediation treatments applied to supra-tidal rocks affected by the Prestige oil spill, and discusses the factors that seem essential for the success of such treatments.

1. INTRODUCTION
To verify the efficiency of 11 different bioremediation approaches, a comparative assay of several formulations was carried out during March-November 2003 on a highly affected rocky area of Sálvora island (National Park of the Galician Atlantic Islands). Treatments included periodical fertilizations in both aqueous (with and without addition of adsorbents: clay, bentonite, sepiolite, vegetable fragments) and oleaginous vehicles, without and with autochthonous or alochthonous microbial reinforcements.

To avoid the main difficulties associated with the comparison among the effectiveness of the different bioremediation methods in a rocky area (the space heterogeneity, the use of indexes valid only during a short period), two resources were applied: 1) To use, for quantification of the process, panels of granitic tiles (1515 cm) impregnated with fuel (3-4 g per tile) from the Prestige and located on the environment affected by the spill. 2) To base the evaluation on the variation with time of the remaining total fuel determined in exhaustive extracts of impregnated tiles, both untreated (control) and subjected to the different treatments. In parallel, the microbiota from tiles subjected to the different treatments was studied by means of traditional culture methods and denaturing gradient gel electrophoresis (DGGE).



2. RESULTS AND DISCUSSION
After a period of 2-3 months, the differences among treated and control tiles only overcame 20% only in three cases (F, I, J, fig. 1), with null or very low statistical significance (=0.05) in the rest. The ulterior evolution of the total remaining fuel in such three cases, during a period of 250 days (fig. 2), confirmed the initial tendencies, revealed an acceleration of the process during the summer period, and definitively demonstrated a substantial advantage for the treatment J: the oleaginous formulation S-200, without microbial reinforcements. Although the process was slightly less advanced in the treated rocky area, due to the permanency of fuel residues in the concavities of the rocks, the general state agreed with the results obtained on the tiles. The S-200 formulation was, this way, the one selected to apply to other rocky coastal areas during 2004, with results that produced a rate of fuel disappearance of 50% of that detected in the actuations carried out in 2003. A possible explanation of this difference is in the aging of the fuel. In fact, we have verified that 48 hours of UV irradiation in the laboratory increases significantly the proportion of hydrocarbons that are strongly adsorbed to the silica-gel (and foregonely, therefore, to the granite surfaces).

On the other hand, the DGGE analysis of the microbiota (fig. 3) showed that, with the exception of the case of S-200, the similarities depended on the temporary factor more than on the formulation applied. Contrary to the rest of the treatments –that reduced the diversity index (Shannon) with respect to the controls–, the S-200 maintained very similar values. However, additional assays carried out with microbial reinforcements isolated from tiles treated with S-200 did not lead to significant improvements.


The preceding results allow us to propose the following conclusions:
1: The key factor for the success of a bioremediation treatment seems to depend on the capacity of the formulation used to retain nutrients and environmental microbiota on the fuel layers that cover the rocks, more than on the addition of microbial reinforcements. Fertilizations in aqueous solutions are not very capable to achieve the retention mentioned, even when adsorbent materials (clay, sepiolite, bentonite or vegetable fragments) are added.
2: On the contrary, the only oleaginous formulation used (S-200) was shown to be able to mix with the fuel layers, to retard fuel hardening and to form surfaces capable of retaining environmental microbiota and to increase the bio-accessibility of the hydrocarbons. It is also possible that the fatty acids present in the formulation act as appropriate co-substrates, which facilitate the co-metabolic degradation of the most recalcitrant hydrocarbons. Finally, it seems clear that the dispersive effect of S-200, although very slow, promotes the migration of the non-biodegradable fuel components (asphaltenes-resins), a process often considered undesirable. But this dispersion (which does not present the negative effect derived of the massive use of dispersant and surfactant products to achieve an environmental make-up) takes place at rates even inferior to those promoted by the wave action, a process considered as positive. On the other hand, it can be pointed out that S-200 was less sensitive than the other formulations to the effects of both pluvial lixiviation, and high summer temperatures in rocks blackened by the fuel.
3: The aging of the fuel retards notably the effectiveness of the bioremediation, and it even hinders pressurized water cleaning. In the laboratory it was verified that the UV radiation increases the proportions of components that are strongly adsorbed by silicates, which could explain, at least partially, such effects.
4: The microbiota captured by the surfaces treated with S-200 shows an acute seasonal variation, and it contains a large proportion of bacteria detectable by DNA, but which cannot be cultured (or we do not know how to culture). This fact explains the absence of significant accelerations obtained in the laboratory with microbial reinforcements isolated from experimental surfaces impregnated with fuel and treated with S-200.
5: At least in rocky substrates, the mechanism of the bioremediation is not assimilable to that of a microbial culture, where the carbon source (here the petroleum) disappears with the kinetics of an auto-catalytic consumption. The process is explained better by means of a model that obeys the following conditions: (1) the contaminated area receives randomly petrolytic organisms the uncontaminated environment, with an approximately constant supply, (2) the viability of the petrolytic organisms in the contaminated area is low, the period of action short, and mortality balances the supply from the source, which prevents the initiation of exponential growth processes and maintains an approximately constant active biomass. This model is consistent, in fact, with the rapid drop in the microbial populations which are added with each application of bacterial reinforcement, and also with the need to repeat said applications, simply in order to achieve an increase in the specific rate (constant) of degradation.






Figure 1: Remaining total fuel (% of the initial level) in tiles subjected to the different treatments assayed (0: control without treatment), at indicated times. Error bars refer to confidence intervals (n=4; a=0.05).

Figure 2: Time-course of remaining total fuel (%) in control tiles (0) and those treated with the three more effective formulations (F, I, J) after 2-3 months. Error bars refer to confidence intervals (n=3; a=0.05).

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