BIP - Production of polyclonal antisera to manganese superoxide dismutase expressed in downy mildew resistant pearl millet and its application for immunodiagnosis

Keywords: Absidia fusca, anthracene, biodegradation, fluoranthene, kinetics.

A strain of Absidia fusca was isolated from a pesticide-contaminated soil (Annaba, Algeria). The biotransformation capability of this strain towards two polycyclic aromatic hydrocarbons (PAHs): anthracene and fluoranthene was compared to that exhibited by another strain of A. fusca isolated from a non-contaminated milieu and considered as a control. The strain isolated from the contaminated soil was more efficient than the control to remove anthracene from the medium, during all the kinetics (90% removed versus 45% after 24 hrs). Concerning fluoranthene, the amount removed by both strains was very high during the first 24 hrs however the control strain was slightly more efficient (94% versus 89%) while the results were similar for the two strains during the rest of the kinetics. On the whole, A. fusca has a potential interest for the bioremediation of PAHs.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are fused ring aromatic compounds formed during the incomplete combustion of almost any organic material and are ubiquitously distributed in the environment. Some of them are considered as dangerous substances because of their mutagenic and carcinogenic potentialities. The presence of PAHs in contaminated soils and sediments poses a significant risk to the environment and human health. Microbial biotransformation is a major environmental process affecting the fate of PAHs in both terrestrial and aquatic ecosystem. The genus Absidia is not known as efficient in the bioremediation of environmental pollutants, only our recent reports underlined the ability of Absidia fusca Linnemann to metabolize herbicides (Bordjiba et al. 2001) and phenolic compounds (Guiraud et al. 2003). In this study, the ability of Absidia fusca to degrade two PAHs: anthracene (AC) and fluoranthene (FA) was investigated. Two strains were compared: one from the CBS-Baarn collection (normal forest soil), the other isolated from a polluted soil, and the results of the degradation kinetics obtained were statistically analyzed.

Materials and Methods

Absidia fusca Linemann (A) was isolated in our laboratory from a polluted soil from Algeria (Bordjiba et al. 2001) while A. fusca Linnemann (B) (CBS 102.35, Baarn, Hollande) was from soil from pine forest (Germany). For biotransformation assays mycelium and spores were aseptically inoculated into sterile Galzy and Slonimski (1957) liquid synthetic medium at pH 4.5, and incubated at 22ºC and 30ºC under shaking, for 2 days. Then AC and FA were added at a final concentration of 0.01 g l^-1. The depletion of the compounds was evaluated after 24, 48, 72, 96 and 120 hrs of cultivation at 22 and 30ºC.

Liquid media with mycelia, containing AC or FA were extracted with one volume of bidistilled ethyl acetate .The extraction was repeated twice. The organic phases were pooled, dried over anhydrous Na₂SO₄ and evaporated to dryness at 40ºC under reduced pressure and the residues were dissolved in acetonitrile. HPLC analysis was performed with a liquid chromatograph (Shimadzu) equipped with a LC 6A pump, an SIL-9A automatic injector and a RF-10AXL spectrofluorescence detector.

Detection was made at 280 nm (λ excitation) and 450 nm (λ emission) for FA and at 250 nm (λ excitation) and 450 nm (λ emission) for AC. Residual amounts of AC and FA were calculated by the integrator connected to the HPLC system, with reference to a standard. The overall biotransformation efficiency (%) was obtained after correction with regard to the abiotic degradation (not exceeding 5%) and the extraction yield (ranging from 95 to 98%). These values were then converted into mg of xenobiotic transformed per g of dry mycelium.

Reported results are means (M), standard errors of the means (SEM) andcoefficient of variation (CV%). For each xenobiotic a multifactorial analysis of variance was run, the factors being: strain S (two levels: A and B), temperature T (two levels: 22ºC and 30ºC) and time t (qualitative factor, five levels: 24 hrs, 48 hrs, 72 hrs, 96 hrs, 120 hrs). In all analyses the significance level α was 0.05 and p values were considered significant at values below or equal to 0.05.

Results and Discussion

The present study reveals the potential of the species A. fusca for the transformation / degradation of two PAHs: AC and FA, and the modification of its efficiency according to the habitat it was isolated from.

For both AC and FA bioremediation assays conducted with A. fusca: the differences in efficiency observed between the strains, the occurrence of new peaks on the HPLC profiles after 24 hrs of incubation, the progressive decrease of these peaks between 24 hrs and 120 hrs, together with the decrease of the peak corresponding to the xenobiotic, were an indication of the biodegradation activity of A. fusca. Identification of the metabolites produced was not undertaken in this work.

Concerning AC, the strain of A. fusca from the contaminated soil (strain A) was considerably more efficient than strain B, since the mean amount of AC transformed was two times higher. The incubation at 30ºC did not have any effect on AC transformation by strain A, while it had a significant inhibitory effect on strain B between 72 and 120 hrs. Concerning FA, the mean amount transformed was slightly but significantly lower for strain A during the first 48 hrs. The transformation rate exhibited by strain A was higher than that observed for strain B. No differences in the transformation rates were obtained as a function of the temperature both for strain A and B.

On the whole, the transformation rate was higher for AC than for FA with both strains (1.5 to 2.6 times). The high levels of transformation observed after 24 hrs was not a specific feature since it has been reported in the literature for numerous fungi and xenobiotics.

In two previous reports we showed that A. fusca can degrade efficiently a wide spectrum of xenobiotics: different classes of herbicides (mostly metribuzin and metobromuron), phenolic compounds (mostly ferulic acid), pentachlorophenol (Bordjiba et al. 2001, Guiraud et al. 2003). In both studies, the biodegradation capabilities were shown to be considerably increased in the strain isolated from a polluted milieu (strain A). Here we observed that A. fusca is also efficient in the degradation of PAHs such as AC and FA, with again an increased capability in strain A at least for AC degradation. Moreover strain A was less sensitive to the temperature changes. The polluted environment has probably favored the selection of a strain expressing low specific but efficient enzymatic systems able to degrade a large panel of molecules. This work underlines the interest of studying the microbial populations able to adapt in polluted ecosystems for bioremediation purpose.

References

BORDJIBA, Ouahiba; STEIMAN, Régine; KADRI, Malika; SEMADI, Ammar and GUIRAUD, Pascale. Removal of herbicides from liquid media by fungi isolated from a contaminated soil. Journal of Environmental Quality, March-April 2001, vol. 30, no. 2, p. 418-426.

GALZY, P. and SLONIMSKI, P. Variations physiologiques de la levure au cours de la croissance sur l'acide lactique comme seule source de carbone. Comptes Rendus de l'Académie des Sciences, December 1957, vol. 245, no. 25, p. 2423-2426.

GUIRAUD, P.; VILLEMAIN, D.; KADRI, M.; BORDJIBA, O. and STEIMAN, R. Biodegradation capability of Absidia fusca Linnemann towards environmental pollutants. Chemosphere, July 2003, vol. 52, no. 4, p. 663-671. [CrossRef]