Biodegradation Of Anthracene And Fluoranthene By Fungi Isolated From An Experimental Constructed Wetland For Wastewater Treatment
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are fused ring aromatic compounds formed during the incomplete combustion of almost any organic material (Menzie et al., 1992). The latter is mainly responsible for their ubiquitous distribution in the environment (Menzie et al., 1992; Cerniglia, 1993; Chaudhry, 1994). Some of them are considered as dangerous substances as a function of their toxic and mutagenic or carcinogenic potentialities (Menzie et al., 1992; Nadon et al., 1995), and 16 are present on the list of priority pollutants established by the US Environmental Protection Agency (EPA). The presence of PAHs in contaminated soils and sediments poses a significant risk to the environment and human health. PAHs are hydrophobic compounds, whose persistence within ecosystems is chiefly due to their low aqueous solubility (Chaudhry, 1994).
Their specific vegetation (macrophytes), microfauna and microflora characterize natural marshlands. As a result, high autotrophic and heterotrophic processes occur in these ecosystems. These intensive biological activities and the consequences observed on water filtering lead to the idea of an application in wastewater treatment (Moshiri, 1993; Brix, 1997). The efficiency of these systems was shown to treat domestic, agricultural or industrial wastewater (Denny, 1997; Lakatos et al., 1997). Removal of nitrogen, phosphate, ammonium … in constructed wetlands have been studied (Drizo et al., 2000; Huang et al., 2000). However, the respective roles of the different compartments and organisms involved remain unclear. Plants as well as microorganisms and soil properties may be involved. Such reconstituted ecosystems involving both plants and microorganisms with successive steps close to natural marshland may be a possibility for the treatment of PAHs-contaminated water.
Microbial biotransformation is a major environmental process affecting the fate of PAHs in both terrestrial and aquatic ecosystem (Kästner and Mahro, 1996). Interest in this area has been catalyzed by the rapid advancement of molecular ecological methodologies. The microbial degradation of PAHs having 2/3 rings is well documented. A large number of bacteria that metabolize PAHs have been isolated (Alcaligenes denitrificans, Rhodococcus sp., Pseudomonas sp., Mycobacterium sp.) (Cerniglia, 1993; Harayama, 1997). A variety of bacteria can degrade certain PAHs completely to CO2 and metabolic intermediates (Kelley et al., 1993; Müncnerova and Augustin, 1994). Knowledge on fungal degradation is limited: PAHs are oxidized to phenolic metabolites by a cometabolic process (Cerniglia, 1993; Harayama, 1997). Non-specific oxidation reaction catalyzed, by extracellular enzymes (Hammel, 1995; Bogan and Lamar, 1996) of white rot fungi, lead to the formation of a variety of quinones and hydroxylated aromatic compounds (Müncnerova and Augustin, 1994). A detailed investigation on fungal metabolism of fluoranthene (FA) was realized in Cunnighamella elegans (Müncnerova and Augustin, 1994). Several preliminary works have been done by our team to evaluate the degradation or depletion of anthracene (AC), FA, and pyrene by selected soil fungi (Krivobok et al., 1998; Salicis et al., 1999; Ravelet et al., 2000).
An experimental constructed wetland was developed in Curienne (Savoie-France), and in order to test the efficiency of the system, assays were conducted with wastewater contaminated by FA. FA is the most abundant PAH in the environment, and is so considered as a pollution indicator (Chaudhry, 1994). Our purpose was to determine the influence on the mycoflora of the increase in FA level both on a qualitative and metabolic point of view. AC is another model compound for the PAHs degradation studies: its structure is found in carcinogenic PAHs such as benzo (a) pyrene and benzo (a) anthracene (Müncnerova and Augustin, 1994). In this study the qualitative analysis of mycoflora present in the PAHs-contaminated pilot-scale-constructed wetland was done and compared to that found in a control wetland. The toxicity of FA and AC on solid media towards micromycetes isolated from these systems was investigated. The possible contribution of the mycoflora to the elimination of PAHs from the contaminated wetland was evaluated by studying the capacity of fungi to deplete FA and AC from a liquid medium. A correlation with their extracellular phenoloxidase activities was researched.
Section snippets
Constructed wetlands and soil samples
Soil samples came from experimental pilot basins simulating small-scale constructed wetlands. The system was installed in Curienne (Savoie, France) where a “filtering macrophyte bed” station for wastewater treatment is located since several years. Two successive basins were composed of 3 layers of gravel with a decreasing granulometry from the bottom to the surface (Fig. 1). The plants introduced were macrophytes, mainly Phragmites australis (cav.) Trin.: Steud., known as very efficient in
Microorganism study in the constructed wetlands
The quantitative estimation of microflora via the number of colonies (cfu) isolated per gram of dry soil showed a significant decrease of total microorganisms (bacteria and fungi) in the soil contaminated by FA (CS) (Fig. 2). On the other hand, the number of fungal colonies was significantly higher in this soil indicating at least a better resistance of some fungi to FA and perhaps the ability of some fungal species to use it as a nutrient. A hypothesis of the proliferation of selected fungal
Conclusions
A pilot-scale-constructed wetland was contaminated by FA for several months, in addition to the PAHs mixture present in the domestic wastewater entering the system. No PAHs were detected when analyzing the effluent water. An analysis of microbial population showed an increase in fungal population in the contaminated system compared to a control pilot wetland. Numerous fungal species were able to resist and adapt to the increase in FA level. Most of the fungi isolated from contaminated and
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