Bacterial communities and enzyme activities of PAHs polluted soils

Abstract

Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H′), the highest genetic biodiversity (H′ = 2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H′ = 0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index = 2.13 was found.

The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria.

From the fastest phenanthrene-degrading culture C_B-BT, representative strains were identified as Achromobacter xylosoxidans (100%), Methylobacterium sp. (99%), Rhizobium galegae (99%), Rhodococcus aetherovorans (100%), Stenotrophomonas acidaminiphila (100%), Alcaligenes sp. (99%) and Aquamicrobium defluvium (100%). DGGE-profiles of culture C_B-BT showed bands attributable to Rhodococcus, Achromobacter, Methylobacterium rhizobium, Alcaligenes and Aquamicrobium.

The isolation of Rhodococcus aetherovorans and Methylobacterium sp. can be consistent with the hypothesis that different phenanthrene-degrading strategies, cell surface properties, or the presence of xenobiotic-specific membrane carriers could play a role in the uptake/degradation of solid phenanthrene.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are widespread in nature (i.e. soil, water and sediments) because of several polluting anthropogenic activities (Samanta et al., 2002). They have been recognised as a potential health risk due to their intrinsic chemical stability, high recalcitrance to different types of degradation and high toxicity to living organisms (Alexander, 1999).

PAHs present in soil may exhibit a toxic activity towards different plants, microorganisms and invertebrates. Microorganisms, being in intimate contact with the soil environment, are considered to be the best indicators of soil pollution. In general, they are very sensitive to low concentrations of contaminants and rapidly response to soil perturbation. An alteration of their activity and diversity may result, and in turn it will reflect in a reduced soil quality (Schloter et al., 2003). Soil enzyme activities are the driving force behind all the biochemical transformations occurring in soil. Their evaluation may provide useful information on soil microbial activity and be helpful to establish effects of soil specific environmental conditions (Dick et al., 1996).

Numerous research efforts are being dedicated to the search of proper remediation technologies to remove as much as possible contaminants from the environment or to transform them into less toxic compounds. Bioremediation appears to be an appealing technology to approach the recovery of PAH-polluted sites (Harayama, 1997). Several microorganisms are capable to mineralise a large variety of PAHs and/or to break down them to their less-toxic metabolites (Cerniglia, 1992). The very low water-solubility of PAHs and the slow mass-transfer rates from solid phase may limit their availability to microorganisms, thus hindering natural attenuation microbial processes. However, some bacteria degrade sorbed PHAs at different rates, indicating organism-specific bioavailability (Grosser et al., 2000).

Bioremediation of PAH contaminated sites rely either on the presence of autochthonous degrading bacteria which capabilities might be stimulated in situ (Margesin and Schinner, 1997), or on the inoculation of selected microorganisms with desired catabolic traits in bioaugmentation techniques (Straube et al., 1999). When microorganisms are added to speed up degradation in contaminated environments, the duration assessment and biological process efficiency depend on the evolution of bacterial communities in terms of composition and catabolic activity. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes represents a powerful tool to study the bacterial community structures in complex environments as well as in enrichment cultures (Muyzer and Smalla, 1998). However, the combination of both culture-independent and culture-dependent techniques might provide useful and complementary information on the structure of microbial communities.

Soils with different pollution history were preliminary characterized in terms of their chemical properties, enzymatic activity and culturable heterotrophic bacteria. Site characterization is a pre-requisite when dealing with any remediation approach of a polluted site (Smith and Mason, 1999). Indeed, chemical and biochemical properties may assist in the analysis of the ability for the soil to be recovered (Margesin et al., 2000). Moreover, the enrichment and selection of bacterial phenanthrene-degrading cultures, capable of degrading solid phenanthrene in batch liquid systems were performed. The kinetics of phenanthrene disappearance by enriched cultures, the comparison of their degradation rates and their species composition were also investigated, as assessed by DGGE analysis of PCR-amplified 16S rDNA gene fragments. The enrichment of such cultures is a necessary step to obtain microorganisms with the desired catabolic traits, usable in the bioaugmentation of polluted soils.