A diversified approach to evaluate biostimulation and bioaugmentation strategies for heavy-oil-contaminated soil

Abstract

A diversified approach involving chemical, microbiological and ecotoxicity assessment of soil polluted by heavy mineral oil was adopted, in order to improve our understanding of the biodegradability of pollutants, microbial community dynamics and ecotoxicological effects of various bioremediation strategies.

With the aim of improving hydrocarbon degradation, the following bioremediation treatments were assayed: i) addition of inorganic nutrients; ii) addition of the rhamnolipid-based biosurfactant M_AT10; iii) inoculation of an aliphatic hydrocarbon-degrading microbial consortium (TD); and iv) inoculation of a known hydrocarbon-degrading white-rot fungus strain of Trametes versicolor.

After 200 days, all the bioremediation assays achieved between 30% and 50% total petroleum hydrocarbon (TPH) biodegradation, with the T. versicolor inoculation degrading it the most. Biostimulation and T. versicolor inoculation promoted the Brevundimonas genus concurrently with other α-proteobacteria, β-proteobacteria and Cytophaga-Flexibacter-Bacteroides (CFB) as well as Actinobacteria groups. However, T. versicolor inoculation, which produced the highest hydrocarbon degradation in soil, also promoted autochthonous Gram-positive bacterial groups, such as Firmicutes and Actinobacteria. An acute toxicity test using Eisenia fetida confirmed the improvement in the quality of the soil after all biostimulation and bioaugmentation strategies.

Introduction

The application of bioremediation technologies to soils contaminated by light oil products, such as petrol or diesel, is feasible. However, decontaminating soils polluted with mineral oils that comprise the heaviest hydrocarbon fractions is still a challenge because of the low bioavailability and complex chemical composition of these products (Lee et al., 2008, Sabaté et al., 2004). In addition, an excessive residual concentration of hydrocarbons and possible oxidative metabolites with unacceptable human health risks may remain in the soil after bioremediation (Nocentini et al., 2000).

The aliphatic fraction of an oil product is formed mainly of alkanes, branched alkanes and isoprenoids, and to a lesser extent by cycloalkanes. Alkanes are more easily biodegraded than branched alkanes and biodegradability decreases with an increase in the number of carbon atoms. This pattern of hydrocarbon biodegradation has been described for bacterial and fungus metabolism (Colombo et al., 1996). Heavy-oil products have a considerable fraction of the so-called unresolved complex mixture (UCM) on the basis of its chromatographic profile. In fact, little is known about the composition of the UCM despite it being the main component of fuel oils (Wang and Fingas, 2003) that harbor branched and cyclic aliphatic and aromatic hydrocarbons, characterized by high resistance to biodegradation (Nievas et al., 2008). Furthermore, increases in the UCM after oil biodegradation processes have been reported in several studies (Ross et al., 2010).

Given this biodegradability pattern, the residual hydrocarbons in a soil contaminated with a heavy-oil product after bioremediation are complex mixtures rich in high-molecular-weight (HMW) hydrocarbons with a substantial proportion of a UCM. Because of this and as it is particularly difficult to decrease the concentration of total petroleum hydrocarbons (TPH) below the limits established by legislation in soils contaminated with heavy-oil products, efforts should be made to minimize the presence of such compounds and to better understand their effect on soil ecotoxicity. To improve understanding and efficacy, both chemical biodegradation and the predominant microbial populations need to be assessed during bioremediation processes. Previous studies have focused on microbial communities responsible for degrading heavy fuel in marine environments (Alonso-Gutierrez et al., 2009) but little is known about oil-degrading communities in industrially polluted soils (MacNaughton et al., 1999, Mishra et al., 2001, Zucchi et al., 2003).

Ecotoxicological tests have successfully been used as a complementary tool to monitor bioremediation efficiency in soil, which is important to assess ecological risks at polluted sites (Wang et al., 2010). However, very few studies combine these toxicological tests with a detailed study of the microbial communities in historically oil-polluted soils (Liu et al., 2010, Sheppard et al., 2011). To ensure proper risk assessment of contaminated sites and the monitoring of bioremediation processes, toxicity assays, chemical analyses and molecular microbial ecology studies of the microbial populations in polluted areas should be combined (Plaza et al., 2010).

Here we evaluated the feasibility of several biostimulation and bioaugmentation agents in soil contaminated with a heavy mineral oil (C₁₅–C₃₅). To this end, we tested the following strategies: i) addition of the biosurfactant M_AT10, obtained by cultivating the strain Pseudomonas aeruginosa AT10 (Abalos et al., 2004); ii) addition of glucose; iii) inoculation of a microbial consortium (TD) that is specialized in the biodegradation of the aliphatic fraction of crude oil (Viñas et al., 2002); and iv) inoculation of a hydrocarbon-degrading strain of the ligninolytic fungus Trametes versicolor (Borràs et al., 2010). In addition, to better understand potential metabolic strategies and their final effects on soil toxicity, we studied toxicity and characterized the microbial community during biodegradation by means of multiple culture-independent techniques.