Bioaugmentation of Native Fungi, an Efficient Strategy for the Bioremediation of an Aged Industrially Polluted Soil With Heavy Hydrocarbons

Abstract

The concurrence of structurally complex petroleum-associated contaminants at relatively high concentrations, with diverse climatic conditions and textural soil characteristics, hinders conventional bioremediation processes. Recalcitrant compounds such as high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) and heavy alkanes commonly remain after standard soil bioremediation at concentrations above regulatory limits. The present study assessed the potential of native fungal bioaugmentation as a strategy to promote the bioremediation of an aged industrially polluted soil enriched with heavy hydrocarbon fractions. Microcosms assays were performed by means of biostimulation and bioaugmentation, by inoculating a defined consortium of six potentially hydrocarbonoclastic fungi belonging to the genera Penicillium, Ulocladium, Aspergillus, and Fusarium, which were isolated previously from the polluted soil. The biodegradation performance of fungal bioaugmentation was compared with soil biostimulation (water and nutrient addition) and with untreated soil as a control. Fungal bioaugmentation resulted in a higher biodegradation of total petroleum hydrocarbons (TPH) and of HMW-PAHs than with biostimulation. TPH (C₁₄-C₃₅) decreased by a 39.90 ± 1.99% in bioaugmented microcosms vs. a 24.17 ± 1.31% in biostimulated microcosms. As for the effect of fungal bioaugmentation on HMW-PAHs, the 5-ringed benzo(a)fluoranthene and benzo(a)pyrene were reduced by a 36% and 46%, respectively, while the 6-ringed benzoperylene decreased by a 28%, after 120 days of treatment. Biostimulated microcosm exhibited a significantly lower reduction of 5- and 6-ringed PAHs (8% and 5% respectively). Higher TPH and HMW-PAHs biodegradation levels in bioaugmented microcosms were also associated to a significant decrease in acute ecotoxicity (EC₅₀) by Vibrio fischeri bioluminiscence inhibition assays. Molecular profiling and counting of viable hydrocarbon-degrading bacteria from soil microcosms revealed that fungal bioaugmentation promoted the growth of autochthonous active hydrocarbon-degrading bacteria. The implementation of such an approach to enhance hydrocarbon biodegradation should be considered as a novel bioremediation strategy for the treatment of the most recalcitrant and highly genotoxic hydrocarbons in aged industrially polluted soils.

Keywords: aged-polluted soil; fungal-bacterial interactions; high molecular weight polycyclic aromatic hydrocarbons; indigenous hydrocarbonoclastic fungi; mycoremediation; native-fungal-bioaugmentation.

FIGURE 1

TPH concentration evolution in microcosm assays along 120 days of incubation.^aData are the means of three independent experiments. ^a,b,cSame lower-case letters indicate lack of statistically significant difference (P < 0.05) between each biostimulation or bioaugmentation treatment. *Asterisk represents the occurrence of significant differences between B and BS treatments with control C (P < 0.05). Values are expressed in terms of dry weight.

FIGURE 2

Alkane fractions in control soil and treatments after 120 days of incubation in microcosms experiments. Same lowercase letters indicate, between microcosms and vs. control soil, respectively, lack of statistically significant difference (P < 0.05).

FIGURE 3

Degradation of PAHs (%) after 120 incubation days in experimental conditions. (Mean values ± SD for three replicates). B, Bioaugmentation; BS, Biostimulation; PHE, (phenanthrene, three rings PAH); PYR, (pyrene, four rings PAH); BAA, [benzo(a) anthracene, four rings PAH]; CHR, (Chrysene, four rings PAH); BF, [benzo(b,k)fluoranthene, five rings PAH]; BAP, [benzo(a)pyrene, five rings PAH]; BP, [benzo(g,h,i)perylene, six rings PAH]. Different lower-case letters within treatments indicate the occurrence of significant differences between them (P < 0.05). Anthracene was at very low values, below the detection limit (i.e., 2 mg Kg^{– 1}), reason why it has not been included in the figure.

FIGURE 4

Microtox ecotoxicity solid phase assays of soil (EC50 values expressed as mg soil L^–1) in the different treatments over 120 days of incubation. Dotted line represents EC value of clean soil (56,682 mg L^–1) collected in the vicinity of polluted site. Brown bar: contaminated control soil; Orange Bar Bioaugmentation; Green Bar (Biostimulation). Different lower-case letters within treatments indicate the occurrence of significant differences between them (P < 0.05).

FIGURE 5

DGGE profiles of bacterial 16S rDNA from microcosmos assays at 0, 30, 60, and 120 days of incubation. A standard ladder (L) has been added at both gel ends in order to check the DNA migration homogeneity. Bacterial bands have been named from B1 to B11.

FIGURE 6

Detrended correspondence analysis (DCA) on the relative DGGE band intensity of bacterial ribotypes from microcosm samples taken at different time and with treatments, as detected from the DGGE profiles (Figure 5). Only those bands with a relative band intensity higher than 1% were considered.