Bioremediation of persistent aromatic pollutants
Author(s)
Stella, Tatiana
Date Issued
June 5, 2014
Type
Doctoral Thesis
Abstract
The remediation of persistent chlorinated aromatic compounds has become a priority of great
relevance due to the teratogenic, carcinogenic and endocrine-disrupting properties of these
xenobiotics. The use of biological methodologies for the clean-up of contaminated sites,
collectively referred to as “bioremediation”, has been gaining an increasing interest in recent years
because it represents an effective, cost-competitive and environmentally friendly alternative to the
physico-chemical and thermal treatments. In this respect, “white rot” fungi, an ecological subgroup
of filamentous fungi, display features that make them excellent candidates to design an effective
remediation technology (“mycoremediation”). In spite of this, fungi have not been widely exploited
for their metabolic capabilities and the mechanism by which they are able to degrade the
aforementioned pollutants has not been fully elucidated yet.
Within this frame, the present Ph.D thesis was aimed at:
i) assessing the efficiency of different mycoremediation strategies for the clean-up of a
polychlorinated biphenyl (PCBs)-contaminated soil;
ii) understanding the fungal degradation pathways of polychlorinated biphenyls and their major
metabolites, namely chlorobenzoic acids (CBAs) and hydroxylated polychlorinated biphenyls (OHPCBs).
i) The combination of chemical, toxicological and molecular biology techniques provided a
comprehensive evaluatation of the technical feasibility of selected remedial strategies. Physicochemical
properties (pH, soil texture, soil organic matter content, ect.) as well as the pollutant
bioavailability of three different PCB-contaminated soil samples from a dumpsite (bulk soil, topsoil
and rhizosphere soil) were assessed before undergoing both bioaugmentation (either with the white
rot fungus Pleurotus ostreatus or Irpex lacteus) and biostimulation (addition of a lignocellulosic
substrate) treatment. The inoculation of P. ostreatus in the rhizosphere soil was the most effective
treatment in terms of PCB degradation and detoxification. The involvement of both intracellular
and extracellular fungal enzymes in the biotransformation of PCBs was demonstrated by the
identification of several PCB degradation intermediates (i.e. chlorobenzoates, chlorobenzaldehydes,
chlorocresols, hydroxylated and methoxylated PCBs). Furthermore, new insights into the microbial
community structure, diversity and dynamics throughout the bioremediation processes were gained
with the combination of two culture-indipendent techniques: phospholipid fatty acids (PLFA) and
454-pyrosequencing analyses. PLFA analysis showed that either the introduction of allochthonous
fungi or the addition of non-inoculated lignocellulosic substrate stimulated the growth of the
resident bacterial populations, while the highest fungal concentration was achieved in P. ostreatustopsoil
microcosms in the incubation middle phase. Metagenomic analysis of bacterial community
revealed that Firmicutes relative abundance increased in Pleurotus ostreatus-bulk and -rhizosphere
soil microcosms; on the other hand, in I.lacteus-augmented microcosms, an initial increase of
Proteobacteria was observed whereas Bacteroidetes became dominant at the end of incubation.
Analysing the fungal community structure in bioaugmented soils, P.ostreatus showed a higher
ability than I. lacteus to compete with the autochthonous soil mycobiota. Indeed, P.ostreatus
sequences accounted to more than 90% of the total fungal amplicons along the whole incubation
period, thus proving the outstanding capability of this fungus to efficiently grow in PCBcontaminated
soils under non-sterile conditions. By contrast, the large majority of fungal sequences
in biostimulated microcosms belonged to the phyla Ascomycota and Zygomycota, with the
exception of the topsoil where members of the phylum Basidiomycota became predominant in the
later phase of the incubation
ii) Microsomal fractions rich in cytochrome P450 monooxygenase (CYP450) activities were
isolated from the white rot fungi Lentinus tigrinus and Pleurotus ostreatus to evaluate their
involvement in the biotransformation of CBAs and PCBs, respectively. In both cases, CYP450 was
firstly detected by carbon monoxide-binding spectrum, and then used to perform in vitro
degradation tests with selected compounds. Such intracellular enzymatic system was able to
degrade either a mixture of CBAs (L. tigrinus) or PCBs (P. ostreatus). Specifically, the
identification of a hydroxylated CBA confirmed the pivotal role of CYP450 in the initial
transformation of CBAs. Moreover, a semi-purified laccase obtained from P. ostreatus was capable
of degrading mono- and dichlorinated hydroxylated biphenyls, at different extent, either under
mediated or non-mediated conditions. The chemical structure of chlorinated organic pollutants,
namely the number and position of substituents, was the main factor affecting the extent of
degradation by both fungal intracellular and extracellular enzymes.
Additional information
Dottorato di ricerca in Scienze ambientali
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