Struttura e funzione delle comunità microbiche in suoli contaminati: effetto degli interventi di micorisanamento
Author(s)
Giubilei, Maria Angela
Date Issued
January 8, 2009
Type
Doctoral Thesis
Abstract
In the recent years, several polluting compounds have been released into the
environment because of industrial and agricultural activities and represent a threat to
humans and other living organisms. These contaminants are either natural compounds
that have been mobilized to a bioavailable form that is toxic to organisms, such as
polycyclic aromatic hydrocarbons (PAHs) present in fossil fuels and heavy metals
present in minerals, or compounds of industrial origin that present chemical structures
alien to the biosphere (xenobiotics), such as polychlorobiphenyls (PCBs). To address
the challenge of cleaning up contaminated sites, there has been an increasing interest in
biological methodologies, collectively indicated as bioremediation, that take advantage
of the astonishing catabolic versatility of microorganisms to degrade or transform
contaminants to less toxic or nontoxic products. A promising bioremediation approach
is represented by the use of white-rot fungi, due to their unspecific, radical-based
degradation machinery that mainly operates in the extracellular environment
(mycoremediation).
A better understanding of the structure and functions of the microbial communities
of a given contaminated site and how they are affected by biotechnological
interventions, such as bioaugmentation with fungi and/or addition of mobilizing agents,
may assist in the design of more appropriate remediation strategies. In these respects,
the increased development of molecular-based techniques, mainly based on the isolation
and analysis of the genome of the entire microbial community in an environmental
sample (the so called ‘metagenome’), has allowed the direct investigation of
microorganisms in their natural environment without laboratory culturing and thus
allowing to mine the whole microbial diversity, including the vast uncultivable
majority.
Considering that white-rot fungi have been successfully employed in several
bioremediation trials but that little is known about whether and how the indigenous
microflora is affected by the massive inoculation with the allochtonous fungi, the aim of
the present Ph.D. thesis project was to investigate the structural and functional
modifications of the microbial communities in contaminated soils following
mycoremediation. In particular, several laboratory-scale trials have been implemented
on historically contaminated soils and model soils (ad hoc contamination) applying
different white-rot fungi, alone or in combination with mobilizing agents.
VII
Combined use of white-rot fungi and mobilizing agents for the remediation of a model
soil artificially contaminated with PAHs:
Bioremediation efficacy by microorganisms is strongly affected by the low water
solubility and bioavailability of PAHs and so the use of additives capable of mobilizing
these contaminants from the soil organic phase to the aqueous one has been shown to
positively influence their degradation. The first part of this thesis work was thus aimed
to evaluate the effect of mobilizing agents (MA) on mycoremediation efficiency and to
better understand the impact of this remediation strategy on the composition and
diversity of the indigenous bacterial community. The study was conducted in a model
system where a pristine soil was spiked with a mixture of seven PAHs (50 mg/g each of
phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[k]fluoranthene and
benzo[a]pyrene), to simulate a recent contamination, and subsequently incubated with
white-rot fungi (Irpex lacteus 617/93, Pleurotus ostreatus 3004, Allescheriella sp.
DABAC 1 and Phlebia sp. DABAC 9) and/or mobilizing agents, namely two
polyoxyethylenepolysorbates (Tween-20 and Tween-80), a plant seed oil characterized
by high concentration of polyunsaturated fatty acid (soybean oil, SO), olive-oil mill
wastewaters (OMW) and randomly methylated -cyclodextrins (RAMEB).
In the first part of this study Phlebia sp. and Allescheriella sp. have been used with
or without the addition of Tween 20, Tween 80 and SO. The two fungal strains
markedly differ for their growth capabilities under non-sterile conditions and without
MAs (3.0 vs. 0.1 μg ergosterol g-1 soil, for Phlebia sp. and Allescheriella sp.,
respectively). However, SO led to a 35-fold increase of Allescheriella sp. growth.
Contaminant degradations by Phlebia sp. and Allescheriella sp. were best supported by
SO and Tween 20, respectively. Interestingly, the highest level of soil detoxification, as
evaluated by assessing dehydrogenase activity, was reached following incubation with
Allescheriella sp. without the addition of any MA. In accordance with these findings,
enumeration of cultivable bacteria and denaturing gradient gel electrophoresis (DGGE)
analysis of PCR-amplified 16S rRNA showed that microbial growth and biodiversity
were positively affected by remediation with white-rot fungi, and especially when
Allescheriella sp. was used.
In the second part of the study two white-rot fungi, namely Irpex lacteus and
Pleurotus ostreatus, were tested alone or in combination with five MAs (Tween-20,
Tween-80, SO, OMW, and RAMEB). Among the different MAs, SO best supported
fungal growth and positively affected PAH degradation and soil detoxification but,
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interestingly, showed a negative effect on both bacterial biomass and biodiversity when
either one of the two fungi was used. It is worth noticing that even if both fungi showed
a general decrease of the total weight of contaminants and an increase of soil
detoxification in combinations with all MAs, their impact on endogenous bacteria was
different. Indeed, when the soil was incubated with P. ostreatus bacterial biodiversity
decreased and the type of MA used did not significantly affect the composition of the
indigenous microflora. By contrast, when I. lacteus was used as inoculum, the diversity
of the bacterial community increased and its structure was influenced by the type of MA
used. Finally, cloning-sequencing of PCR-amplified 16S rRNA genes suggested the
presence of well known PAH-degrading bacterial species belonging to the Bacillus e
Burkholderia genera.
Bioremediation of a historically contaminated soil with Botryosphaeria rhodina and
Pleurotus pulmonarius:
Two white-rot fungi, namely Botryosphaeria rhodina DABAC P82 and Pleurotus
pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic
hydrocarbons in an aged contaminated soil from the ACNA site (Cengio, SV, Italy), a
former industrial area, decommissioned in 1994, where large-scale production of a wide
array of organic chemicals had taken place for more than 100 years. The soil was
characterized by the concomitant presence of both aromatic hydrocarbons, including
chlorinated benzenes and anilines, thiophenes and polyaromatic hydrocarbons, and
heavy metals (mainly Hg, Cu and As). Previous studies had demonstrated the
unfeasibility of biostimulating the indigenous microflora to bioremediate this soil and
thus mycoremediation with white-rot fungi was attempted. To evaluate the role of the
indigenous microflora on the overall process, incubations were performed on both
fumigated and non-fumigated soils.
Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in
the non-fumigated soil while the growth of P. pulmonarius showed an opposite
response. Interestingly, degradation performances and detoxification by both fungi were
markedly higher in the non-fumigated soil than those observed in the fumigated one
suggesting an apparent synergistic effect occurring between the bioaugmented fungi and
indigenous soil microflora. Heterotrophic bacterial counts in nonfumigated soil
inoculated with either B. rhodina or P. pulmonarius were significantly higher than those
of the corresponding incubation control. To evaluate the effects of fungal
IX
bioremediation on the bacterial community structure, numerical analysis of DGGE
profiles of PCR-amplified 16S rRNA genes was performed. The bacterial community in
the original non-amended ACNA soil was characterized by a low biodiversity. The
amendment with sterilized maize stalks led to an augmentation of species richness and
to marked changes in the community structure. Bioaugmentation with both fungi further
increased the community diversity and resulted either in the appearance or in the
enrichment of dominant hydrocarbon-degrading species as suggested by cloning and
sequencing of 16S rRNA genes.
Structural and functional modifications of the indigenous bacterial community during
bioremediation of a historically contaminated soil with Panus tigrinus:
Bioremediation of the aged contaminated soil ACNA was further investigated using
the white-rot fungus Panus tigrinus. In this part of the thesis project a laboratory-scale
trial was setup to monitor the structural and functional modifications of the indigenous
bacterial community during mycoremediation. Analyses were performed at the
beginning of the experiment (t0) and after 7, 15, 30 and 60 days of incubation (t7, t15, t30
and t60).
Amendment of contaminated soil with milled maize stalks proved to be valuable for
the fungus growth under non-sterile conditions: the matrix was rapidly colonized within
the first two weeks, thus indicating good tolerance towards both organic contaminants
and heavy metals and capability to compete with the autochthonous microflora.
Moreover, and in spite of the non-favorable pH (around 7.4) and the presence of heavy
metals, P. tigrinus produced interesting lignin-modifying enzyme activities such as
laccase and Mn-peroxidase. Although bioremediation experiments were prolonged for
only 60 days, GC-MS analysis of the soil treated with P. tigrinus indicated removal or
strong reduction of several contaminants such as naphthalene, 1,2,3,4-
tetrachlorobenzene, 2,4- and 2,6-dichloroaniline, 2,3,4,5,6-pentachloroaniline,
diphenylether and 1,1’-binaphtalene. Heterotrophic bacterial counts and DGGE analysis
showed that the size and the diversity of the endogenous bacterial populations increased
during fungal bioaugmentation. Cluster analysis of DGGE fingerprints indicated that
bacterial populations in the incubation control and in the fungal-augmented soil during
the first fifteen days of incubation remained quite similar. On the contrary, after 30 and
60 days of incubation, a higher degree of diversification between incubation controls
and fungal treatments was evident suggesting a specific effect of P. tigrinus inoculation
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on the endogenous bacterial community. Finally, to evaluate the impact of
mycoremediation on the bacterial community function, Real-Time PCR experiments
were performed targeting genes involved in the degradation of aromatic contaminants.
In particular, it was found that the copy number of both naphthalene dioxygenase and
catechol 2,3-dioxygenase genes tended to decrease over time in soils incubated with P.
tigrinus. This finding suggested the occurrence of a functional shift in soil microbial
populations, probably as a consequence of aromatic contaminant degradation, and thus
leading to a reduction in the relative abundance of specialized bacterial groups. Cloning
and sequencing of 16S rRNA genes further confirmed this hypothesis showing that
following remediation both specialized and opportunistic bacteria belonging to several
different genera were present.
Combined use of Panus tigrinus and soybean oil for the bioremediation of a soil
historically contaminated with polychlorinated biphenyls:
Based on the encouraging results obtained with the use of white-rot fungi for the
remediation of PAH-contaminated soils, the aim of the last part of the thesis work was
to evaluate a similar mycoremediation approach for the reclamation of a soil historically
contaminated with polychlorinated biphenyl (PCB). Indeed, PCBs are toxic xenobiotics
of great ecological concern present in large amounts throughout the environment, and in
particular in soils and sediments. A laboratory-scale mycoremediation trial with P.
tigrinus, alone or in combination with SO as mobilizing agent, was established and
analyses were performed at the beginning of the experiment (t0) and after 30 (t30) and 60
(t60) days of incubation. Colonization of P. tigrinus was clearly visible and also
confirmed by ergosterol quantification. Interestingly, ergosterol slightly increased in
non-inoculated incubation controls as well, suggesting the presence of a viable
indigenous fungal community. Extracellular enzymatic activities were detected in soil
inoculated with P. tigrinus. In particular, hydrolase activities peaked after 30 days of
incubation especially in the absence of SO. On the contrary, the use of the MA had
opposite effects on lignin-modifying enzyme activities: SO inhibited the production of
laccase but stimulated that of Mn-peroxidase. GC-MS analysis of PCB concentration in
the different soil samples showed that, as expected, there was a significant degradation
activity carried on by the endogenous microflora but that, interestingly, the addition of
the mobilizing agent negatively affected this activity. On the contrary, mycoremediation
with P. tigrinus did not lead to a significant PCB depletion. Nevertheless, heterotrophic
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bacterial counts and DGGE analysis of both 16S and 18S rRNA genes showed that the
size and the diversity of the bacterial and fungal communities increased following
incubation with the fungus and the mobilizing agents, suggesting a significant effect of
these biological interventions on the endogenous microflora. Real-Time PCR
quantification of catabolic genes, namely biphenyl dioxygenase and catechol 2,3-
dioxygenase, showed that the number of gene copies remained unchanged during the
incubation period, suggesting that treatment with P. tigrinus and/or SO did not affect
the function of the endogenous microbial community.
In conclusion, the results obtained during this thesis project confirmed the
importance of using white-rot fungi, alone or in combination with mobilizing agents, to
decontaminate and detoxify PAH-contaminated soils. On the contrary, more
investigations are needed to assess the validity of a similar strategy for the
bioremediation of soils polluted with PCBs. The detailed, culture-independent,
molecular-based analyses of soil microflora revealed that mycoremediation, both with
and without the use of mobilizing agents, had a profound effect on the structure and
function of the endogenous microbial communities. In particular, and with the only
exception of the use of Phlebia sp. in combination with SO, all fungi and all mobilizing
agents stimulated bacterial growth and enhanced bacterial biodiversity. Furthermore,
when the mycoremediation of the soil was successful, also the function of the bacterial
community seemed to undergo a shift toward a less specialized role. Taken together
these results point out the importance of gaining insights about the ecological effects of
biotechnological interventions, particularly those involving bioaugmentation with fungi,
on the endogenous microbial populations in order to design more integrated, successful
bioremediation strategies.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
Subjects
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