Aspetti di ecologia microbica della valorizzazione degli scarti dei frantoi oleari: dinamiche delle comunità microbiche durante il processo di compostaggio e applicazione in strategie di biorisanamento
Author(s)
Scargetta, Silvia
Date Issued
June 25, 2013
Type
Doctoral Thesis
Abstract
The management of agro-industrial waste is one of the problems related to economic
development that now more than ever must become a sustainable economic development.
For this reason in our country, second producer of olive oil, the composting of olive mill
waste (OMW) represents a form of valorisation of this waste.
Among the many uses of this
kind of compost, one that may be valuable and economically convenient is its application
in bioremediation strategies. Indeed, the application of organic amendments in
bioremediation can facilitate the degradation of organic pollutants present in soil. In
particular, compost may represent a source of nutrients, which can stimulate the
degradative activity of the indigenous microbiota, and a source of an exogenous microbial
population with great metabolic potential that can be integrated in the polluted soil. In
composting, as well as in bioremediation, microorganisms are the key to ensuring the
success or failure of the process. It is therefore clear that microbial ecology studies can
help to understand the mechanisms that underlie these processes, providing the tools to
intervene and optimize the strategy applied.
The main aim of the thesis project was to deepen our understanding of microbial ecology
aspects of the valorisation of OMW with a particular regard to the application in
bioremediation strategies.
The experimental approach used for microbial ecology studies in the different experiments
involved both culture-dependent and culture-independent methods. The culture-dependent
methods, which were based on the cultivation of fungi and bacteria in appropriate media,
were used for viable count and evaluation of enzymatic activities. Microbial isolates of
particular interest were also identified by sequencing their ribosomal genes (rRNA). These
techniques were integrated with cultivation-independent approach such as molecular
techniques, based on metagenomic DNA. In particular, these were used to study the
biodiversity of bacterial and fungal community using denaturing gradient gel
electrophoresis (DGGE) analyses of rRNA gene fragments (16S for bacteria and 18S
rRNA for fungi) that provided the fingerprinting of communities. Numerical analysis of
the DGGE fingerprints provided an immediate comparison of the community similarity
and allowed the computation of ecological indexes. The abundance of bacterial and fungal
populations was evaluated with qPCR analyses of rRNA genes.
The first part of the thesis work has focused on the study of the composting process of
humid husk from a two-phase olive mill (TPOMW) and has been carried out in
collaboration with the Institute for Agriculture and Forest Systems in the Mediterranean
(CNR, Perugia). The composting process was monitored for 140 days and in this period
samples at different stages were collected to evaluate the organic matter transformation
and to study the microbial populations involved in this process. The chemical parameters
showed the typical trend of composting, in which temperature showed an initial activation
phase, followed by a thermophilic phase, result of an intense microbial activity, and a final
mesophilic phase during which the temperature decreased and remained stable until the
end of the process. During the composting process high mineralization and humification of
carbon, loss of nitrogen and complete degradation of polyphenols led to the waste
biotransformation into a high-quality compost. Microbiological analyses, carried out for
the assessment of microbial biodiversity and abundance, showed that many bacterial and
fungal populations were dominant and were observed only in specific phases, indicating
the presence of specialized microorganisms with different roles during the
biotransformation of this waste. In particular, the thermophilic phase, in which the highest
degrading activity was recorded, was characterized by the highest biodiversity, while in
correspondence of the chemical stabilization of the process, the communities were
characterized by a lower biodiversity with the community structure that tended to stabilize.
It’s interesting to note that most of the tannin-degrading bacteria were isolated during the
thermophilic phase, suggesting a pivotal role for these populations in the of organic matter
transformation. The identification of fungal populations during the process showed their
strategic role in the process, particularly during the thermophilic phase where
thermotolerant and thermophilic species were found. Interestingly, many of these species
are already known for their ability to degrade complex substances such as lignin or
cellulose.
As a direct consequence of the study about microbial populations involved in the
composting of OMW, bacterial communities of different compost obtained from the same
type of product varying the technological parameters, were also analysed. The aim of this
study was to explore the possible relationship between microbial diversity, composting
technologies and chemical features of the compost produced. The DGGE analysis of the 15
composts showed a great variability of the fingerprinting of bacterial communities.
Interestingly, bacterial diversity seemed negatively correlated with the pH but positively
with the C/N ratio, mainly due to a negative correlation with total nitrogen. Co-inertia
analysis of the co inertia showed that the diversity of compost binds to the maturation
process. Indeed, compost with a low complexity of bacterial community indicated a
stabilization of the process, and a maturation of the composted matrix characterized by
values of pH, nitrogen and C/N ratio of a typical mature compost. This analysis confirms
and strengthens the results obtained during the monitoring of the composting process in
which microbial biodiversity was closely linked to the level of maturity of the matrix.
Taken together these indications suggest the use of a possible additional index for the
assessment of the maturity of the compost based on bacterial diversity.
The second part of the thesis work was aimed to evaluating the effects of compost addition
on the indigenous microbiota of contaminated environments, with particular attention to
the fate of the compost microbial communities. With this aim, the TPOMW compost
characterized during the first part of the thesis was used, together with other kind of
composts, to implement two lab-scale bioremediation trials involving a soil historically
contaminated with diesel oil and a drug-spiked sludge.
In the first bioremediation trial, the diesel oil contaminated soil was amended with the
TPOMW compost and two spent mushroom composts (SMC A and SMC P) from the
cultivation of Agaricus bisporus and Pleorotus ostreatus, and incubated for 120 days.
Samples were taken for analyses at regular intervals. The effects of the TPOMW compost
were compared to those of SMC. To study the ecological effects of this remediation
strategy on the indigenous microbial communities, all the composts were used both
sterilized and unsterilized, with the aim of simulating biostimulation (nutrients presents in
composts) and bioaugmentation (biomass of composts), respectively. The DGGE analysis
indicated that adding the compost to the contaminated soil dramatically changed the
bacterial and fungal communities that showed complex and stable fingerprints during all
the incubation period. On the contrary, when the soil was amended with the sterilized
composts the microbial fingerprints showed an evolution from a very simple community
(typical of contaminated soils) to a complex community, rich in biodiversity. qPCR
quantification of bacterial and fungal populations confirmed that the composts provided to
the soil a very rich biomass, while the sterilized composts were able to stimulate the
growth of microbial populations. Taken together, these data suggested that the amendment
with composts, even of very different kind (namely TPOMW compost and SMC), could be
regarded as a bioaugmentation strategy where very complex exogenous communities were
able to win the competition with the endogenous soil microbiota and completely colonize
the contaminated soil. On the contrary, the biostimulation approach, simulated by the
addition of sterilized composts, seemed to stimulate the growth of the endogenous
microbiota that, theoretically, might be the more suitable to achieve the degradation of the
contaminants. Even if chemical data regarding contaminant removal are, at the moment,
not available, it is possible to draw the hypothesis that the addition of compost to
contaminated soil may not be a suitable strategy for bioremediation as the complex
biomass present in this kind of amendment tend to inhibit the potentially active microbial
populations.
The second lab-scale bioremediation trail was based on a sewage sludge contaminated with
several drug residues. To better monitor the capacity of the amendments to stimulate
remediation, the sludge was spiked with known concentrations of carbamazepine, a highly
persistent antiepileptic drug commonly found in this kind of matrix. To investigate the
microbiological effects on indigenous microbiota during the bioremediation of the spiked
sludge, two different kinds of composts derived of OMW were used: TPOMW compost
obtained from normal composting process (and already used in the first bioremediation
trial) and a OMW compost, named “big bag compost” produced with an innovative
procedure involving a stabilization of the waste and a static composting process. As in the
first trial, the composts were used non-sterilized and sterilized to understand if the putative
bioremediation effects were due to either the nutrients or the microbial communities added
with the amendments. During the 30 days of trial the microbial communities dynamics
were monitored by the DGGE and qPCR, and together with carbamazepine concentrations.
The use of compost in a contaminated matrix, did not seem to produce satisfactory results,
because the community of compost tended to colonize the substrate, inhibiting the
endogenous microflora that can, potentially, degrade the contaminant. On the contrary, the
supply of nutrients, which are supplied with sterile compost, it seemed to adequately
stimulate indigenous populations by promoting the expression of their degradative
potential, resulting in the removal, even partial, of the contaminant in a relatively shorter
compared to a natural attenuation. As regards fungal communities, while in the control and
in the treatments in which sterilized composts were used was not detected the presence of
any fungal population, if not those attributable to a subsequent colonization of environment
microorganisms, in the sludge treated with the non-sterilized compost, fungal community
suffered a sharp decline of biodiversity probably due to the high concentrations of the drug.
Given these results, and compared with those of bacterial communities, we can assume that
the fungal communities did not have a strong importance on the activity of degradation of
the drug.
This Ph.D. thesis work provided a good demonstration of how microbial ecology studies
can support the optimization of agro-industrial waste valorisation and the definitions of
optimal bioremediation strategies. In particular, it was demonstrated how during the
composting process of OMW, a rapid succession of several specialized microbial
populations drives the organic matter biotransformation that ensures the physico-chemical
changes related to the production of a good quality amendment. It was also shown that
great care should be taken when planning the application of this kind of composts in the
bioremediation of contaminated environments. The rich and complex microbiota that
characterizes these amendments may, in fact, inhibit the degradative potential of
endogenous populations, while biostimulation strategies based on the addition of nutrients
may represent a successful remediation approach.
Additional information
Dottorato di ricerca in Scienze ambientali
Subjects
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