Hydrocarbon (HC) pollution is a worldwide threat to marine natural ecosystems due to the increasing exploitation of underground marine petroleum deposits in several areas and to the high traffic of oil tankers and the presence of submarine pipes that are main transport routes for crude oil and refined products. HCs spread in the marine environment is mainly due to accidental oil spills or inadequate practices and their release affects marine ecosystems causing severe ecological and economical damages. The Mediterranean Sea is particularly endangered by hydrocarbon pollution because of its physical nature – it is an enclosed basin with a slow water exchange – and because it hosts about 20% of the global oil tanker traffic in its waters and tens of oil-related sites along its coastline. The conventional remediation strategies, comprising chemical and physical methods, are extremely expensive and invasive, therefore the development of cheaper and eco-friendly approaches is crucial to preserve human and ecosystem health. In this perspective, bioremediation (i.e. the use of living organisms to remove pollutants from a contaminated area) is a promising technology which, taking advantage of microbes’ metabolic potential to degrade a wide range of pollutants, can both reduce the costs and may represent a permanent solution. Nevertheless, there is still a scarce knowledge of the processes and the microorganisms involved in the clean-up of hydrocarbons from marine environments, hence some problems still exist concerning the in-field application of bioremediation. The aim of the present PhD thesis was to: i) investigate the overall prokaryotic diversity of pristine and oil polluted sites across the whole Mediterranean Sea; ii) depict the phylogenetic and functional diversity of hydrocarbonoclastic bacteria inhabiting pristine and polluted sites; iii) establish a large collection of bacteria showing degrading activities toward hydrocarbon compounds; iv) set up microcosm experiments to investigate the potential of bacterial bioaugmentation in bioremediation processes under laboratory scale conditions, v) test the degrading potential of selected bacterial strains and consortia under different pressure values, simulating different depths along the water column. The diversity of planktonic bacterial communities in the Mediterranean Sea was firstly evaluated on open seawater samples collected at different depths in a transect covering the main oil tanker route across the whole basin, from the Levantine Sea to the Gibraltar strait. Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the microbiome inhabiting deep and surface water samples were sharply separated. Furthermore, the composition of the bacterial communities described in the surface layers of the water columns at different sampling stations has been significantly correlated, beside to their geographical position and depth, to the temperature and salinity values recorded for each sample. Denaturing Gradient Gel Electrophoresis (DGGE) and ARISA fingerprinting were also applied to depict the bacterial composition of highly polluted sediments collected at the Ancona harbor (Italy) and El-Max district (Egypt), showing the significant influence of the different pollutants’ concentration (i.e. hydrocarbons, heavy metals) in the selection of peculiar bacterial assemblages . This molecular approach led to the identification of bacterial species potentially useful for site-tailored bioremediation purposes. A large collection of hydrocarbon degrading bacterial strains was hence established from enrichments using contaminated sediments as inoculum and diesel, crude oil and naphthalene as unique carbon sources. The cultivation approaches adopted to enrich and isolate hydrocarbonoclastic bacteria from chronically polluted area, like the Ancona harbor, permitted to evaluate the influence of different hydrocarbon pollutants used as single carbon source in the selection of specific marine bacteria populations. The results obtained taking advantage of DGGE fingerprinting and 16S rRNA pyrosequencing applied on the enrichments showed that, under laboratory conditions, the supply of different hydrocarbon compounds led to the selection of different, and specialized, bacterial communities. A total of 248 bacterial strains have been isolated from open sea surface water collected along oil tanker routes and the chronically polluted sediments, and have been identified by 16S rRNA gene sequencing. Alcanivorax and Marinobacter, two ubiquitous marine hydrocarbonoclastic genera, were the most abundant within the established collection, representing respectively 67% and 23% of the isolates. Due to the great importance of the Alcanivorax genus for hydrocarbon remediation of marine polluted sites, all the isolates belonging to this genus were investigated at a finer level in terms of phylogenetic and functional diversity. This sub-collection, comprising 179 isolates belonging to the 4 species A. borkumensis, A. jadensis, A. venustensis and A. dieselolei, were genotyped using two different fingerprinting techniques: Internal Transcribed Spacer (ITS)-PCR and BOX-PCR. The combination of the applied techniques allowed the identification of 85 genotypes, distributed among the different sites investigated, showing clear evidence of geographic divergence. The functional diversity of these strains was furthermore investigated through the PCR amplification of the alkB gene, encoding for an alkane monooxigenase involved in the first step of hydrocarbons degradation, and subsequent Restriction Fragment Length Polymorphism (RFLP) analysis of the amplicons, allowing the identification of 16 different polymorphisms. The results demostrated the existence of a high degree of geographical divergence within the Alcanivorax genus, suggesting a potentially high metabolic diversity that could be exploited for site-tailored bioremediation interventions. Recently, the Deepwater Horizon break in the Gulf of Mexico (2010) and the subsequent huge oil spill occurred at a depth of 1500 meters, highlighted the need to get more insight on bioremediation processes occurring at high depth. This accident represents a milestone and shed a light on the importance to investigate the effect of pressure, an environmental parameter that might hamper the activity of oil-degrading strains, on growth and degradation capabilities. The capability of selected hydrocarbonoclastic strains, belonging to the species A. jadensis, A. dieselolei and M. hydrocarbonoclasticus, to adapt and degrade a model alkane molecule (dodecane) at high pressure was therefore tested. The growth of the strains at increasing hydrostatic pressure and their physiologic activities were evaluated, comparing the results with the type strain A. borkumensis SK2. Overall, the results showed a detrimental effect of pressure for all the strains in terms of growth rates, O2 consumption and CO2 production. The potential adaptation of A. borkumensis and A.dieselolei was evaluated also with less recalcitrant carbon source than alkanes (pyruvate), without showing substantial differences, except for the higher consumption of pyruvate by A. borkumensis SK2. This investigation pinpointed that the tested bacteria can survive at high hydrostatic pressures, even though both their growth and degradation capability were mostly inhibited with the increase in hydrostatic pressure. Moreover, aiming to create a baseline for future transcriptomic analyses, the complete genome of this 4 strains was sequenced and annotated: all the strains owned multiple copies of the genes involved in the degradation of hydrocarbons (alkane monoossigenase, alk and cytochrome p450, cyp450), apparently belonging to different families, highlighting the great functional potential of these strains. A second sub-collection of hydrocarbonoclastic bacteria, isolated from chronically polluted sediments, was screened for the presence of functional genes involved in the degradation/detoxification of specific pollutants (alkanes and heavy metals), the ability to grow on different HCs and the ability to produce biosurfactant and from biofilm. The results showed that several isolates, mainly belonging to the Marinobacter genus, were positive for the investigated traits, hence they could be potentially exploited for autochthonous bioaugmentation (ABA) purposes in the sites of provenience. Finally, the bacterial community response in a biodegradation process based on an ex-situ landfarming set-up was evaluated. Landfarming was performed, using a combination of biostimulation and bioaugmentation, to remediate oil-polluted sediment collected at Elefsina bay (Greece). This work was realized to determine the effect of bioaugmentation by four allochthonous oil-degrading bacterial consortia, previously isolated from 4 polluted areas located in the Southern Mediterranean, in relation to the degradation efficiency of the indigenous community. DGGE fingerprinting analysis allowed the characterization of the bacterial community dynamics, evaluating the dominant taxa through time and at each treatment. The results showed that the added allochthonous bacteria quickly perished and were rarely detected, furthermore their addition induced minimal shifts in the community structure. These data, together with the measurement of HC degradation over the experimental time, suggested that, during the landfarming, biodegradation was mostly performed by the autochthonous populations rather than by the allochthonous ones. Furthermore, biostimulation, in contrast to bioaugmentation, was proved to enhance the HCs degradation when compared to the control treatment. To conclude, the results obtained this Ph.D. project emphasized the high bacterial diversity of the Mediterranean Sea in both pristine and polluted sites and the occurrence of distribution patterns which were significantly related to several environmental parameters, including the concentration of hydrocarbons and heavy metals. Moreover, this study confirmed the great potential of the Mediterranean Sea as a source of bacterial strains harbouring degradation capabilities toward different hydrocarbon molecules and, through the ex-situ application of different bioremediation strategies (bioaugmentation and biostimulation), it demonstrated the great importance of autochthonous microbial community in remediating polluted environments.

POLLUTED MARINE ECOSYSTEMS: RESERVOIR OF MICROBIAL RESOURCES FOR HYDROCARBON BIOREMEDIATION / M. Barbato ; coordinatore: D. Daffonchio ; supervisor: D. Daffonchio. - : . DIPARTIMENTO DI SCIENZE PER GLI ALIMENTI, LA NUTRIZIONE E L'AMBIENTE, 2014 Dec 18. ((27. ciclo, Anno Accademico 2014. [10.13130/barbato-marta_phd2014-12-18].

POLLUTED MARINE ECOSYSTEMS: RESERVOIR OF MICROBIAL RESOURCES FOR HYDROCARBON BIOREMEDIATION

M. Barbato
2014

Abstract

Hydrocarbon (HC) pollution is a worldwide threat to marine natural ecosystems due to the increasing exploitation of underground marine petroleum deposits in several areas and to the high traffic of oil tankers and the presence of submarine pipes that are main transport routes for crude oil and refined products. HCs spread in the marine environment is mainly due to accidental oil spills or inadequate practices and their release affects marine ecosystems causing severe ecological and economical damages. The Mediterranean Sea is particularly endangered by hydrocarbon pollution because of its physical nature – it is an enclosed basin with a slow water exchange – and because it hosts about 20% of the global oil tanker traffic in its waters and tens of oil-related sites along its coastline. The conventional remediation strategies, comprising chemical and physical methods, are extremely expensive and invasive, therefore the development of cheaper and eco-friendly approaches is crucial to preserve human and ecosystem health. In this perspective, bioremediation (i.e. the use of living organisms to remove pollutants from a contaminated area) is a promising technology which, taking advantage of microbes’ metabolic potential to degrade a wide range of pollutants, can both reduce the costs and may represent a permanent solution. Nevertheless, there is still a scarce knowledge of the processes and the microorganisms involved in the clean-up of hydrocarbons from marine environments, hence some problems still exist concerning the in-field application of bioremediation. The aim of the present PhD thesis was to: i) investigate the overall prokaryotic diversity of pristine and oil polluted sites across the whole Mediterranean Sea; ii) depict the phylogenetic and functional diversity of hydrocarbonoclastic bacteria inhabiting pristine and polluted sites; iii) establish a large collection of bacteria showing degrading activities toward hydrocarbon compounds; iv) set up microcosm experiments to investigate the potential of bacterial bioaugmentation in bioremediation processes under laboratory scale conditions, v) test the degrading potential of selected bacterial strains and consortia under different pressure values, simulating different depths along the water column. The diversity of planktonic bacterial communities in the Mediterranean Sea was firstly evaluated on open seawater samples collected at different depths in a transect covering the main oil tanker route across the whole basin, from the Levantine Sea to the Gibraltar strait. Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the microbiome inhabiting deep and surface water samples were sharply separated. Furthermore, the composition of the bacterial communities described in the surface layers of the water columns at different sampling stations has been significantly correlated, beside to their geographical position and depth, to the temperature and salinity values recorded for each sample. Denaturing Gradient Gel Electrophoresis (DGGE) and ARISA fingerprinting were also applied to depict the bacterial composition of highly polluted sediments collected at the Ancona harbor (Italy) and El-Max district (Egypt), showing the significant influence of the different pollutants’ concentration (i.e. hydrocarbons, heavy metals) in the selection of peculiar bacterial assemblages . This molecular approach led to the identification of bacterial species potentially useful for site-tailored bioremediation purposes. A large collection of hydrocarbon degrading bacterial strains was hence established from enrichments using contaminated sediments as inoculum and diesel, crude oil and naphthalene as unique carbon sources. The cultivation approaches adopted to enrich and isolate hydrocarbonoclastic bacteria from chronically polluted area, like the Ancona harbor, permitted to evaluate the influence of different hydrocarbon pollutants used as single carbon source in the selection of specific marine bacteria populations. The results obtained taking advantage of DGGE fingerprinting and 16S rRNA pyrosequencing applied on the enrichments showed that, under laboratory conditions, the supply of different hydrocarbon compounds led to the selection of different, and specialized, bacterial communities. A total of 248 bacterial strains have been isolated from open sea surface water collected along oil tanker routes and the chronically polluted sediments, and have been identified by 16S rRNA gene sequencing. Alcanivorax and Marinobacter, two ubiquitous marine hydrocarbonoclastic genera, were the most abundant within the established collection, representing respectively 67% and 23% of the isolates. Due to the great importance of the Alcanivorax genus for hydrocarbon remediation of marine polluted sites, all the isolates belonging to this genus were investigated at a finer level in terms of phylogenetic and functional diversity. This sub-collection, comprising 179 isolates belonging to the 4 species A. borkumensis, A. jadensis, A. venustensis and A. dieselolei, were genotyped using two different fingerprinting techniques: Internal Transcribed Spacer (ITS)-PCR and BOX-PCR. The combination of the applied techniques allowed the identification of 85 genotypes, distributed among the different sites investigated, showing clear evidence of geographic divergence. The functional diversity of these strains was furthermore investigated through the PCR amplification of the alkB gene, encoding for an alkane monooxigenase involved in the first step of hydrocarbons degradation, and subsequent Restriction Fragment Length Polymorphism (RFLP) analysis of the amplicons, allowing the identification of 16 different polymorphisms. The results demostrated the existence of a high degree of geographical divergence within the Alcanivorax genus, suggesting a potentially high metabolic diversity that could be exploited for site-tailored bioremediation interventions. Recently, the Deepwater Horizon break in the Gulf of Mexico (2010) and the subsequent huge oil spill occurred at a depth of 1500 meters, highlighted the need to get more insight on bioremediation processes occurring at high depth. This accident represents a milestone and shed a light on the importance to investigate the effect of pressure, an environmental parameter that might hamper the activity of oil-degrading strains, on growth and degradation capabilities. The capability of selected hydrocarbonoclastic strains, belonging to the species A. jadensis, A. dieselolei and M. hydrocarbonoclasticus, to adapt and degrade a model alkane molecule (dodecane) at high pressure was therefore tested. The growth of the strains at increasing hydrostatic pressure and their physiologic activities were evaluated, comparing the results with the type strain A. borkumensis SK2. Overall, the results showed a detrimental effect of pressure for all the strains in terms of growth rates, O2 consumption and CO2 production. The potential adaptation of A. borkumensis and A.dieselolei was evaluated also with less recalcitrant carbon source than alkanes (pyruvate), without showing substantial differences, except for the higher consumption of pyruvate by A. borkumensis SK2. This investigation pinpointed that the tested bacteria can survive at high hydrostatic pressures, even though both their growth and degradation capability were mostly inhibited with the increase in hydrostatic pressure. Moreover, aiming to create a baseline for future transcriptomic analyses, the complete genome of this 4 strains was sequenced and annotated: all the strains owned multiple copies of the genes involved in the degradation of hydrocarbons (alkane monoossigenase, alk and cytochrome p450, cyp450), apparently belonging to different families, highlighting the great functional potential of these strains. A second sub-collection of hydrocarbonoclastic bacteria, isolated from chronically polluted sediments, was screened for the presence of functional genes involved in the degradation/detoxification of specific pollutants (alkanes and heavy metals), the ability to grow on different HCs and the ability to produce biosurfactant and from biofilm. The results showed that several isolates, mainly belonging to the Marinobacter genus, were positive for the investigated traits, hence they could be potentially exploited for autochthonous bioaugmentation (ABA) purposes in the sites of provenience. Finally, the bacterial community response in a biodegradation process based on an ex-situ landfarming set-up was evaluated. Landfarming was performed, using a combination of biostimulation and bioaugmentation, to remediate oil-polluted sediment collected at Elefsina bay (Greece). This work was realized to determine the effect of bioaugmentation by four allochthonous oil-degrading bacterial consortia, previously isolated from 4 polluted areas located in the Southern Mediterranean, in relation to the degradation efficiency of the indigenous community. DGGE fingerprinting analysis allowed the characterization of the bacterial community dynamics, evaluating the dominant taxa through time and at each treatment. The results showed that the added allochthonous bacteria quickly perished and were rarely detected, furthermore their addition induced minimal shifts in the community structure. These data, together with the measurement of HC degradation over the experimental time, suggested that, during the landfarming, biodegradation was mostly performed by the autochthonous populations rather than by the allochthonous ones. Furthermore, biostimulation, in contrast to bioaugmentation, was proved to enhance the HCs degradation when compared to the control treatment. To conclude, the results obtained this Ph.D. project emphasized the high bacterial diversity of the Mediterranean Sea in both pristine and polluted sites and the occurrence of distribution patterns which were significantly related to several environmental parameters, including the concentration of hydrocarbons and heavy metals. Moreover, this study confirmed the great potential of the Mediterranean Sea as a source of bacterial strains harbouring degradation capabilities toward different hydrocarbon molecules and, through the ex-situ application of different bioremediation strategies (bioaugmentation and biostimulation), it demonstrated the great importance of autochthonous microbial community in remediating polluted environments.
DAFFONCHIO, DANIELE GIUSEPPE
DAFFONCHIO, DANIELE GIUSEPPE
bioremediation; Mediterranean Sea; bacterial communities; hydrocarbons; marine pollution
Settore AGR/16 - Microbiologia Agraria
POLLUTED MARINE ECOSYSTEMS: RESERVOIR OF MICROBIAL RESOURCES FOR HYDROCARBON BIOREMEDIATION / M. Barbato ; coordinatore: D. Daffonchio ; supervisor: D. Daffonchio. - : . DIPARTIMENTO DI SCIENZE PER GLI ALIMENTI, LA NUTRIZIONE E L'AMBIENTE, 2014 Dec 18. ((27. ciclo, Anno Accademico 2014. [10.13130/barbato-marta_phd2014-12-18].
Doctoral Thesis
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