Final Report Summary - ULIXES (Unravelling and exploiting Mediterranean Sea microbial diversity and ecology for Xenobiotics’ and pollutants’ clean up)
Executive Summary:
The project ULIXES aimed to unravel, categorize, catalogue, exploit and manage the microbial diversity available in the Mediterranean Sea for addressing bioremediation of polluted marine sites. The idea behind ULIXES is that the multitude of diverse environmental niches of the Mediterranean Sea contains a vast array of microorganisms and their components (e.g. catabolic enzymes) or products (e.g. biosurfactant) that can be exploited in pollutant- and site-tailored bioremediation approaches. Within the three-years EU project, the ULIXES consortium conducted different lines of research, aiming to study the microbial communities dwelling polluted and pristine sites across the Mediterranean Sea and the Gulf of Aqaba (Red Sea), taking advantage of both cultivation dependent and independent approaches. All the partners involved in Work Package (WP) 2 performed sampling campaigns, both on coastal and open sea sites, in order to collect sediment and seawater samples for the research purposes detailed in the project DoW. Starting from these samples, a large collection of microbes, including bacteria and fungi, was established. Such collection can be divided in four collections, depending on the type of activities and pollutants (hydrocarbons, halogenated compounds, heavy metals) targeted by the enriched mixed cultures and isolated bacteria. The collections have been screened for different features, allowing the identification of promising microbial strains in term, for example, of biofilm formation, biosurfactant production, biopalladium precipitation and metal mobilisation. A subset of microbes in the ULIXES collections were used within the WP7 to setup innovative processes and products for bioremediation purposes, such as the reductive dechlorination of polychlorinated compounds (PCBs) by using biopalladium particles produced and activated by marine bacteria. Moreover, a selection of the microbial consortia and the bacterial isolates obtained in the frame of WP4 activities were subsequently tested by partners involved in WP8 for their potential application in ex situ mesocosms and/or in situ treatments of polluted sediments.
Additional lines of investigation, tightly related to those previously described, were the study of overall prokaryotic community inhabiting polluted and pristine sites located across the Mediterranean Sea and the Gulf of Aqaba. These studies involved both the use of molecular ecology techniques and the OMICs approach, and provided data on the prokaryotic communities’ structure, diversity and abundance, which concurred to establish the widest OMIC-based datasets obtained from the Mediterranean Sea, with special focus on the Southern Mediterranean polluted sites, and a map of the basin biodiversity. The overall data showed peculiar microbial communities in the Southern Mediterranean Sea sites adapted to the particular environmental conditions of the Southern latitudes of the basin. The obtained data represent a novel contribute to the knowledge of microbial diversity in polluted sites. Indeed, the Southern Mediterranean coast, has been widely overlooked by the OMICs-based research on oil-degrading microbial networks, though it represent one of the major oil-producing areas on our planet. The data contribute to fill the gap of knowledge on the microbial diversity in oil polluted sites and features potentially exploitable novel resources for bioremediation purposes, one of the main objectives of the ULIXES project. Besides the RTD activities the ULIXES consortium established an intense dissemination plan, which was realized accordingly. A website was established within the first six months of the project, and several databases, newsletters, leaflet and brochure were prepared and disseminated both through digital and printed copies. The results of the performed research resulted in the publication, at the time of preparation of the present report, of 21 scientific papers in different international peer-reviewed journals, including high-impact factors and open-access journals. Several other manuscripts are actually in preparation and will be published in the next months under the auspices of the ULIXES project.
Project Context and Objectives:
The Mediterranean basin is one of the areas in the world where, since millennia, a large human population lives and intense economic activities occur. Natural resources of the basin have been long exploited resulting in profound impacts on the environment. The recent extensive development of aquaculture, the exploitation of resources from the subsurface including mineral, oil and gas extraction, the intense maritime traffic and the presence of many industrial poles on both the north and south sides of the basin, have resulted in a rapid increase of the number of polluted coastal and marine areas that should require extensive and careful strategies for remediation and clean up.
The Mediterranean Sea has a high exposure risk to many chemicals including oil hydrocarbons, halogenated compounds and heavy metals, with a pollution threat enhanced by the semi-enclosed nature of the basin. The size of the potential pollution load in the Mediterranean Sea can be appreciated by considering the figures related to the annual traffic and load of crude oil and petroleum products. The European coastal zone contains approximately 7% of the world coastline yet houses around 15% of the world population and generates nearly 16% of the total world trade. More than a half of all European coastal zones are located in semi-enclosed seas, so that pollutants are trapped (e.g. typical flushing time in Mediterranean is of the range of 70-90 years) and tend to accumulate. There are already more than 40 oil-related sites (e.g. pipeline terminals, refineries, offshore platforms) distributed along the Mediterranean coastal zone, from and to which estimates of 55,000,000 and 15,000,000 metric tons, respectively of crude oil and petroleum products, are annually loaded, unloaded and transported by oil tankers. Despite the relative little size of the basin, accounting for less than 1% of the earth’s marine surface, it has been estimated that more than 20% of the global maritime traffic yearly passes through it.
Many sites already experienced pollution events and several environmental disasters occurred in the Mediterranean Sea, comprising oil spills from pipelines and oil tankers shipwrecks. In 2000, pollution caused only by the illegal discharge of oil-contaminated bilge and ballast waters was estimated to reach 600,000 tones for the Mediterranean Sea alone, i.e. the equivalent of the pollution caused by the 1999 Erika shipwreck every week. The sinking of the Prestige off the coast of Spain in November 2002 and of the Erika off the coast of France in 1999, which were transporting 77,000 and over 10,000 tones of heavy fuels respectively, caused serious localized damage to the marine environment and to entire coastal regions and marine-related industries. The pollution of a relatively restricted basin like the Mediterranean Sea could lead to big socio-economic problems that could strongly affect the life style of the inhabitants. Besides the effects on biodiversity and wild life, marine oil spill disasters profoundly impact the inhabitants of the affected areas due to reductions of tourism and fishing activities and the consequent loss of jobs and heritage.
In a so densely inhabited area like the Mediterranean basin, the management of remediation efforts should be designed taking in consideration all the economic and political resources available in the basin, in a way that both the sides, the European one and the African and Middle-East south side, may benefit of a more careful and safe global management. The Mediterranean basin should need a ‘global’ remediation intervention that would involve simultaneously all the countries facing and surrounding the basin. Such a global approach would guarantee the best management of industrial development, minimizing the impact. A careful analysis of the available literature data showed that polluted sites on the Southern side of the Mediterranean Sea have been until now overlooked by studies based on state of the art techniques and methods. Besides access to North side sites, the ULIXES project had the necessary facilities and access to several major polluted sites on the Southern Mediterranean coasts of Morocco, Tunisia, and Egypt and to some polluted sites in the Aqaba Gulf (Red Sea, Jordan), a reference site to envision the perspective of the ongoing global warming on the Mediterranean Sea. The study of the microbial resource with potential for bioremediation from the selected North and South sites is of great interest to understand the overall bioremediation potential in the basin.
Bioremediation represents an environmental friendly approach for the clean-up of polluted environments including marine sediments and water. Applications of bioremediation technologies in marine environments have been proposed in a large range of cases like, for example, oil polluted marine water and beaches due to accidental oil spills from ships or pipelines, or for the removal of halogenated compounds from polluted sediments. The proposed clean up approaches include both in situ or ex situ technologies and can take large advantages from biostimulation, defined as “the act of adding materials to contaminated environments to cause an acceleration of the natural biodegradation processes”. Bioremediation practices can be differentiated in “biostimulation” and “bioaugmentation”, the latter being the addition of selected microbial species capable of degrading the pollutants which often provide interesting results when based on the addition of microbes obtained and enriched from the same site to be remediated. The low level of nutrients typical of the oligotrophic waters of the Mediterranean Sea and the low availability of hydrocarbon pollutants, may strongly limit the successful performance of biodegradation. Several studies showed that during the biostimulation of contaminated sediments the removal of the pollutants can be boosted by adding different electron donors/acceptors to stimulate the autochthonous microbial communities. Besides nutrients, biodegradation processes by naturally occurring microorganisms are enhanced by the in situ production or the addition of biosurfactants like rhamnolipids and other microbial surfactants and/or lipophilic fertilizers of natural origin (uric acid and lecithin). For instance in the case of polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons, the addition of the methylated cyclodextrins, microbial and phytogenic surfactants, resulted in marked improvement of pollutant bioavailability and of the final biodegradation in actual site solid matrices even in cases of aged contaminations. Biostimulation with nutrients and biosurfactants enables naturally occurring microbes to adapt better and faster to the polluted environment resulting in shorter lag phase and faster biodegradation of crude oil and hydrophobic (chlorinated) organo-aromatic pollutants, thus making it an effective ‘tool’ for combating oil spills. When biostimulation is not efficient in the removal of the pollutants, bioaugmentation procedures can be adopted by introducing in the polluted sediments or water specific consortia of biodegrading microbes, previously enriched under laboratory conditions. Effective sustainable strategies to clean up marine polluted ecosystems hence require the definition of the complex relationships among biodegradation rates, the different categories of contaminant molecules, and the microbial community composition and dynamics.
In the above context, the ULIXES project aimed at the exploration and exploitation for bioremediation purposes of the microbial natural resources (biodegrading/detoxifying microbes and their enzymes and metabolites) present in a series of selected polluted sites from the North and the South sides of the Mediterranean basin. Eight EU and four Southern Mediterranean laboratories were cooperating with a scientific approach starting with the isolation of pollutant degrading/detoxifying microbes and microbial consortia and their metabolites and the use of cultivation independent methods to investigate the phylogenetic and functional diversity of the overall microbiome inhabiting polluted seawater and sediments. Special attention has been posed to pollutions determined by oil hydrocarbons, PCBs and heavy metals, three pollutant categories included in the priority list of high impact pollutants in the EU and most of the countries in the world. For a comprehensive characterization of the polluted sites “meta-omics” techniques were also applied to enhance knowledge about the metabolic pathways involved in the remediation processes. Following such efforts in identifying suitable microbial resources, ULIXES aimed at developing and implementing novel processes at reactor scale as well as in pilot ex situ and in situ field treatments at some selected sites.
The combined cultivation dependent and independent approaches applied in ULIXES provided insights on the taxonomic and metabolic diversity of biodegrading microbial communities inhabiting the selected polluted sites, including seashore sands, coastal lagoons and oil refinery sediments. The characterization of the microbial diversity in those sites and the setup of novel clean up strategies and their field tests represent the major contributes of the ULIXES project to fill a knowledge gap for supporting the ecological sustainability of the Mediterranean Sea.
Such objectives were complemented by the implementation of a strategy of dissemination of the scientific results targeting the academia as well as a series of public and private stakeholders operation in the sector of oil management.
According to all the considerations at the basis of the ULIXES concept and the general objectives of the project, a list of specific objectives can be resumed as follows:
1. To establish a detailed description of the microbial diversity of culturable and unculturable microorganisms associated to the ULIXES polluted sites in the Mediterranean Sea basin. Such investigation included a wide series of polluted sites (17 sites plus specific areas in some of the sites).
2. To define the proof of concept for defining the first map of the microbial diversity associated to polluted sites in the Mediterranean Sea.
3. To establish a collection of microbial isolates and mixed microbial cultures with catabolic/detoxifying capabilities of aliphatic and aromatic hydrocarbons, halogenated solvents and PCBs and heavy metals.
4. To establish a collection of microbial isolates capable of biopalladium nanoparticles precipitation and activation by new hydrogen donors.
5. To establish a collection of microbial isolates capable of producing robust and versatile hydroxylases and/or biosurfactants and/or capable of biofilm formation.
6. To identify new functions by employing high-throughput methods, including microarray, metagenomics and metaproteomic methods, and establishing a set of genes coding for biodegradation enzymes from two major families, namely oxidoreductases (EC1) which catalyse oxidoreduction reactions and hydrolases (EC 3) which catalyses a number of hydrolytic activities with potential use in biodegradation. The major groups of activities considered were mono- and dioxygenases, hydroxylases, hydratases and dehydratases, haloacid and haloalkane dehalogenases and meta-cleavage hydrolases, to cite the most important.
7. To develop processes for the reductive dehalogenation of chlorinated compounds. Model compounds were the chlorinated aliphatic solvents and PCBs. The choice of these two compound classes was made on the base of their repartition in marine systems: chlorinated solvents in water and PCBs in sediments.
8. To develop processes of PCB reductive dehalogenation exploiting the catalitic propoerties of bio-palladium and other nanoparticles in marine sediments.
9. To define a fate model in polluted sediments for metal and metalloids in function of biodegradation yield.
10. To simulate bioremediation models from experimental microcosms, mesocosms and pilot ex situ and in situ field studies. Such activity was conducted in different sites from the North and South sides of the Mediterranean Sea as well as in the Gulf of Aqaba in the Red Sea.
A further objective of ULIXES was to establish and implement a strategy of dissemination of the realized research activities and scientific results. Such dissemination was based on a series of actions and tools including the establishment of a database of stakeholders and SMEs in the field of oil management, a web site, leaflet and communication to the scientific community through conferences and scientific papers. The dissemination activity was culminating with the organization of the ULIXES International Conference at the end of the project, “MedRem-2014, Microbial Resource Management For Polluted Marine Environments And Bioremediation, organized in Hammamet, Tunisia on January 2014.
Project Results:
WP2. Sampling and chacterization of the Mediterranean Sea sites.
WP2 activities consisted of the sampling and geochemical characterization of the different polluted area (Figure 1) located both in the Mediterranean basin and in the Gulf of Aqaba (Red Sea, Jordan).
According to the aims of ULIXES and the activities foreseen in WP2, seventeen polluted sites have been considered as sampling targets of ULIXES.
These include: i) a series of coastal sites like industrial poles in marine lagoons, polluted sites in coastal lakes/lagoons in the south coast of the Mediterranean Sea (Tunisia and Morocco), industrial harbors and polluted seashores. ii) A series of open sea sites, both shallow and deep. iii) A series of deep-sea natural hydrocarbon-seeping sites. Twelve coastal sites have been considered. Seven sites are located in the coastline of European countries (Greece, two sites; Italy, four sites). Four coastal sites are located in the South coast of the Mediterranean Sea (Egypt, one site; Tunisia, two sites; Morocco, one site). One site is located in the Red Sea (Jordan) and it has been chosen as a model for the possible future transition of the Mediterranean Sea toward a sub-tropical environmental condition determined by the ongoing global warming.
Three cruises, during 2011-2012, were performed to sample both epi- and bathypelagic waters at transect of major tanker routes and deep sea natural hydrocarbon seeping sites. The sampling activities performed on board of the oceanographic vessels included the collection of polluted sediments, water column and water-brine interfaces of the Deep Hypersaline Anoxic Basins (DHABs) in the Eastern Mediterranean Sea.
Excluding site 6 (the Milazzo harbour, Tyrrhenian Sea, Italy), according to what proposed in the Project DoW, all of the sites indicated (Figure 1) were sampled and characterized. Furthermore, additional samples were collected from three additional sites (harbour of Messina, Italy; Oued Hamdoun-Monastir, Tunisia and harbour of Gela, Italy) and they were analyzed.
A total of 106 sediments and seawater samples were collected (102 during the first 18 months, 4 during the last 18 months) by different Partners (Table 1 and 2). At the same time 4 oceanographic cruises and 1 direct sampling in open sea were organized for the collection of samples in both the Eastern and Western basins of the Mediterranean Sea (Table 3 and 4).
Task 2.1: Sampling organization and geochemical characterization of the coastal target sites.
Sampling activities in the El-Max district, Egypt (site 1)
The sampling area was divided into highly polluted and moderately polluted sites at El Max district bay at Alexandria (Egypt). MUCSAT partner performed all the sampling campaigns realized in this area. MUCSAT performed chemical characterization of the sediment and water samples, which were also sent to other ULIXES partners (UMIL, CSISC, TUC) for analyses complementary to the cultivation approach adopted at MUCSAT.
Sampling activity on the Elefsina (site 2) and Zakynthos area (site 3), Greece
Different seawater samples were collected on 06/04/2011 and 31/08/2011 respectively from Elefsina Gulf and the wetlands of Kery (Zakynthos) by TUC. The geochemical and microbiological characteristics of the samples collected at the Elefsina Gulf, a seashore close to oil refinery (Attika, Aegean Sea), have been carried out. On 14th January 2013, additional sampling efforts were devoted by TUC to collect polluted sediment sample (c.a. 30 L) from a coast adjacent to an oil refinery unit in the Elefsina Gulf. Such sediment was used to set up a landfarming experiments within the WP8 activities.
Sampling activity on the Venice Lagoon (site 4), Italy
Seven samples of surface sediments were collected by UNIBO with a Van Veen Grab along the Brentella canal in the first industrial area of Porto Marghera (Venice lagoon, Italy) for investigating the structure of the bacterial communities and establishing PCB dechlorinating enrichment cultures.
Sampling activity on the Ancona harbour (site 5), Italy
Samples collection from the Ancona harbour coastal site was performed on 12/04/2011 onboard of the R/V “Calipso” by ETS, during a sampling campaign named ULX_AN1. A second sampling campaign was realized during October 2012 aiming to collect additional samples. Sediment and seawater samples were collected in pollutant-impacted and control stations, located respectively inside and outside the harbour area.
Sampling activities in the area of Priolo Gargallo, Italy (site 7)
Sediment and tar samples were collected by IAMC in the harbour of Priolo Gargallo (Siracusa, Italy. In particular, a total of 10 cores sediments (1 meter long, 20 cm Ø) were collected at a depth of 6 meters from two stations (SY01 and SY03) located inside the harbour.
Sampling activities in the Messina harbour, Italy (additional site 1 selected by IAMC partner) Sediment samples were collected by IAMC in the harbour of Messina (“Mare Sicilia” station), a site located within a zone impacted by heavy ship traffic, both for touristic and industrial activities.
Sampling activity on the Bizerte Lagoon, Tunisia (site 8)
Sediments and seawater were collected from UTUN Partner from different sites located in the northern coast of Tunisia near the STIR (Oil Refinery Industry) company harbor at Bizerte region. The purpose of STIR is the refining of crude oil in order to satisfy the needs of the national market of oil products. The sampled sites present a marked pollution due to the release of the refinery wastewater containing residual hydrocarbons.
Sampling activity on the Sebkha of Soliman, Tunisia (site 9)
The Sebkha of Soliman is an organochlorinated compounds contaminated area located in south-western peninsula of Cap Bon (Tunisia). This site is highly exposed to different types of pollutants from agricultural and industrial activities located in the region. The contamination is easily detectable by eye both in the water and in the sediment samples. The sampling activities were performed by UTUN partner.
Sampling activity on Oued Hamdoun-Monastir, Tunisia (additional site 2 selected by partner UTUN)
The Oued Hamdoun-Monastir is an area polluted by organophosphorous molecules. A water sample has been collected from the site Oued Hamdoun-Monastir in order to isolate chlopyrifos ethyl degrading bacteria.
Sampling activities in the Gulf of Aqaba, Jordan (site 10)
The Aqaba Gulf was sampled by YU Partner in coordination with the Authorities of the oil terminal and the harbor of Aqaba (Jordan). Water and sediment samples were collected during three campaigns: May 2011, July 2011, and December 2011. During the first sampling campaign YU scientists collected samples of seawater and sediments from four sites in the Gulf of Aqaba-Jordan: Marine Science Station (MS), phosphate port (P), oil terminal (O) and fertilizers terminal (F). The samples collected by YU have been sent for additional analyses to other partners (e.g. CSIC, UMIL, TUC).
Sampling activities in the lagoon of Mar Chica, Morocco (site 11)
The Lagoon of Mar Chika lagoon is located on the northwest coast of Morocco in front of the Alboran Sea. The lagoon is isolated from the Mediterranean by a strip of fine sand and dunes, while it had a communication channel to the sea through the key of Boucana. Despite the biological, ecological and landscape importance of Mar Chika lagoon, it is exposed to continuous pollution by the town of Nador, and also by agriculture and industrial activities. Water and sediment samples have been collected from the lagoon by the Moroccan partner UH2C.
Sampling activities in the refinery of Gela, Italy (additional site 3 selected by IAMC partner)
Seawater and sediment samples were collected, in June 2013, by IAMC-CNR in another area located in the proximity to the refinery of Gela (Caltanissetta, Sicily, Italy). This additional area was chosen following an oil spill occurred from a pipeline of the “Topping1” installation of the ENI Refinery during July 2013. The spill released around 5,000 liters of heavy crude oil in the marine environment.
Task 2.2: Organization of the cruise for sampling open and deep Mediterranean Sea sites
Sampling activities in the Haven tanker shipwreck site, Harbour of Arenzano, Italy (site 12)
Tar samples were collected, by scuba of IAMC partner, around the harbor of Arenzano (Genova, Italy). In this area, on 11st April 1991, the Amoco Milford Haven tanker (a cargo load of 230.000 tons of crude oil) exploded during a routine operation and spilled over 45 million gallons of petroleum into the Mediterranean Sea. The samples have been used for the activities foreseen within WPs 3-4-5-6.
Ulixes_M11 Oceanographic cruise (R/V Meteor)
The Ulixes M11 oceanographic cruise has been carried out on the R/V METEOR, from 03th to 28th April 2011, interesting most of the basins located in the Mediterranean Sea. The UMIL partner was invited to participate to the M84/3 Meteor cruise by Dr. Toste Tanhua (Kiel University, Germany) and Dr. Dagmar Hainbucher (Hamburg University, Germany) in the frame of a previously established collaboration. During the sampling activities different water samples were collected and subsequently analyzed within the WPs 3-4.
Ulixes_U11-1 Oceanographic cruise (R/V Urania, September 2011)
The Ulixes U11-1 oceanographic cruise has been carried out on the R/V URANIA, from 09th to 23th September 2011, interesting different Deep Hypersaline Anoxic basins (DHABs) located in the Eastern Mediterranean Sea. During the U11-1 cruise, the UMIL team collected deep seawater, seawater-brine interface, brine and sediment samples from the Urania, and Tyro DHABs.
Ulixes_UEU Oceanographic cruise (R/V Urania, April 2012)
The Ulixes_UEU oceanographic cruise has been carried out on the April 2012, interesting different area of Central Western Mediterranean Sea. During the UEU cruise, the IAMC team collected different samples of surface and deep seawater from different station.
Ulixes_UBO Oceanographic cruise (R/V Urania, November 2011)
The Ulixes_BO oceanographic cruise has been carried out on the November 2011, interesting different area of Central Western Mediterranean Sea.
Besides the microbiological studies, all the samples have been characterized for a series of geochemical parameters according to their nature and the type of investigations ongoing. Overall, the sampling performed during ULIXES project allowed to establish an accurate mapping of chemical, physical and microbiological characteristic of different sediments and seawater present in the Mediterranean Sea basin.
WP3 Microbial diversity exploration in the Mediterranean Sea sites
The general objective of WP3 was the characterization of the phylogenetic and functional diversity of the microbial communities inhabiting the polluted sites studied within ULIXES by applying molecular biology-based cultivation independent approaches. The application of different molecular ecology techniques allowed dissecting the composition of the microbiome dwelling in the selected polluted sites. Direct counting methods based on fluorescence microscopy after staining with fluorescent dyes were used to evaluate the microbial abundance in natural and polluted ecosystems located across the Mediterranean basin.
Task 3.1 Determining a detailed description of site microbial diversity
An array of molecular techniques was used to study highly contaminated sites, offshore surface and deep seawater and on the metagenome extracted from pollutant-exposed microcosms aiming to depict the structure of the inhabiting microbiomes from a phylogenetic perspective. A database of microbial species present in the different investigated sites was established in the frame of Task 3.1. This database encompasses data on the i) community structure, ii) community diversity and iii) microbial abundance. The community structure dataset was mainly represented by the results obtained through the application of Automated Ribosomal Interspacer Sequence Analysis (ARISA) fingerprinting by UBANGOR, ETS and UMIL partners. ARISA fingerprinting was applied on the metagenomic DNA extracted from the sediments collected at the Ancona harbour, Priolo Gargallo bay and the Haven sites and from deep and surface seawater samples in different Mediterranean Sea sites. The ARISA results allowed identifying which are the environmental factors (in particular referring to the concentrations of different pollutants) contributing to drive the structure and diversity of the bacterial communities dwelling the sediment of the control and impacted stations, in particular for those in the Ancona harbour site. Through the ARISA fingerprinting, we also provided a comprehensive depiction of bacterioplankton community structure across the water column over a longitudinal transect covering the whole Mediterranean Sea. Our investigation demonstrated that the overall planktonic bacterial community inhabiting epipelagic seawater was spatially patterned in the Mediterranean Sea, exhibiting different populations in the Eastern and Western basins.
Furthermore, ARISA fingerprinting was applied on the enrichment cultures obtained from the samples collected from different sites located in particular at Elefsina Bay (Greece), El-Max district (Egypt) and the Gulf of Aqaba (Jordan) to estimate the shifts of the bacterial community composition and richness during incubation in the presence of pollutants. ARISA fingerprinting permitted to assess the evolution of the laboratory enriched microbial communities, considering also those microbes that cannot be isolated in pure culture. This approach provided a wider picture of the microbial resources that were selected under specific conditions and gave information about the whole community composition, contributing to setup the best cultivation condition for selecting the most promising biodegrading microbial consortia (WP4). A similar molecular study was conducted by applying Denaturing Gradient Gel Electrophoresis (DGGE) to assess the role of the site, the sampling, the pressure imposed by different pollutants and the cultivation strategy in the selection of HC degrading bacteria from the sediment of the Ancona harbour, evidencing that all these factors concur in shaping different bacterial communities.
A second part of the database was devoted to summarise the different bacterial species detected after sequencing PCR-DGGE bands, 16S rRNA clone libraries and 16S rRNA PCR-barcoded fragments following deep pyrosequencing. DGGE analysis allowed to describe the microbial diversity on the metagenome directly extracted from the environmental samples and it was performed both on seawater and sediment samples collected across the Mediterranean and the Red Sea. The contaminated sediments collected from the Priolo Gargallo bay, the Messina harbour, the Haven shipwreck area, and different stations located in the Gulf of Aqaba (Jordan) and El-Max district (Egypt) were tested by DGGE showing the prevalence of bacteria belonging to the class Proteobacteria whereas the control sediments collected at the Marine Science Station (Jordan) were mainly colonized by Bacilli. DGGE band sequences retrieved from the water samples collected along the oil-tanker routes showed the presence of Cyanobacteria in addition to the alpha-proteobacteria subclass, as expected for surface open seawater.
16S rRNA libraries of the 16S rRNA gene amplified from the cDNA obtained after retrotranscription from the total sediment RNA, were constructed from the polluted sediment and tar samples collected at the Haven shipwreck site. The phylogenetic affiliation of two hundreds 16S-cDNA clone inserts showed the presence of a rich bacterial community. Despite all the analysed Haven samples shared a similar community at higher phylogenetic rank level, a more detailed analysis of the microbiome structure revealed that some of the tar samples host peculiar groups of bacteria related to Cycloclasticus and Nitrospira genera. These bacterial genera are widespread in hydrocarbon polluted sediments and thus potentially exploitable for bioremediation purposes. In particular, bacteria related to genus Cycloclasticus are known as key microorganisms involved in the aerobic breakdown of polycyclic aromatic hydrocarbons (PAHs).
The microbial species diversity dataset includes also information on the microbial abundance obtained by DAPI staining on i) the collected surface and bottom seawater along the oil-tanker routes in the Mediterranean Sea and ii) the Ancona harbour sediment samples. Microbial abundance was evaluated by applying two diverse direct counting methods based on fluorescence microscopy, namely DAPI and Sybr Green staining. This approach allowed to determine the total cell count in deep and surface waters from different stations located in the Eastern and Western Mediterranean Sea and to assess the total microbial abundance and bacterial biovolume for the sediment samples collected from the Ancona harbour (Italy).
In addition to the investigation of taxonomic diversity of the target polluted sites, Task 3.1 aimed to study the functional diversity of biodegrading microbial communities present in polluted seawater and sediment samples through a cultivation-independent approach. Firstly, an accurate bibliographic research was performed to survey the available literature data looking for specific PCR primer sets and protocols that were modified, when necessary, through the PCR amplification on a subset of the environmental samples and bacterial isolates. In particular, we focused on alkane hydroxylases. These genes are divided into three different clusters: group I (alkB-I) codifies for enzyme that degrade short chain length alkanes (C6-C12), group II (alkM) codifies for enzyme that degrade medium chain length alkanes (C12-C18) and the group III (alkB-III) that codifies for enzyme involved in the degradation of long chain length alkanes (>C16). In addition to the alk gene, specific PCR assays were set up to amplify the ndo gene, which codifies for the naphthalene dioxygenase, an enzyme involved in the degradation of polycyclic aromatic hydrocarbon (PAHs). Additional catabolic genes investigated within the WP3 were those encoding the dissimilatory sulfite reductase (drsB) and the reductive dehalogenase (dca-rd). The catabolic dsrB gene encodes for a key enzyme involved in the anaerobic sulfate reduction metabolisms and is frequently used for investigating the diversity within the sulfate-reduction bacterial community. Seawater and marine sediments generally show high sulfate concentrations. The interest devoted in the ambit of the Task 3.1 to the dsrB analysis was related to the study of anaerobic dehalogenating microcosms (WP4) to enrich bacteria able to biodegrade organohalide compounds, like for example 1,2-dichloroethane (1,2-DCA). In fact, under the anaerobic condition used to study the reductive dechlorination of 1,2-dichloroethane (1,2-DCA), sulfate reduction represents a metabolism that may compete with reductive dechlorination for the electron donors available. Similarly, dca-rd (DCA- dechlorinating reductase) genes were PCR amplified from the metagenome extracted from the 1,2-DCA dechlorinating microcosms established from the sediment collected at the El-Max district (Egypt) to follow the evolution of the 1,2-DCA dechlorinating bacterial communities.
Task 3.2 Establishing a database and a map of the diversity associated to polluted sites
To design a map describing the most important microbial diversity information we applied pyrosequencing analysis on a set of samples, chosen after an initial wider screening involving a higher number of samples compared to those presented here. Overall, the samples included in the map have been chosen basing on the quality of the extracted metagenome and for their geographical distribution in the Mediterranean Sea. Most of them have been collected from strategic areas, widely interested from the production, transformation and transport of oil, hence potentially subjected to a higher risk of pollution. To create the biodiversity map we focus on the prokaryotic communities, analyzing both the bacterial and archaeal 16S rRNA gene using universal primer. Seven out of the eight sites analyzed by pyrosequencing are located in the Mediterranean coastline (Italy, Greece, Morocco, Tunisia, Egypt) while the eighth sample come from the Aqaba Gulf. For each sample, the rarefaction curves indicated a good species sampling coverage. Pyrosequencing results showed that each of the sediment hosted a peculiar bacterial community. Overall, the bacterial communities showed a higher diversity compared to the archaeal communities, which were characterized by higher evenness values, especially in the sites located in the Aqaba Gulf, the Haven shipwreak and the harbour of Messina. The depiction of archaeal communities showed the predominant role of methanogenic communities in the polluted sediments of the sites located along the Italian coastline. In the case of the Aqaba Gulf’s sample the pyrosequencing data on the archaeal community showed that about 88% of the retrieved sequences were unknown, likely being unreliable at the kingdom level. For this reason, we indicated as the most abundant group the genus Nitrosopumilus even if it represent only 10% of the total archaeal sequences. In the case of the bacterial communities, we could obtain the description of the taxonomic groups at the family level. A collaborative manuscript including both the pyrosequencing obtained at UMIL and the metagenomic data obtained by CSIC partner is under preparation at the moment. The whole dataset on the characterization of the bacterial communities in the different polluted sites studied will be included in that publication. The established microbial ecology map of the Mediterranean Sea provides a link between the geography of the Mediterranean Sea and the information on the taxonomic composition of the bacterial and archaeal communities elaborated during the project, showing that it is possible to trace the prokaryotic diversity in the Mediterranean basin.
An additional activity conducted within Task 3.2 was the establishment of databases of protein and nucleotide sequences of different catabolic genes involved in the biodegradation of hydrocarbon compounds, aiming to develop a functional gene array termed BiodegChip. The study was realized at UTUN to develop a DNA microarray focused on the study of catabolic genes of aromatic BTEX pollutants in complex microbial communities. BTEX is an acronym that indicates unsubstituted or alkyl-substituted monoaromatic hydrocarbons including benzene, toluene, ethylbenzene, and o-, m-, and p-xylene. A total of 3558 protein sequences of validly described members of 20 different catabolic protein families were collected from reference pathways. These protein sequences constitute the protein sequence database. The majority of the different catabolic protein families considered in the present study contains a large number of protein sequences, which have to be used independently as seeds for search using BLASTP against 200 non redundant protein and environmental sequence databases (GeneBank, RefSeq, EMBL, DDBJ and PDB). For this reason, for all the protein families containing > 50 protein sequences, a phylogenetic analysis is recommended using ClustalX and MUSCLE conducted in MEGA5. The evolutionary histories of proteins were inferred using the neighbour-joining method and the p-distance model. In the second step, all representative protein sequences from the divergent branches were used to perform TBLASTN searches. In order to design probes for the BiodegChip array, we thereby built a databases of corresponding DNA sequences of the created catabolic protein database, which is used for the physical construction of the microchip detection system for biodegradation genes. All the sequences that constitute the database for the BiodegChip have been included in the Deliverable 3.3.
WP4. Establishment of collections of microbial isolates and metabolites
Different enrichment and culturing techniques were used for establishing different large collections of microbial isolates having different features for potential application in bioremediation. Isolates were obtained using both non selective rich media and mineral media supplemented with different pollutant mixtures as unique carbon source. Sub-collections were created basing on the screening of isolates for specific activities.
Pollutant degrading bacteria were isolated by the different ULIXES partners from several contaminated sites in the Med Sea and the Gulf of Aqaba:
MUCSAT and UMIL isolated from the seashore of El Max district (site 1);
TUC established microbial cultures from the Elefsina Gulf seashore close to oil refinery (site 2) and from the wetlands of Keri in Zakynthos (site 3);
UNIBO established mixed PCB dechlorinating anaerobic cultures from the Venice lagoon (site 4);
UMIL and ETS established microbial cultures from the Ancona harbour (site 5);
IAMC and UMIL cultured from Milazzo harbour (site 6), Augusta bay (site 7) and several surface open sea sites located across the oil tanker routes in the Mediterranean basin (site 17);
UTUN isolated microorganisms from the Bizerte lagoon (site 8) and the Sebkha of Soliman (site 9) from the Oued Hamdoun site close to Monastir and from SidiThabet in Tunisia;
YU and UMIL analyzed samples from Aqaba Gulf polluted seashores (site 10);
UH2C established a collection of isolates from the lagoon of Mar Chica (site 11);
IAMC and UMIL analyzed samples from natural deep-sea hydrocarbon seepage sites (Amsterdam, Kula, Napoli and Olimpi mud volcanoes and the hypersaline anoxic lakes Tyro and Urania, sites 13-16), and from the tanker Haven sunk offshore Genoa (site 12).
The ULIXES isolates database, available to the public at the URL http://www.lab-biotech.com/data/isolates.php includes several collections details.
Four tasks related to the construction of specific sub-collections were planned in WP4 including: (i) collection 1, hydrocarbon degrading microbes (including 3 sub-collections for biofilm and EPS formation, biosurfactant production, and high molecular weight hydrocarbon degradation); (ii) collection 2, halogenated compounds degrading microbes; (iii) collection 3, biopalladium nanoparticles precipitating prokaryotes; and (iv) collection 4, metal biostabilizing prokaryotes.
Task 4.1. Collection 1: Hydrocarbon degrading microbes
To isolate and characterize hydrocarbon (HC) degrading microbes, isolation procedures were mainly based on the use of minimal media supplemented with the targeted pollutant(s). UMIL established a bacteria collection of 235 isolates by enrichment culture using diesel and crude oil as unique carbon sources. Alcanivorax and Marinobacter were shown to be predominant representing respectively 67% and 23% of the whole collection. A sub-collection of 179 Alcanivorax isolates was further characterized by DNA fingerprinting using ITS and BOX-PCR. The genotyping study showed that all the detected genotypes were retrieved exclusively from one site, hence demonstrating the occurrence of a geographic divergence at the strain level. The used approach confirmed the huge biodiversity hosted in the Mediterranean Sea in terms of microbial resources to be exploited for oil remediation. From the contaminated sites 8 and 9, UTUN isolated a collection of 185 hydrocarbonoclatic bacteria using BHMS and MMC media supplemented with crude oil, pyrene, phenanthrene or naphthalene as the sole carbon source. Beside molecular identification (16S rRNA sequencing and ITS-PCR), the collection was characterized in term of biotechnological potential (i.e. biosurfactant production, emulsification activity, HC degradation), which allowed the selection of 16 “efficient” isolates. These selected bacteria were used to setup designed consortia in microcosm experiments and they showed interesting bioremediation potential. Beside several isolates from UMIL and UTUN, TUC constructed several reconstituted and natural consortia and established a collection of biosurfactant producing strains (sub-collection 1B), on which they evaluated biofilm formation, and EPS production (sub-collection 1A). To investigate the latter feature, several enriched and designed consortia were used in different conditions in presence of diverse carbon sources. The results showed that HC biodegradation efficiency, in the presence or absence of biofilm, varied according to the specific used consortium/strain. Within sub-collection 1B, biosurfactant production, characterization and purification were investigated in pure strains and mixed cultures, on which 16S rRNA pyrosequencing community screening was realized. The results showed that biosurfactant production remained constant in low concentration over time and that it was independent from the total culture biomass. Partner YU established a collection of 75 hydrocarbonoclastic isolates from which they selected the most efficient to be applied in WP8 in bioremediation experiments to test the clean-up potential of oil polluted water and sediment samples through microcosm experiments and by in situ-like processes through on-site mesocosms at oil contaminated sites in the Gulf of Aqaba. From the Mar Chica lagoon, shown to be contaminated by heavy metals (site 11), UH2C performed isolation trials, showing that several strains were able to grow in the presence of heavy metals, PAH and chlorinated compounds and, in particular, three isolates were selected for the degradation of anthracene. Finally, sub-collection 1C established by IAMC, includes bacteria selected on sterile mineral medium able to degrade high molecular weight hydrocarbons, such as squalene, pyrene, dibenzothiophene. A total of 40 isolates were obtained from polluted sediments and seawater, tar sample and mesocosms samples, and were assigned mainly to Gammaproteobacteria (Alcanivorax, Cycloclasticus, Marinobacter, Neptunomonas). These strains are of high interest in the frame of the ULIXES project and could be exploited for future bioremediation purposes.
Task 4.2. Collection 2: Halogenated compounds degrading microorganisms
To establish this collection, research efforts were realized mainly by UNIBO, UMIL and UTUN. Samples from contaminated sediments of the Venice lagoon (UNIBO) have been used to develop anaerobic slurry primary microcosms for the enrichment of anaerobic PCB-dechlorinating cultures on which DGGE analysis were performed to follow the evolution of the bacterial community diversity during the dechlorination process. The results of the enrichments indicated that two Chloroflexi phylotypes represent two dehalorespirers having different PCB dechlorination specificities. Using q-PCR, UNIBO researchers showed also that the growth of both phylotypes was associated to PCB dechlorination and that two strains identified as Dehalobium chlorocoercia and an uncultured Chloroflexi sp., were the responsible for dechlorination activities in the different sediments. UMIL focused on the selection of 1,2-dichloroethane (1,2-DCA) degrading cultures by establishing anaerobic microcosms. Molecular analysis performed on the metagenome extracted from the setup microcosms revealed the selection of peculiar bacterial communities from each sediment and confirmed that the enrichment of such peculiar bacterial community was driven by the sediment type used as inocula. Partner UTUN selected dimethoate-degrading microbes from polluted environmental samples, leading to the isolation of 35 strains that were assigned to three phylogenetic groups (Firmicutes, Bacteroidetes and Proteobacteria) and represented potential candidate for dimethoate bioremediation processes.
Task 4.3. Collection 3: Biopalladium nanoparticles precipitating prokaryotes
Thirty facultative anaerobic marine strains and sixteen aerobic strains have been isolated by partner UGENT from contaminated marine sediments collected from different sites (Priolo Gargallo bay and Mar Chica Lagoon). Eight strains showed the ability of concurrent biohydrogen production and Pd precipitation. X-ray diffraction (XRD) and thin section transmission electron microscopy (TEM) analysis indicated the presence of metallic Pd nanoparticles of various sizes (5-20 nm) formed either in the cytoplasm, in the periplasmic space, or extracellularly. Subsequently, the isolates were screened for their ability to generate bio-hydrogen (to activate the Bio-Pd) and to produce bio-surfactants to increase the solubility of pollutants. Promising results were obtained, indicating the selection of microbial resources potentially exploitable for interesting bioremediation applications.
Task 4.4. Collection 4: Metal biostabilizing prokaryotes
From the sediments collected from the Ancona harbour, partner ETS established a collection of metal biostabilizing microbes. For this, five experimental sets, with the addition of organic substrates, were studied in comparison with non-amended controls. The isolation of seventy-two bacteria was performed independently from sediments collected from each of the different microcosm setup. The time course incubations resulted in a significant reduction of total hydrocarbon concentrations in all the experimental systems, coupled with significant changes of metal speciation in sediments. In particular, the fraction of Cu, Pb, Ni and Cr extractable with standard techniques was reduced (on average 13% for Cu, 11% for Pb and 3% for Cr) indicating the potential of these strains to positively influence the mobilization of the investigated heavy metals.
Overall the mixed cultures and the bacteria isolated in the ambit of WP4 constitute a promising resource that was exploited in the ambit of the more applicative WPs of the ULIXES project, namely WP7 and WP8.
WP5. Metabolome analysis of the microbiome associated to selected polluted sites
During the ULIXES project, we completed the following activities within the WP5, which produce a number of outcomes:
• An in silico array was produced and amended in the ULIXES project. The array, which contains a set of 5,315 gene sequences encoding enzymes participating in biodegradation steps, allows by using a simple BLASTP search (score > 45 and E-value < 10e-3) inferring, identifying and quantifying the types and diversity of such genes from any kind of sequence data.
• A total of approximately 310 Mbp high quality sequences have been obtained, by 454 and Illumina Hiseq 2000 sequencing, either from raw or microcosm samples from sediment samples from four major oil refinery polluted Mediterranean Sea sites on the coastlines of Morocco (lagoon of Mar Chica), Tunisia (Bizerte lagoon), Egypt (El-Max site), Greece (Eleysina bay), and Italy (Gulf of Genoa, strait of Messina and Syracuse, and Ancona port) as well as on the coastline of the Red Sea (Gulf of Aqaba along the Jordanian coast). The sequences were obtained from the original samples from the sites as well as from microcosms enriched on specific compounds. All the obtained sequences have been deposited in public databases with the following accession numbers: IDs PRJNA222659, PRJNA222657, PRJNA222660, PRJNA222661, AZIB00000000, AZIC00000000, AZID00000000, AZIE00000000, AZIF00000000, PRJNA222667, PRJNA222664, PRJNA222665, PRJNA222666, PRJNA222663, AZIG00000000, AZIH00000000, AZII00000000, AZIJ00000000, and AZIK00000000. According to these figures, the ULIXES project contributed to provide, to the best of our knowledge, the biggest set of metagenomic sequences from the Mediterranean and the Aqaba Gulf in the Red Sea deposited in databases. Moreover, the overall dataset contributed to increase the knowledge on the genetic diversity and functions of the microbiomes associated to the polluted sites from the Southern side of the Mediterranean Sea, that have been rarely considered in previous metagenomic studies. For samples locations see Figure 2.
• At least 83,000 16S rRNA gene fragments obtained by 454 pyro-sequencing or Illumina Hiseq 2000 sequencing have been identified from sediment samples on the coastlines of the Mediterranean and Red Sea sites (see above). Accordingly, at least 18,435 operational taxonomic units (equivalent to distinct species) were identified. This constitutes, to the best of our knowledge, the biggest set of environmental species within the Mediterranean and the Aqaba Gulf in the Red Sea deposited in databases.
• Meta-genomic (theoretical) and metabolomic (experimental) evidences for the metabolism of 31 distinct pollutant substrates and/or intermediates including alkanes and PAH-like derivatives have been successfully identified in the ULIXES project. A total of 223,416 out of a total of 10,907,240 (or 2.05%) potential protein-coding genes, covering 56 types of major activities, were found as being involved in such processes.
• Three protocols have been developed to: reconstruct biodegradation networks from DNA (1) and 16S rRNA (2) meta-genome sequences, as well as meta-proteomic and meta-metabolomic data (3). Graphical representation of these networks was developed using the igraph package for the R platform and in house algorithms. Additionally, a fourth graphical method was produced by using the Ocean Data View software, in which spatial distributions of percentages of genes encoding enzymes participating in biodegradation steps are plotted as iso-surface distributions. These tools were crucial for inferring putative and active biodegradation reactions in the network, and pathway organizations at the global, organismal and spatial levels.
• A total of 18 uric acid and ammonium-amended microcosms have been produced and analyzed in terms of: (1) community structure; (2) geochemical parameters; (3) presence of uric acid degrading members of the Halomonas genus; (4) the comparison of diversity and distribution of catabolic genes for alkane and aromatic degradation as consequence of the applications of ammonium and uric acid. Results highlighted that Halomonas spp. members may promote biodegradation efficiency by converting uric acid into ammonium which can be readily utilized by marine hydrocarbonoclastic consortia. Additionally, examining the sediment of Ancona port (northern Italy) we demonstrated that while ammonium improved the presence of microbes degrading phthalate, gallate, catechol to 2-hydroxymuconate semialdehyde and toluene (via anaerobic pathway), uric acid improved the presence of dinitrotoluene- and halogenated catechol-degrading microbes.
WP6. Microbial metagenomics and proteomics of the polluted sites
During the ULIXES project, we completed the following activities within the WP6, which produce a number of outcomes, which are highlighted below. To note is that the activities and results within this WP derived from sequences resources and material derived from sediment samples from four major oil refinery polluted Mediterranean Sea sites on the coastlines of Morocco (lagoon of Mar Chica), Tunisia (Bizerte lagoon), Egypt (El-Max site), and Italy (Gulf of Genoa, strait of Messina and Syracuse, and Ancona port) as well as on the coastline of the Red Sea (Gulf of Aqaba along the Jordanian coast), as well as from the polyaromatic hydrocarbon degradation Cycloclasticus sp. ME7.
• A total of 42 metagenomic libraries (28 fosmid libraries and 14 cosmid libraries) containing a total of 1.4 million clones (about 1.25 million in fosmids and about 150,000 in cosmids) have been produced.
• At least, 1,221 out of approx. 1.2 millions clones encoding enzymes of interest have been identified by screening of all 42 libraries.
• 34 fosmid clones have been fully characterized in respect to their substrate profile.
• A total of 306,948 potential protein-coding genes (cut-off of 20 amino acid-long sequences) have been identified within the ULIXES meta-genomic resources. They encode alpha/beta hydrolase family proteins and extradiol dioxygenases.
• At least 173 out of 306,948 genes encoding biotech-relevant enzymes of interest amended in ULIXES project have been cloned and expressed using the Ek/LIC 46 based expression vector in Escherichia coli BL22.
• Apart from activity-based enzyme mining, we have generated five metagenome sequencing datasets (by HiSeq Illumina sequencing) from crude-oil enrichment cultures amended with uric acid or ammonium. Resulting data was mined for genes encoding oxidoreductases and hydrolases with a subset of 50 enzymes that were cloned in E.coli.
• At least 74 biotech-relevant enzymes have successfully expressed and fully characterized with regard to substrate spectra and temperature range. A battery of at least circa 100 different substrates has been used to evaluate the biochemical properties. Characterized enzymes showed elevated optimum temperatures along the temperature gradient of Mediterranean and Red Sea (with lower optimum temperatures in Northern Mediterranean and elevated optimum temperatures in Red Sea).
• A crystal structure has been solved, deposited in the Protein Databank (PDB) and published. The structural co-ordinates reported for Cycloclasticus sp. ME7 CCSP0084 alpha/beta hydrolase appears in the PDB under accession code 4I3F.
• At least six mutant proteins were generated. Mutations were introduced into plasmid p15TV-Lic containing CCSP0084 gene encoding a dual esterase: MCP hydrolase from the α/β hydrolase family from Cycloclasticus sp. ME7. The biochemical analysis of the corresponding mutant variants was undertaken with four distinct substrates and the potential role of those residues in determining substrate profile was determined. Through this analysis, we were able to provide first indications about the residues dictating the evolution between esterases and meta-cleavage product alpha/beta hydrolases, that is, the evolution between a non-degradative and a degradative enzyme.
• At least, a set of 188 gene clusters presumably participating in the production of known secondary metabolite compound classes (poliketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, bata-lactams, butyrolactones, siderophores, melanins and others) have been identified using AntiSMASH (ANTIbiotics & Secondary Metabolite Analysis Shell; http:// antismash.secondarymetabolites.org). To note is the high number of interesting multifunctional genes of polyketide synthases (PKS) or non-ribosomal peptide synthases (NRPS) which are the main responsible for the biosynthesis of industrial cytotoxic compounds (for details see Figure 3).
To note is that within WP6, ULIXES project has contributed to the full characterization of the biggest set of proteins from the Mediterranean and Aqaba Gulf in the Red Sea, which constituted the biggest set of purified protein from a single marine location. In addition, it has delivered the biggest set of expression clone libraries around polluted sites in the Mediterranean and Aqaba Gulf in the Red Sea. Finally, within WP6 ULIXES consortium has demonstrated, for first time, that polluted sites around the Mediterranean and Red Sea may contain the higher number of gene clusters encoding secondary metabolite compound classes, namely, polyketides and non-ribosomal peptides, reported for free-living bacteria in a marine sample.
WP7. Establishing new product/processes for bioremediation
Task 7.1. Using hydrocarbon degradation enzymes and microorganisms to obtain valuable chemicals (fine chemicals and biosurfactant)
The main objective of the ULIXES research within the Task 7.1 was the development the biosynthesis of valuable fine chemicals and new biosurfactants at the laboratory scale by means of the hydrocarbon degrading microbes obtained in WP4. The achievement of this objective implied: (i) production and optimization of in-house protocols that allow investigating the reactivity-profile of enzymes produced in the frame of ULIXES project in order to develop novel and/or optimized biotransformation processes, (ii) the selection of marine bacteria able to produce biosurfactants to be tested in lab scale bioprocesses for the ex-situ bioremediation of marine sediments contaminated by hydrophobic chlorinated contaminants. ULIXES provided the first experimental evidences to demonstrate the biotechnological potential of MCP hydrolases, previously thought to be restricted to biodegradation pathways, has to be reassessed and that C-C meta-cleavage product hydrolases may be useful for important transformations, such as chiral resolutions and lactone hydrolysis. The discovery and success biotech testing of dual esterases - MCP hydrolases from the alpha/beta hydrolase family could be considered as a top achievement in relation to the development of new biocatalysis processes using enzymes previously considered to have low biotech applicability.
Additionally, ULIXES provided a set of at least 31 enzymes for chiral synthon production using the uncommon substrate specificities and enantioselective enzymes. Accordingly, these enzymes can be used for novel biocatalysis processes related to chiral ester resolutions and the concomitant production of pharmaceuticals. Finally, ULIXES has provided a set of at least 40 enzymes that have a substrate preference distinct from other reported alpha/beta hydrolase, as they are catalytically most active in the hydrolysis of poly-aromatics. These enzymes offer a new window for the stereo-assembly of multi-aromatic molecules useful in biology, chemistry and material science, for which enzyme processes have not yet being established. The hydrolases are catalytically active towards PAH esters, which can lead to the potential applicability to generation a unique set of complex aromatic molecules with yet unknown properties. We believe that some of the enzymes may open new opportunities to design a-la-carte enantiomeric forms of complex aromatic molecules, due to the regio- and enantio-selective properties of these enzymes. Such derivatives are known to have industrial relevance for the production of microelectronic components with photolytic properties, printed circuit boards, silk screen printing devices, optical disks, black matrix of colour tubes, dyes, flavouring compounds, plasticizers, perfumes, pesticides, mold growth biomarkers, insect repellent, fibers and films productions, and solid rocket propellants, to cite some reported applications.
Finally, isolates able to produce surface active molecules in different marine mineral media were screened, and they belong to the genera Alkanivorax, Paracoccus, Sulfitobacter, Shewanella, Pseudoalteromonas and Roseovarius. The latter three isolates showed high rate of growth, biosurfactant productivity and stability, and were thus compared in terms of ecotoxicity of the surface active molecule produced towards the marine bacterium Vibrio fisheri. The biosurfactant produced by the Shewanella sp. showed the lowest toxicity and was identified as the most promising biosurfactant for biostimulation applications. Therefore, it was selected for enhancing the bioavailability of hydrophobic chlorinated pollutants in the ex-situ bioremediation of marine sediments in lab-scale bioreactors.
Task 7.2. Development of small scale bioprocesses for the reductive dehalogenation of chlorinated compounds
The main objective of this task was to develop small scale bioprocesses for the reductive dehalogenation of chlorinated compounds at laboratory scale, by the means of the microbial consortia obtained in WP4. The up-scaling of the 1,2-DCA dehalogenating enriched cultures (see Deliverable 4.2) to optimize the conditions for the reductive dechlorination of this pollutant were unsuccessful. To overcome the instability of the established consortia from the marine polluted sites in the framework of ULIXES project, an alternative plan was setup with new dehalogenating enrichment cultures. These consortia were gradually adapted to the salinity value typical of seawater (about 3%). Unfortunately, the process of adaptation to higher salinity values stopped at the first step. In addition, the enriched bacteria did not show 1,2-DCA degradation activities at 0.25% salinity value in the medium even if they were highly efficient in the 1,2-DCA removal at 0% salinity value. Further efforts should be conducted in order to discover promising bacterial consortia involved in the dechlorination of 1,2-DCA in marine ecosystems with the optimization of reductive dechlorination activity against 1,2-DCA.
Even though the 1,2-DCA degrading enriched cultures could not be successfully used for the cleanup of this chlorinated solvent form seawater and sediment, the aim of D7.4 was still achieved. In fact, we achieved the setup of batch and/or continuous anaerobic bioreactors for dehalogenation of chlorinated solvents in marine water and slurry small-scale anaerobic reactors for the dechlorination of PCB mixtures in real sediments of the Venice Lagoon.
The bioremediation process for PCB contaminated sediments was tested in anaerobic shaken reactors and static microcosms supplemented with the selected sediment from the Venice lagoon (sediment E) in WP4. Within WP7, different strategies for the enhancement of PCB dechlorination activities were investigated: i) bioaugmentation of sediment with a marine PCB dechlorinating culture, ii) biostimulation of PCB dechlorinating microbes by providing an electron donor, iii) PCB mobilization by employing biosurfactant, and combinations of all these activities. The most active PCB dechlorinating culture was obtained from marine sediments of the Venice lagoon (i.e. culture from sediment D). The combination of this consortium with a suitable (i.e. lactate) electron donor allowed to achieve the bioaugmentation and biostimulation purposes in PCB dechlorination activity. The biosurfactant produced by the marine isolate XP8 were suitable for increasing the PCB bioavailability. Biostimulation process realized using lactate had no effect on PCB dechlorination by the indigenous microbial community of sediment; on the contrary, the bioaugmentation with the selected PCB dechlorinating culture promoted dechlorination of the PCB, which was further enhanced by biostimulation with lactate. Dechlorination process in the bioaugmented culture supplemented with the selected biosurfactant was partially inhibited. This might be due to some toxic effect of biosurfactant on the dechlorinating microorganisms. The same behaviors were observed in both shaken bioreactors and in static microcosms, where the absence of mixing resulted in lower PCB dechlorination rates and extents. In conclusion, bioaugmentation combined with biostimulation was found to be the most effective strategy to promote PCB reductive dechlorination for ex-situ treatments of PCB contaminated marine sediments.
Task 7.3. Development of novel biopalladium nanoparticles dechlorination processes
Bio-palladium (bio-Pd) nanoparticles combined with a hydrogen donor in order to produce reactive radicals, showed highly catalytically activity in multiple remediation processes, such as the dechlorination of halogenated compounds. The required H2 for activation of bio-Pd catalysts could be provided directly by the microbes, through the fermentative conversion of organic compounds. The general objective of Task 7.3 was to develop bio-Pd nanoparticles able to dehalogenate toxic compounds, such as PCBs and Cl-solvents (TCE), polluting marine environments.
In the framework of WP7, UGent has delivered the following objectives:
(i) Identification and application of 46 indigenous Mediterranean marine isolates for the synthesis of very small, narrow sized palladium nano-catalysts.
(ii) Optimised fermentative production of hydrogen to activate the nano-catalysts, using one of the above microorganisms.
(iii) Complete dechlorination of TCE in synthetic marine water, marine water and sandy marine sediments slurries, using the formed bio-Pd-NPs.
(iv) Enhanced catalytic activity of Bio-Pd nanoparticles for the reductive dehalogenation of TCE, upon modification of the nanoparticle size.
(v) Bio-Pd nanoparticles were able to dechlorinate both PCB20 and Aroclor 1254 PCBs in marine water, where PCBs can be fully dechlorinated to the less harmful biphenyl.
(vi) Activation of bio Pd and further dehalogenation of TCE using either source of H2 (bogenic or exogenous).
(vii) Immobilized catalysts in organic polymers were successfully degraded TCE in marine water and marine sediment slurries.
The newly biogenic nanoparticles developed within the ULIXES WP7 can open a new perspective for future marine nano-based remediation applications.
Task 7.4. Definition of a fate model for metal and metalloids in polluted sediments
The partner EcoTS studied the hydrocarbon biodegradation, mobilization and stabilization of metals through biostimulation experiments. The experimental design included the addition of substrates (sodium acetate and lactose) and mineral nutrients. The biodegradation of hydrocarbons was more efficient after 60 days of incubation, while best results were obtained in presence of acetate and nutrients plus acetate. These treatments resulted in a significant increasing of total bacterial abundance and biomass compared to the control system. The geochemical fractionation of heavy metals in the sediments during the biostimulation experiments was studied, considering the exchangeable and carbonate bound fractions, iron and manganese oxides fraction, organic and sulfide fraction, and the residual fraction. Results indicated a range of different behaviors in terms of mobility/bioavailability of each metal in the sediment matrix. The best performances in terms of immobilization of metals (25% of heavy metals immobilized after 60 days incubation) were achieved with the addition of nutrients and substrates. The rates of mobilization/immobilization of each metal were statistically analyzed for the production of a fate model, as well as to select the best treatment strategies for contaminated sediments. The ecotoxicological assays (Microtox, algal growth inhibition and immobilization of crustaceans), was performed to assess the eco-compatibility of sediment treatments, and suggested a slight reduction of toxicity in systems displaying the best performances of metal immobilization.
The overall results indicated that the biostimulation of autochthonous bacterial community can be an effective strategy for the immobilization of heavy metals in marine sediments. It is noteworthy that specific treatments have to be carefully designed to obtain good performances and re-use options.
WP8. Testing new product/processes by pilot bioremediation treatments in the field
The work package 8 included four different tasks that were carried out by four different partners of the ULIXES consortium, namely YU, MUCSAT, TUC, IAMC.
The overall aim of WP8 was to test selected microbial resources previously selected within the WP4 in a pilot and field scales. Furthermore, UMIL and ETS partners supported the monitoring activity of part of the setup bioremediation processes, investigating respectively the bacterial community structure during the experiments and assessing the eco-toxicity of the treated sediments. Each task was devoted to test microbial resources in the four different sites, all located in industrial areas contaminated by hydrocarbons, chosen as representatives of different latitudes and climatic conditions in the Mediterranean and Red Sea, the latter constituting a reference example of the potential climate situation of the Mediterranean in the next years following the global warming trend.
The WP8 activities were finalized to:
i) Simulate bioremediation models from experimental microcosms, mesocosms and pilot in situ field studies;
ii) Verify the efficiency of the whole approach adopted with ULIXES, i.e. whether the microbial resources retrieved with the microbiological surveys can be effective new products to improve the effectiveness of bioremediation up to the field scale.
Task 8.1. Treatment at the site 1, El-Max district, Alexandria, Egypt
MuCSAT partner performed several preliminary activities aimed the setup of bioremediation treatment of site 1, El-Max district. Five different natural consortia were obtained and characterized from highly polluted stations located at El-Max bay. Moreover, during the consortia growth under laboratory conditions, analysis of the remaining crude oil components were performed in order to choose the most suitable consortium to be applied for the in situ biodegradation experiment. The consortium R, previously obtained from station R, was selected for the in situ biodegradation of crude oil into seawater. A study of different immobilization supporters were performed using three different immobilization material that could be used as bacterial supporter to carry on the bacteria during the in situ biodegradation of crude oil. The results showed good degradation rate: about 99% of Unresolved Complex Mixture (UCM) and 94% of the Total Resolved Peak ratio (TRPs) were removed after 28 days of exposure of the cells to the oil in the seawater using specific devices (D8.4). An experimental trial was performed to study the effectiveness of immobilization of the cells on enhancement of the oil degradation. The results showed that the immobilization of cells generally increased the biodegradation ability, as the residual Total Petroleum Hydrocarbons (TPH) decreased in presence of the immobilized cells compared with the treatment performed using the free cells.
The Plackett-Burman experimental design method was applied to test the effect, in term of oil biodegrading capabilities, of six bacterial isolates used in a consortium. The data showed that the combined use of Pseudomonas plecoglossici and Bacillus aerophilis for the remediation of crude light petroleum pollution enhanced the degradation capability. Future experiments may reveal the optimization of the content of such mixture and formulate it further for in situ bioremediation purposes.
Task 8.2. Treatment at the site 2, the Elefsina Gulf, Attika, Aegean Sea, Greece
TUC partner initially proposed a plan that involved a two-phase experimental design. Phase A consisted of a small scale in situ aeration experiment and an ex situ landfarming mesocosm experiment. Phase B included a larger scale in situ aeration approach, combining the results obtained from phase A. Unfortunately, excavation activities were realized at the target contaminated site and the water depth increased to 90-95 m. Therefore, it was not possible to perform the in situ aeration experiments initially foreseen in the DoW. As a contingency plan, TUC extended the landfarming experiments, doubling the originally planned experimental time (8 weeks instead of the proposed 4) and performed an additional accurate set of chemical (sediment hydrolytic activity), microbiological (total bacterial population monitoring using flow cytometry) and eco-toxicological analyses. The results obtained by TUC for the landfarming ex situ treatment showed that the addition of allochthonous degraders, i.e. allochthonous bioaugmentation, did not enhance biodegradation even though the added degrading consortia had been acclimated in sediment slurry before their application to the sediment and their initial CFUs were 10-fold higher than those of the autochthonous viable bacteria. The degradation patterns of saturated and aromatic compounds, total bacterial population numbers and sediment hydrolytic activity of the bioaugmentated treatments were similar to those of the respective control treatment. On the other hands, biostimulation proved to be necessary, as the above parameters were significantly lower in the treatment. Moreover, the early peaks in total bacterial populations and sediment hydrolytic activity that were observed in the treatment receiving the dead bacterial biomass, indicated that the addition of organic nutrients may accelerate the degradation process. Additional microbiological analyses were performed at UMIL to dissect the composition of the bacterial community in the original sediment and to follow the evolution of the bacterial community structure during the different landfarming treatment experiments. To clarify the role of biostimulation on the autochthonous bacterial communities the sediments have been analysed through Denaturing Gradient Gel Electrophoresis (DGGE), a method that allow both the identification of the inoculated consortia and the study of the overall bacterial community.
Task 8.3. Treatment at the site 10, the Aqaba Gulf, Red Sea, Jordan
Partner YU performed several experiments to enrich consortia obtained from several polluted and unpolluted sites located in the Aqaba Gulf: the marine science station, the phosphate loading berth, the oil terminal and the industrial jetty site. These sites were sampled in the frame of the WP2 activities, aiming to assess the efficiency of different natural consortia and to have a snapshot of the microbial community structure. In the YU laboratory, different experiments were setup using 500 ml flasks, 5000 ml vessels and 12000 ml aquaria, as part of WP4, to enrich the natural consortia and to increase their biomass to be used later as inocula for the large tanks (2000 L). An on-site experiment was held at the shores of the marine science station (MSS) using large tanks (2000 L). The seawater was collected from the MSS site and used as media to support the microbial growth, supplying 0.5% crude oil as the sole carbon source. Chemical and microbiological analyses were carried out during the course of the experiment in order to monitor the progress of microbial growth and asses the crude oil biodegradation, monitoring several parameters for each consortium. The bacterial strains isolated from petroleum polluted sites in the Gulf of Aqaba were predominated by Gammaproteobacteria, which is a common phylum in marine environments. Fifty bacteria isolated from all the sampling sites were positive to the biosurfactant production tests; forty-eight isolates showed the ability to form biofilm and forty-six were able to produce exopolysaccharides. The removal efficiencies of n-alkanes were about 80-90% within 7 days of incubation, where the consortium FW showed the highest degradation activity. The removal efficiency in the presence of individual hydrocarbons phenanthrene, fluoranthene and pyrene by two of the tested consortia reached the highest level after growing the mixed cultures for 16 days. The oxygen concentration had a significant effect on the degradation of hydrocarbons. A correlation could be inferred between the removal of hydrocarbon, the increase in microbial load (CFU) and the CO2 production. Additionally, the samples provided by MUCSAT and TUC for field testing, were enriched and tested in the lab as a first stage. In experiments conducted, there was a clear difference in the n-alkanes removal efficiencies between the MUCSAT and TUC consortia.
Task 8.4. Treatment at the site 12, shipwreck site of the oil tanker Haven, Genoa Gulf, Italy
Partner IAMC-CNR have carried out a series of biostimulation approaches and obtained a better knowledge on the structure and dynamics of indigenous bacterial communities in parallel with the biodegradation processes. The mesocosms experiments were initially considered as preliminary test before the in situ treatment on site 12, corresponding to the oil tanker Haven shipwreck. In this context, sampling activities were performed in the area of the Haven shipwreck where different tar samples were collected for the cultivation of microbial consortia capable of petroleum degradation and for the description of the inhabiting microbial community achieved by molecular methods. However, despite numerous attempts, IAMC-CNR failed to obtain the official permission signed by the Italian Ministry for Environment for the realization of the field experiments on the tanker Haven shipwreck site. The realization of bioremediation experiment would have nevertheless been very challenging and difficult to perform due to significant depth of the shipwreck (about 80 meters) and the difficulties in obtaining the official permission from the local authority, the Italian Naval Authority and the Municipality of Arenzano (Genova), to enter the site. As a contingency plan, IAMC-CNR proposed the treatment of a chronically contaminated and easily accessible site located in the harbour of Messina. Almost 2 tons of contaminated sand sediments were collected and placed in 14,000 L mesocosms. The sediments for the in situ bioremediation study were further polluted with crude oil and the effect of different biostimulation approaches were validated in mesocosms experiments, monitoring in parallel the community structure and dynamics of indigenous bacterial communities during the biodegradation processes. Moreover, in June 2013 Partner IAMC-CNR suggested and included another area for in situ bioremediation treatments, namely the shoreline site located in the proximity to the refinery of Gela (Caltanissetta, Sicily, Italy). The new proposed area represents the most attractive scenario for the realization of in situ experiments. In fact, on 4th June 2013, an oil spill from the pipeline of “Topping1” installation of ENI Refinery of Gela released in the marine environment around 5,000 liters of heavy crude oil. Although quick action were undertaken by the Italian Naval Authority and the ENI S.p.A to combat with the spill, significant part of oil was spilled out, thus contaminating both open sea and the beach. Differently from what previously occurred for the Haven shipwreck site, IAMC-CNR succeeded to obtain the official permission signed by Italian Authorities to monitor the recovery process of the shoreline. Partner IAMC-CNR installed in the contaminated beach of Gela a 10 m2 area for trial field and environmental monitoring. This experimental area, indeed, represented a unique opportunity to work in the area affected by an actual oil spill, where direct application of specific strategies for environmental recovery could be placed, monitored and validated. In this new experimental area partner IAMC-CNR carried out two different strategies for recovery of polluted sediments. In particular, partner IAMC-CNR performed in situ biostimulation (addition of inorganic nutrients) and bioaugmentation (addition of inorganic nutrients plus biomass of indigenous Marinobacter sp. cells previously isolated from the same site).
Potential Impact:
Overall, the ULIXES project had a high environmental added value since it especially focuses on the exploitation of microorganisms that represent a renewable and sustainable resource for the clean-up and bioremediation of the polluted coastal marine environments in the Mediterranean Sea. Bioremediation and enhanced natural attenuation based on microbial products can be, with no doubts, considered as an environmentally friendly process. The approach applied in the ambit of the ULIXES research had the main goal of setting up pollutant- and site-tailored bioremediation processes for remediating marine ecosystems in a sustainable manner, and to develop innovative processes for environmental protection. To pursue this aim a holistic approach, comprising microorganisms cultivation, molecular ecology, OMICs based analyses and development of innovative bioprocesses and field pilot studies, has been applied by the ULIXES partners, in accordance to their specific well established expertise and available facilities. All the activities foreseen in the ULIXES DoW have been performed, leading to the achievement of the foreseen deliverables and the obtainment of most of the expected results. The establishment of a large collection of biodegrading strains and mixed cultures had been finalized (WP4). The ULIXES microbial collection encompasses bacteria of high interest due to their ability to form biofilm, produce biosurfactants and degrade high molecular weight hydrocarbons. Moreover, bacteria able to mobilize different metals, to degrade halogenated compounds and to produce activated bio-palladium nanoparticles have been isolated from the samples collected by the ULIXES partners across the Mediterranean Sea. The ULIXES microbial collection constitutes a massive reservoir of genetic resources that has been organized in a database available to the whole consortium. The collection has been already partially exploited within the project for the setup of bioremediation processes (WPs 7-8), and could be easily consulted by all the partners for further experiments beyond the project end, which can allow the set-up of new biodegrading microbial inocula for the treatment of polluted seawater and sediments. Until now, according to the result of a detailed survey of the scientific literature on marine bioremediation studies, minor attention was devoted by the international scientific community on the study of environmental pollution and safety in the Southern Mediterranean countries. In this contest, the EU project ULIXES represented a novel and tangible effort to integrate the North and South sides of the Mediterranean basin for the setup and improvement of clean up strategies exploiting natural microbiological products and the so called Microbial Resource Management. In the frame of the ULIXES project, the consortium realized the widest metagenomic and 16SrRNA pyrosequencing dataset of the Mediterranean Sea and the Gulf of Aqaba in the Red Sea produced until now (WPs 3-5-6). This dataset encompasses both taxonomic and functional biodiversity data and its sequences will become available to the public as soon as common manuscripts (which involve the whole consortium and are under preparation) will be published. The performed OMICs analyses allowed to get a detailed biodegradation metabolome map of each hydrocarbon-polluted site, correlated with a microbial biodiversity metagenomic map obtained through metagenomics and 16S rRNA pyrosequencing. To achieve a detailed picture of the diversity of the biodegradation potentials in each site, and in particular in the Southern Mediterranean, was one of the key objectives of the ULIXES research. This result boosts the filling of the knowledge gap on the bioremediation potential in a yet overlooked region, the Southern Mediterranean that is one of the major oil-producing areas on Earth, identifying site-specific metabolic routes and genes of potential biotechnological application.
Overall, the results showed that the three-years EU project ULIXES led to the consolidation of a consortium of partners that proficiently cooperated in the planned research activity. The consortium, within the ULIXES project, has published a high number of scientific papers. Moreover, several studies will be finalized after the official end of the project leading to additional results that will provide a high quality contribute to the scientific research on the bioremediation field.
In addition to the scientific achievements obtained within the ULIXES project, the research results had also consequences potentially relevant for social and political aspects of the research, which have to be taken into account when evaluating their implementation. The involvement of scientists from the two sides of the Mediterranean Sea promoted an active exchange of information and knowledge between partners of the different countries. This proactive cooperation resulted also in short visit/training periods of young scientists in the partner’s laboratories, an activity that will be possibly continued beyond the official end of the project, according to the availability of internal funding opportunities of the involved laboratories. The dissemination activities, in addition to the scientific publications, included several activities like the establishment of the project website (www.ulixes.unimi.it) the participation to national and international conferences, the establishment of different databases, the preparation of leaflet, brochure, newsletters, and the organization of a ULIXES final conference (MedRem-2014). MedRem-2014, the final ULIXES conference held in Hammamet, Tunisia on 16-18 January 2014, put together scientists from 26 countries, including the participation of companies and stakeholders operating in the sector of energy and oil and gas extraction and management. The feedback obtained during the conference together with the stakeholder database created under the ULIXES project indicate a high potential for the implementation of the ULIXES results as well as leave the concrete possibility that the project may have potential future influence on the oil management policy in the countries of the Southern Mediterranean, towards more environmental-friendly strategies. Overall, the ULIXES consortium positively evaluated the implementation of the obtained results, which will have an impact primarily at the research level but also on the social, economic and cultural activities by promoting a more conscious behavior in respect of ecosystem protection.
The research activities carried out within the WP7, on the other hand, shed a light on new opportunities to use bacteria as i) nano-engineer to produce nanoparticles that can be applied in the remediation of polluted matrices and ii) key player of dehalogenating processes. These results gave a contribute to the set-up of innovative processes, and can be implemented during the management of polluted sediment and water of both marine and freshwater origin, as reported for example in the following publications of the ULIXES project:
• De Corte S., Hennebel T., De Gusseme B., Verstraete W., Boon N. (2011) Bio-palladium: from metal recovery to catalytic applications. Microbial Biotechnology 5:5-17.
• De Corte S., Hennebel T., Fitts J.P. Sabbe T., Bliznuk V., Verschuere S., van der Lelie D., Verstraete W., Boon, N. (2011). Biosupported bimetallic Pd/Au nanocatalysts for dechlorination of environmental contaminants. Environmental Science & Technology 45:8506-8513.
• De Corte S., Sabbe T., Hennebel T., Vanhaecke L., De Gusseme B., Verstraete W., Boon N. (2012) Doping of biogenic Pd catalysts with Au enables dechlorination of diclofenac at environmental conditions. Water Research 46:2718-2726.
• Hennebel T., De Corte S., Verstraete W., Boon N. (2012) Microbial production and environmental applications of Pd nanoparticles for treatment of halogenated compounds. Current Opinion in Biotechnology 23:1–7.
• Hosseinkhani B, Hennebel T and Boon N. (2014) Biogenic hydrogen production in marine environments for hydrogen driven remediation strategies. New biotechnology (Under revision).
• Hosseinkhani B., Hennebel T., Van Nevel S., Verschuere S., Yakimov M.M. Cappello S., Blaghen M., Boon N. (2014) Biogenic nanopalladium based remediation of chlorinated hydrocarbons in marine environments. Environmental Science &Technology 48:550-557.
• Zanaroli G., Balloi A., Negroni A., Borruso L., Daffonchio D., Fava F. (2012) A Chloroflexi bacterium dechlorinates polychlorinated biphenyls in marine sediments under in situ-like biogeochemical conditions. Journal of Hazardous Materials 209-210:449-457.
• Zanaroli G., Negroni A., Vignola M., Shu H-Y., Fava F. (2012) Enhancement of microbial reductive dechlorination of polychlorinated biphenyls (PCBs) in a marine sediment by nanoscalezerovalent iron (NZVI) particles, Journal of Chemical Technology and Biotechnology 87:1246-1253.
Additional publications acknowledging ULIXES show that the discovered microbial resources (e.g. bacteria, biosurfactant, enzyme) can be implemented in tailored bioremediation techniques for either the in situ or ex situ treatment marine waters and sediments polluted with petroleum hydrocarbons, halogenated compounds and heavy metals, providing input to the discussion about the biostimulation/bioaugmentation effectiveness. These studies, which have been also complemented by synthetic ecology studies to address general principles of the system functionality under invasion/augmentation processes, provide novel perspectives for the realization of field bioremediation treatments. See among others the following publications of the ULIXES project:
• Alcaide M., Tornés J., Stogios P.J. Xu X., Gertler C., Di Leo R., Bargiela R., Lafraya A., Guazzaroni M.E. López-Cortés N., Chernikova T.N. Golyshina O.V. Nechitaylo T.Y. Plumeier I., Pieper D.H. Yakimov M.M. Savchenko A., Golyshin P.N. Ferrer M. (2013) Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/β hydrolase family. Biochemical Journal 454(1):157-66. doi: 10.1042/BJ20130552.
• De Roy K., Marzorati M., Negroni A., Thas O., Balloi A., Fava F., Verstraete W., Daffonchio D., Boon N. (2013) Environmental conditions and community evenness determine the outcome of biological invasion. Nature Communication, 4:1383.
• Cappello S., Genovese M., Della Torre C., Crisari A., Hassanshahian M., Santisi S., Calogero R., Yakimov M.M. (2012) Effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics during assays of oil spill bioremediation: A microcosm study. Marine Pollution Bullettin 64:2820-2828.
• Cappello, S., Antonella C., Hassanshahian M., Genovese M., Santisi S., Yakimov, M.M. (2012) Effect of a bioemulsificant Exopolysaccharide (EPS2003) on abundance and vitality of marine bacteria. Water, Air, & Soil Pollution, 223:3903-3909.
• Amer R., El-Gendy N. Sh., Taha T., Farag S. and Abdel Fattah Y. Biodegardation of crude oil by using indigenous bacterial consortium isolated from contaminated area in Egyptian Mediterranean ecosystem. Environmental Science Process and Impact. (Under revision)
• Mahjoubi M., Jaouani A., Guesmi A., Ben Amor S., Jouini A., Cherif H., Najjari A., Boudabous A., Koubaa N ., Cherif A. (2013) Hydrocarbonoclastic bacteria isolated from petroleum contaminated sites in Tunisia: isolation, identification and characterization of the biotechnological potential. New Biotechnology, 30:723-733.
• Nikolopoulou M., Eickenbusch P., Pasadakis N., Venieri D., Kalogerakis N. (2013) Microcosm evaluation of autochthonous bioaugmentation to combat marine oil spill. New Biotechnology, 30:734-742.
• Nikolopoulou, M., Pasadakis N., Kalogerakis N. (2013) Evaluation of autochthonous bioaugmentation and biostimulation during microcosm-simulated oil spills, Mar. Pollut. Bull. 72:165–173.
• Gertler C., Nather D.J. Cappello S., Gerdts G., Quilliam R.S. Yakimov M.M. Golyshin P.N. (2012) Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study. FEMS Microbiol Ecol 81:520-536.
In the context of treatment application for field bioremediation intervention, a patent authorization has been requested to the Hellenic Industrial Property Organisation and, at the moment, it is under evaluation.
Genomic studies, as those reported below, were also performed during the ULIXES project, providing novel information on the functional capabilities of the bacteria involved in oil hydrocarbons remediation.
• Guazzaroni M.E. Herbst F.A. Lores I., Tamames J., Peláez A.I. López-Cortés N., Alcaide M., Del Pozo M.V. Vieites J.M. von Bergen M., Gallego J.L. Bargiela R., López-López A., Pieper D.H. Rosselló-Móra R., Sánchez J., Seifert J., Ferrer M. (2012) Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation. The ISME Journal 7:122-136.
• Kube M., Chernikova T.N. Al-Ramahi Y., Beloqui A., Lopez-Cortez N., Guazzaroni M.E. Heipieper H.J. Klages S., Kotsyurbenko O.R. Langer I., Nechitaylo T.Y. Lünsdorf H., Fernández M., Juárez S., Ciordia S., Singer A., Kagan O., Egorova O., Alain Petit P., StogiosP., Kim Y., Tchigvintsev A., Flick R., Denaro R., Genovese M., Albar J.P. Reva O.N. Martínez-Gomariz M., Tran H., Ferrer M., Savchenko A., Yakunin A.F. Yakimov M.M. Golyshina O.V. Reinhardt R., Golyshin P.N. (2013) Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispiraantarctica. Nature Communications. 4:2156.
Overall, the consortium positively evaluated the implementation of the results of the project. Moreover, several studies will be finalized after the official end of the project leading to additional results that will provide a high quality contribute to the scientific research on the bioremediation field. These studies will be submitted as publication, which complete list (at the end of the project) is included in the Deliverable 9.7.
List of Websites:
www.ulixes.unimi.it
The project ULIXES aimed to unravel, categorize, catalogue, exploit and manage the microbial diversity available in the Mediterranean Sea for addressing bioremediation of polluted marine sites. The idea behind ULIXES is that the multitude of diverse environmental niches of the Mediterranean Sea contains a vast array of microorganisms and their components (e.g. catabolic enzymes) or products (e.g. biosurfactant) that can be exploited in pollutant- and site-tailored bioremediation approaches. Within the three-years EU project, the ULIXES consortium conducted different lines of research, aiming to study the microbial communities dwelling polluted and pristine sites across the Mediterranean Sea and the Gulf of Aqaba (Red Sea), taking advantage of both cultivation dependent and independent approaches. All the partners involved in Work Package (WP) 2 performed sampling campaigns, both on coastal and open sea sites, in order to collect sediment and seawater samples for the research purposes detailed in the project DoW. Starting from these samples, a large collection of microbes, including bacteria and fungi, was established. Such collection can be divided in four collections, depending on the type of activities and pollutants (hydrocarbons, halogenated compounds, heavy metals) targeted by the enriched mixed cultures and isolated bacteria. The collections have been screened for different features, allowing the identification of promising microbial strains in term, for example, of biofilm formation, biosurfactant production, biopalladium precipitation and metal mobilisation. A subset of microbes in the ULIXES collections were used within the WP7 to setup innovative processes and products for bioremediation purposes, such as the reductive dechlorination of polychlorinated compounds (PCBs) by using biopalladium particles produced and activated by marine bacteria. Moreover, a selection of the microbial consortia and the bacterial isolates obtained in the frame of WP4 activities were subsequently tested by partners involved in WP8 for their potential application in ex situ mesocosms and/or in situ treatments of polluted sediments.
Additional lines of investigation, tightly related to those previously described, were the study of overall prokaryotic community inhabiting polluted and pristine sites located across the Mediterranean Sea and the Gulf of Aqaba. These studies involved both the use of molecular ecology techniques and the OMICs approach, and provided data on the prokaryotic communities’ structure, diversity and abundance, which concurred to establish the widest OMIC-based datasets obtained from the Mediterranean Sea, with special focus on the Southern Mediterranean polluted sites, and a map of the basin biodiversity. The overall data showed peculiar microbial communities in the Southern Mediterranean Sea sites adapted to the particular environmental conditions of the Southern latitudes of the basin. The obtained data represent a novel contribute to the knowledge of microbial diversity in polluted sites. Indeed, the Southern Mediterranean coast, has been widely overlooked by the OMICs-based research on oil-degrading microbial networks, though it represent one of the major oil-producing areas on our planet. The data contribute to fill the gap of knowledge on the microbial diversity in oil polluted sites and features potentially exploitable novel resources for bioremediation purposes, one of the main objectives of the ULIXES project. Besides the RTD activities the ULIXES consortium established an intense dissemination plan, which was realized accordingly. A website was established within the first six months of the project, and several databases, newsletters, leaflet and brochure were prepared and disseminated both through digital and printed copies. The results of the performed research resulted in the publication, at the time of preparation of the present report, of 21 scientific papers in different international peer-reviewed journals, including high-impact factors and open-access journals. Several other manuscripts are actually in preparation and will be published in the next months under the auspices of the ULIXES project.
Project Context and Objectives:
The Mediterranean basin is one of the areas in the world where, since millennia, a large human population lives and intense economic activities occur. Natural resources of the basin have been long exploited resulting in profound impacts on the environment. The recent extensive development of aquaculture, the exploitation of resources from the subsurface including mineral, oil and gas extraction, the intense maritime traffic and the presence of many industrial poles on both the north and south sides of the basin, have resulted in a rapid increase of the number of polluted coastal and marine areas that should require extensive and careful strategies for remediation and clean up.
The Mediterranean Sea has a high exposure risk to many chemicals including oil hydrocarbons, halogenated compounds and heavy metals, with a pollution threat enhanced by the semi-enclosed nature of the basin. The size of the potential pollution load in the Mediterranean Sea can be appreciated by considering the figures related to the annual traffic and load of crude oil and petroleum products. The European coastal zone contains approximately 7% of the world coastline yet houses around 15% of the world population and generates nearly 16% of the total world trade. More than a half of all European coastal zones are located in semi-enclosed seas, so that pollutants are trapped (e.g. typical flushing time in Mediterranean is of the range of 70-90 years) and tend to accumulate. There are already more than 40 oil-related sites (e.g. pipeline terminals, refineries, offshore platforms) distributed along the Mediterranean coastal zone, from and to which estimates of 55,000,000 and 15,000,000 metric tons, respectively of crude oil and petroleum products, are annually loaded, unloaded and transported by oil tankers. Despite the relative little size of the basin, accounting for less than 1% of the earth’s marine surface, it has been estimated that more than 20% of the global maritime traffic yearly passes through it.
Many sites already experienced pollution events and several environmental disasters occurred in the Mediterranean Sea, comprising oil spills from pipelines and oil tankers shipwrecks. In 2000, pollution caused only by the illegal discharge of oil-contaminated bilge and ballast waters was estimated to reach 600,000 tones for the Mediterranean Sea alone, i.e. the equivalent of the pollution caused by the 1999 Erika shipwreck every week. The sinking of the Prestige off the coast of Spain in November 2002 and of the Erika off the coast of France in 1999, which were transporting 77,000 and over 10,000 tones of heavy fuels respectively, caused serious localized damage to the marine environment and to entire coastal regions and marine-related industries. The pollution of a relatively restricted basin like the Mediterranean Sea could lead to big socio-economic problems that could strongly affect the life style of the inhabitants. Besides the effects on biodiversity and wild life, marine oil spill disasters profoundly impact the inhabitants of the affected areas due to reductions of tourism and fishing activities and the consequent loss of jobs and heritage.
In a so densely inhabited area like the Mediterranean basin, the management of remediation efforts should be designed taking in consideration all the economic and political resources available in the basin, in a way that both the sides, the European one and the African and Middle-East south side, may benefit of a more careful and safe global management. The Mediterranean basin should need a ‘global’ remediation intervention that would involve simultaneously all the countries facing and surrounding the basin. Such a global approach would guarantee the best management of industrial development, minimizing the impact. A careful analysis of the available literature data showed that polluted sites on the Southern side of the Mediterranean Sea have been until now overlooked by studies based on state of the art techniques and methods. Besides access to North side sites, the ULIXES project had the necessary facilities and access to several major polluted sites on the Southern Mediterranean coasts of Morocco, Tunisia, and Egypt and to some polluted sites in the Aqaba Gulf (Red Sea, Jordan), a reference site to envision the perspective of the ongoing global warming on the Mediterranean Sea. The study of the microbial resource with potential for bioremediation from the selected North and South sites is of great interest to understand the overall bioremediation potential in the basin.
Bioremediation represents an environmental friendly approach for the clean-up of polluted environments including marine sediments and water. Applications of bioremediation technologies in marine environments have been proposed in a large range of cases like, for example, oil polluted marine water and beaches due to accidental oil spills from ships or pipelines, or for the removal of halogenated compounds from polluted sediments. The proposed clean up approaches include both in situ or ex situ technologies and can take large advantages from biostimulation, defined as “the act of adding materials to contaminated environments to cause an acceleration of the natural biodegradation processes”. Bioremediation practices can be differentiated in “biostimulation” and “bioaugmentation”, the latter being the addition of selected microbial species capable of degrading the pollutants which often provide interesting results when based on the addition of microbes obtained and enriched from the same site to be remediated. The low level of nutrients typical of the oligotrophic waters of the Mediterranean Sea and the low availability of hydrocarbon pollutants, may strongly limit the successful performance of biodegradation. Several studies showed that during the biostimulation of contaminated sediments the removal of the pollutants can be boosted by adding different electron donors/acceptors to stimulate the autochthonous microbial communities. Besides nutrients, biodegradation processes by naturally occurring microorganisms are enhanced by the in situ production or the addition of biosurfactants like rhamnolipids and other microbial surfactants and/or lipophilic fertilizers of natural origin (uric acid and lecithin). For instance in the case of polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons, the addition of the methylated cyclodextrins, microbial and phytogenic surfactants, resulted in marked improvement of pollutant bioavailability and of the final biodegradation in actual site solid matrices even in cases of aged contaminations. Biostimulation with nutrients and biosurfactants enables naturally occurring microbes to adapt better and faster to the polluted environment resulting in shorter lag phase and faster biodegradation of crude oil and hydrophobic (chlorinated) organo-aromatic pollutants, thus making it an effective ‘tool’ for combating oil spills. When biostimulation is not efficient in the removal of the pollutants, bioaugmentation procedures can be adopted by introducing in the polluted sediments or water specific consortia of biodegrading microbes, previously enriched under laboratory conditions. Effective sustainable strategies to clean up marine polluted ecosystems hence require the definition of the complex relationships among biodegradation rates, the different categories of contaminant molecules, and the microbial community composition and dynamics.
In the above context, the ULIXES project aimed at the exploration and exploitation for bioremediation purposes of the microbial natural resources (biodegrading/detoxifying microbes and their enzymes and metabolites) present in a series of selected polluted sites from the North and the South sides of the Mediterranean basin. Eight EU and four Southern Mediterranean laboratories were cooperating with a scientific approach starting with the isolation of pollutant degrading/detoxifying microbes and microbial consortia and their metabolites and the use of cultivation independent methods to investigate the phylogenetic and functional diversity of the overall microbiome inhabiting polluted seawater and sediments. Special attention has been posed to pollutions determined by oil hydrocarbons, PCBs and heavy metals, three pollutant categories included in the priority list of high impact pollutants in the EU and most of the countries in the world. For a comprehensive characterization of the polluted sites “meta-omics” techniques were also applied to enhance knowledge about the metabolic pathways involved in the remediation processes. Following such efforts in identifying suitable microbial resources, ULIXES aimed at developing and implementing novel processes at reactor scale as well as in pilot ex situ and in situ field treatments at some selected sites.
The combined cultivation dependent and independent approaches applied in ULIXES provided insights on the taxonomic and metabolic diversity of biodegrading microbial communities inhabiting the selected polluted sites, including seashore sands, coastal lagoons and oil refinery sediments. The characterization of the microbial diversity in those sites and the setup of novel clean up strategies and their field tests represent the major contributes of the ULIXES project to fill a knowledge gap for supporting the ecological sustainability of the Mediterranean Sea.
Such objectives were complemented by the implementation of a strategy of dissemination of the scientific results targeting the academia as well as a series of public and private stakeholders operation in the sector of oil management.
According to all the considerations at the basis of the ULIXES concept and the general objectives of the project, a list of specific objectives can be resumed as follows:
1. To establish a detailed description of the microbial diversity of culturable and unculturable microorganisms associated to the ULIXES polluted sites in the Mediterranean Sea basin. Such investigation included a wide series of polluted sites (17 sites plus specific areas in some of the sites).
2. To define the proof of concept for defining the first map of the microbial diversity associated to polluted sites in the Mediterranean Sea.
3. To establish a collection of microbial isolates and mixed microbial cultures with catabolic/detoxifying capabilities of aliphatic and aromatic hydrocarbons, halogenated solvents and PCBs and heavy metals.
4. To establish a collection of microbial isolates capable of biopalladium nanoparticles precipitation and activation by new hydrogen donors.
5. To establish a collection of microbial isolates capable of producing robust and versatile hydroxylases and/or biosurfactants and/or capable of biofilm formation.
6. To identify new functions by employing high-throughput methods, including microarray, metagenomics and metaproteomic methods, and establishing a set of genes coding for biodegradation enzymes from two major families, namely oxidoreductases (EC1) which catalyse oxidoreduction reactions and hydrolases (EC 3) which catalyses a number of hydrolytic activities with potential use in biodegradation. The major groups of activities considered were mono- and dioxygenases, hydroxylases, hydratases and dehydratases, haloacid and haloalkane dehalogenases and meta-cleavage hydrolases, to cite the most important.
7. To develop processes for the reductive dehalogenation of chlorinated compounds. Model compounds were the chlorinated aliphatic solvents and PCBs. The choice of these two compound classes was made on the base of their repartition in marine systems: chlorinated solvents in water and PCBs in sediments.
8. To develop processes of PCB reductive dehalogenation exploiting the catalitic propoerties of bio-palladium and other nanoparticles in marine sediments.
9. To define a fate model in polluted sediments for metal and metalloids in function of biodegradation yield.
10. To simulate bioremediation models from experimental microcosms, mesocosms and pilot ex situ and in situ field studies. Such activity was conducted in different sites from the North and South sides of the Mediterranean Sea as well as in the Gulf of Aqaba in the Red Sea.
A further objective of ULIXES was to establish and implement a strategy of dissemination of the realized research activities and scientific results. Such dissemination was based on a series of actions and tools including the establishment of a database of stakeholders and SMEs in the field of oil management, a web site, leaflet and communication to the scientific community through conferences and scientific papers. The dissemination activity was culminating with the organization of the ULIXES International Conference at the end of the project, “MedRem-2014, Microbial Resource Management For Polluted Marine Environments And Bioremediation, organized in Hammamet, Tunisia on January 2014.
Project Results:
WP2. Sampling and chacterization of the Mediterranean Sea sites.
WP2 activities consisted of the sampling and geochemical characterization of the different polluted area (Figure 1) located both in the Mediterranean basin and in the Gulf of Aqaba (Red Sea, Jordan).
According to the aims of ULIXES and the activities foreseen in WP2, seventeen polluted sites have been considered as sampling targets of ULIXES.
These include: i) a series of coastal sites like industrial poles in marine lagoons, polluted sites in coastal lakes/lagoons in the south coast of the Mediterranean Sea (Tunisia and Morocco), industrial harbors and polluted seashores. ii) A series of open sea sites, both shallow and deep. iii) A series of deep-sea natural hydrocarbon-seeping sites. Twelve coastal sites have been considered. Seven sites are located in the coastline of European countries (Greece, two sites; Italy, four sites). Four coastal sites are located in the South coast of the Mediterranean Sea (Egypt, one site; Tunisia, two sites; Morocco, one site). One site is located in the Red Sea (Jordan) and it has been chosen as a model for the possible future transition of the Mediterranean Sea toward a sub-tropical environmental condition determined by the ongoing global warming.
Three cruises, during 2011-2012, were performed to sample both epi- and bathypelagic waters at transect of major tanker routes and deep sea natural hydrocarbon seeping sites. The sampling activities performed on board of the oceanographic vessels included the collection of polluted sediments, water column and water-brine interfaces of the Deep Hypersaline Anoxic Basins (DHABs) in the Eastern Mediterranean Sea.
Excluding site 6 (the Milazzo harbour, Tyrrhenian Sea, Italy), according to what proposed in the Project DoW, all of the sites indicated (Figure 1) were sampled and characterized. Furthermore, additional samples were collected from three additional sites (harbour of Messina, Italy; Oued Hamdoun-Monastir, Tunisia and harbour of Gela, Italy) and they were analyzed.
A total of 106 sediments and seawater samples were collected (102 during the first 18 months, 4 during the last 18 months) by different Partners (Table 1 and 2). At the same time 4 oceanographic cruises and 1 direct sampling in open sea were organized for the collection of samples in both the Eastern and Western basins of the Mediterranean Sea (Table 3 and 4).
Task 2.1: Sampling organization and geochemical characterization of the coastal target sites.
Sampling activities in the El-Max district, Egypt (site 1)
The sampling area was divided into highly polluted and moderately polluted sites at El Max district bay at Alexandria (Egypt). MUCSAT partner performed all the sampling campaigns realized in this area. MUCSAT performed chemical characterization of the sediment and water samples, which were also sent to other ULIXES partners (UMIL, CSISC, TUC) for analyses complementary to the cultivation approach adopted at MUCSAT.
Sampling activity on the Elefsina (site 2) and Zakynthos area (site 3), Greece
Different seawater samples were collected on 06/04/2011 and 31/08/2011 respectively from Elefsina Gulf and the wetlands of Kery (Zakynthos) by TUC. The geochemical and microbiological characteristics of the samples collected at the Elefsina Gulf, a seashore close to oil refinery (Attika, Aegean Sea), have been carried out. On 14th January 2013, additional sampling efforts were devoted by TUC to collect polluted sediment sample (c.a. 30 L) from a coast adjacent to an oil refinery unit in the Elefsina Gulf. Such sediment was used to set up a landfarming experiments within the WP8 activities.
Sampling activity on the Venice Lagoon (site 4), Italy
Seven samples of surface sediments were collected by UNIBO with a Van Veen Grab along the Brentella canal in the first industrial area of Porto Marghera (Venice lagoon, Italy) for investigating the structure of the bacterial communities and establishing PCB dechlorinating enrichment cultures.
Sampling activity on the Ancona harbour (site 5), Italy
Samples collection from the Ancona harbour coastal site was performed on 12/04/2011 onboard of the R/V “Calipso” by ETS, during a sampling campaign named ULX_AN1. A second sampling campaign was realized during October 2012 aiming to collect additional samples. Sediment and seawater samples were collected in pollutant-impacted and control stations, located respectively inside and outside the harbour area.
Sampling activities in the area of Priolo Gargallo, Italy (site 7)
Sediment and tar samples were collected by IAMC in the harbour of Priolo Gargallo (Siracusa, Italy. In particular, a total of 10 cores sediments (1 meter long, 20 cm Ø) were collected at a depth of 6 meters from two stations (SY01 and SY03) located inside the harbour.
Sampling activities in the Messina harbour, Italy (additional site 1 selected by IAMC partner) Sediment samples were collected by IAMC in the harbour of Messina (“Mare Sicilia” station), a site located within a zone impacted by heavy ship traffic, both for touristic and industrial activities.
Sampling activity on the Bizerte Lagoon, Tunisia (site 8)
Sediments and seawater were collected from UTUN Partner from different sites located in the northern coast of Tunisia near the STIR (Oil Refinery Industry) company harbor at Bizerte region. The purpose of STIR is the refining of crude oil in order to satisfy the needs of the national market of oil products. The sampled sites present a marked pollution due to the release of the refinery wastewater containing residual hydrocarbons.
Sampling activity on the Sebkha of Soliman, Tunisia (site 9)
The Sebkha of Soliman is an organochlorinated compounds contaminated area located in south-western peninsula of Cap Bon (Tunisia). This site is highly exposed to different types of pollutants from agricultural and industrial activities located in the region. The contamination is easily detectable by eye both in the water and in the sediment samples. The sampling activities were performed by UTUN partner.
Sampling activity on Oued Hamdoun-Monastir, Tunisia (additional site 2 selected by partner UTUN)
The Oued Hamdoun-Monastir is an area polluted by organophosphorous molecules. A water sample has been collected from the site Oued Hamdoun-Monastir in order to isolate chlopyrifos ethyl degrading bacteria.
Sampling activities in the Gulf of Aqaba, Jordan (site 10)
The Aqaba Gulf was sampled by YU Partner in coordination with the Authorities of the oil terminal and the harbor of Aqaba (Jordan). Water and sediment samples were collected during three campaigns: May 2011, July 2011, and December 2011. During the first sampling campaign YU scientists collected samples of seawater and sediments from four sites in the Gulf of Aqaba-Jordan: Marine Science Station (MS), phosphate port (P), oil terminal (O) and fertilizers terminal (F). The samples collected by YU have been sent for additional analyses to other partners (e.g. CSIC, UMIL, TUC).
Sampling activities in the lagoon of Mar Chica, Morocco (site 11)
The Lagoon of Mar Chika lagoon is located on the northwest coast of Morocco in front of the Alboran Sea. The lagoon is isolated from the Mediterranean by a strip of fine sand and dunes, while it had a communication channel to the sea through the key of Boucana. Despite the biological, ecological and landscape importance of Mar Chika lagoon, it is exposed to continuous pollution by the town of Nador, and also by agriculture and industrial activities. Water and sediment samples have been collected from the lagoon by the Moroccan partner UH2C.
Sampling activities in the refinery of Gela, Italy (additional site 3 selected by IAMC partner)
Seawater and sediment samples were collected, in June 2013, by IAMC-CNR in another area located in the proximity to the refinery of Gela (Caltanissetta, Sicily, Italy). This additional area was chosen following an oil spill occurred from a pipeline of the “Topping1” installation of the ENI Refinery during July 2013. The spill released around 5,000 liters of heavy crude oil in the marine environment.
Task 2.2: Organization of the cruise for sampling open and deep Mediterranean Sea sites
Sampling activities in the Haven tanker shipwreck site, Harbour of Arenzano, Italy (site 12)
Tar samples were collected, by scuba of IAMC partner, around the harbor of Arenzano (Genova, Italy). In this area, on 11st April 1991, the Amoco Milford Haven tanker (a cargo load of 230.000 tons of crude oil) exploded during a routine operation and spilled over 45 million gallons of petroleum into the Mediterranean Sea. The samples have been used for the activities foreseen within WPs 3-4-5-6.
Ulixes_M11 Oceanographic cruise (R/V Meteor)
The Ulixes M11 oceanographic cruise has been carried out on the R/V METEOR, from 03th to 28th April 2011, interesting most of the basins located in the Mediterranean Sea. The UMIL partner was invited to participate to the M84/3 Meteor cruise by Dr. Toste Tanhua (Kiel University, Germany) and Dr. Dagmar Hainbucher (Hamburg University, Germany) in the frame of a previously established collaboration. During the sampling activities different water samples were collected and subsequently analyzed within the WPs 3-4.
Ulixes_U11-1 Oceanographic cruise (R/V Urania, September 2011)
The Ulixes U11-1 oceanographic cruise has been carried out on the R/V URANIA, from 09th to 23th September 2011, interesting different Deep Hypersaline Anoxic basins (DHABs) located in the Eastern Mediterranean Sea. During the U11-1 cruise, the UMIL team collected deep seawater, seawater-brine interface, brine and sediment samples from the Urania, and Tyro DHABs.
Ulixes_UEU Oceanographic cruise (R/V Urania, April 2012)
The Ulixes_UEU oceanographic cruise has been carried out on the April 2012, interesting different area of Central Western Mediterranean Sea. During the UEU cruise, the IAMC team collected different samples of surface and deep seawater from different station.
Ulixes_UBO Oceanographic cruise (R/V Urania, November 2011)
The Ulixes_BO oceanographic cruise has been carried out on the November 2011, interesting different area of Central Western Mediterranean Sea.
Besides the microbiological studies, all the samples have been characterized for a series of geochemical parameters according to their nature and the type of investigations ongoing. Overall, the sampling performed during ULIXES project allowed to establish an accurate mapping of chemical, physical and microbiological characteristic of different sediments and seawater present in the Mediterranean Sea basin.
WP3 Microbial diversity exploration in the Mediterranean Sea sites
The general objective of WP3 was the characterization of the phylogenetic and functional diversity of the microbial communities inhabiting the polluted sites studied within ULIXES by applying molecular biology-based cultivation independent approaches. The application of different molecular ecology techniques allowed dissecting the composition of the microbiome dwelling in the selected polluted sites. Direct counting methods based on fluorescence microscopy after staining with fluorescent dyes were used to evaluate the microbial abundance in natural and polluted ecosystems located across the Mediterranean basin.
Task 3.1 Determining a detailed description of site microbial diversity
An array of molecular techniques was used to study highly contaminated sites, offshore surface and deep seawater and on the metagenome extracted from pollutant-exposed microcosms aiming to depict the structure of the inhabiting microbiomes from a phylogenetic perspective. A database of microbial species present in the different investigated sites was established in the frame of Task 3.1. This database encompasses data on the i) community structure, ii) community diversity and iii) microbial abundance. The community structure dataset was mainly represented by the results obtained through the application of Automated Ribosomal Interspacer Sequence Analysis (ARISA) fingerprinting by UBANGOR, ETS and UMIL partners. ARISA fingerprinting was applied on the metagenomic DNA extracted from the sediments collected at the Ancona harbour, Priolo Gargallo bay and the Haven sites and from deep and surface seawater samples in different Mediterranean Sea sites. The ARISA results allowed identifying which are the environmental factors (in particular referring to the concentrations of different pollutants) contributing to drive the structure and diversity of the bacterial communities dwelling the sediment of the control and impacted stations, in particular for those in the Ancona harbour site. Through the ARISA fingerprinting, we also provided a comprehensive depiction of bacterioplankton community structure across the water column over a longitudinal transect covering the whole Mediterranean Sea. Our investigation demonstrated that the overall planktonic bacterial community inhabiting epipelagic seawater was spatially patterned in the Mediterranean Sea, exhibiting different populations in the Eastern and Western basins.
Furthermore, ARISA fingerprinting was applied on the enrichment cultures obtained from the samples collected from different sites located in particular at Elefsina Bay (Greece), El-Max district (Egypt) and the Gulf of Aqaba (Jordan) to estimate the shifts of the bacterial community composition and richness during incubation in the presence of pollutants. ARISA fingerprinting permitted to assess the evolution of the laboratory enriched microbial communities, considering also those microbes that cannot be isolated in pure culture. This approach provided a wider picture of the microbial resources that were selected under specific conditions and gave information about the whole community composition, contributing to setup the best cultivation condition for selecting the most promising biodegrading microbial consortia (WP4). A similar molecular study was conducted by applying Denaturing Gradient Gel Electrophoresis (DGGE) to assess the role of the site, the sampling, the pressure imposed by different pollutants and the cultivation strategy in the selection of HC degrading bacteria from the sediment of the Ancona harbour, evidencing that all these factors concur in shaping different bacterial communities.
A second part of the database was devoted to summarise the different bacterial species detected after sequencing PCR-DGGE bands, 16S rRNA clone libraries and 16S rRNA PCR-barcoded fragments following deep pyrosequencing. DGGE analysis allowed to describe the microbial diversity on the metagenome directly extracted from the environmental samples and it was performed both on seawater and sediment samples collected across the Mediterranean and the Red Sea. The contaminated sediments collected from the Priolo Gargallo bay, the Messina harbour, the Haven shipwreck area, and different stations located in the Gulf of Aqaba (Jordan) and El-Max district (Egypt) were tested by DGGE showing the prevalence of bacteria belonging to the class Proteobacteria whereas the control sediments collected at the Marine Science Station (Jordan) were mainly colonized by Bacilli. DGGE band sequences retrieved from the water samples collected along the oil-tanker routes showed the presence of Cyanobacteria in addition to the alpha-proteobacteria subclass, as expected for surface open seawater.
16S rRNA libraries of the 16S rRNA gene amplified from the cDNA obtained after retrotranscription from the total sediment RNA, were constructed from the polluted sediment and tar samples collected at the Haven shipwreck site. The phylogenetic affiliation of two hundreds 16S-cDNA clone inserts showed the presence of a rich bacterial community. Despite all the analysed Haven samples shared a similar community at higher phylogenetic rank level, a more detailed analysis of the microbiome structure revealed that some of the tar samples host peculiar groups of bacteria related to Cycloclasticus and Nitrospira genera. These bacterial genera are widespread in hydrocarbon polluted sediments and thus potentially exploitable for bioremediation purposes. In particular, bacteria related to genus Cycloclasticus are known as key microorganisms involved in the aerobic breakdown of polycyclic aromatic hydrocarbons (PAHs).
The microbial species diversity dataset includes also information on the microbial abundance obtained by DAPI staining on i) the collected surface and bottom seawater along the oil-tanker routes in the Mediterranean Sea and ii) the Ancona harbour sediment samples. Microbial abundance was evaluated by applying two diverse direct counting methods based on fluorescence microscopy, namely DAPI and Sybr Green staining. This approach allowed to determine the total cell count in deep and surface waters from different stations located in the Eastern and Western Mediterranean Sea and to assess the total microbial abundance and bacterial biovolume for the sediment samples collected from the Ancona harbour (Italy).
In addition to the investigation of taxonomic diversity of the target polluted sites, Task 3.1 aimed to study the functional diversity of biodegrading microbial communities present in polluted seawater and sediment samples through a cultivation-independent approach. Firstly, an accurate bibliographic research was performed to survey the available literature data looking for specific PCR primer sets and protocols that were modified, when necessary, through the PCR amplification on a subset of the environmental samples and bacterial isolates. In particular, we focused on alkane hydroxylases. These genes are divided into three different clusters: group I (alkB-I) codifies for enzyme that degrade short chain length alkanes (C6-C12), group II (alkM) codifies for enzyme that degrade medium chain length alkanes (C12-C18) and the group III (alkB-III) that codifies for enzyme involved in the degradation of long chain length alkanes (>C16). In addition to the alk gene, specific PCR assays were set up to amplify the ndo gene, which codifies for the naphthalene dioxygenase, an enzyme involved in the degradation of polycyclic aromatic hydrocarbon (PAHs). Additional catabolic genes investigated within the WP3 were those encoding the dissimilatory sulfite reductase (drsB) and the reductive dehalogenase (dca-rd). The catabolic dsrB gene encodes for a key enzyme involved in the anaerobic sulfate reduction metabolisms and is frequently used for investigating the diversity within the sulfate-reduction bacterial community. Seawater and marine sediments generally show high sulfate concentrations. The interest devoted in the ambit of the Task 3.1 to the dsrB analysis was related to the study of anaerobic dehalogenating microcosms (WP4) to enrich bacteria able to biodegrade organohalide compounds, like for example 1,2-dichloroethane (1,2-DCA). In fact, under the anaerobic condition used to study the reductive dechlorination of 1,2-dichloroethane (1,2-DCA), sulfate reduction represents a metabolism that may compete with reductive dechlorination for the electron donors available. Similarly, dca-rd (DCA- dechlorinating reductase) genes were PCR amplified from the metagenome extracted from the 1,2-DCA dechlorinating microcosms established from the sediment collected at the El-Max district (Egypt) to follow the evolution of the 1,2-DCA dechlorinating bacterial communities.
Task 3.2 Establishing a database and a map of the diversity associated to polluted sites
To design a map describing the most important microbial diversity information we applied pyrosequencing analysis on a set of samples, chosen after an initial wider screening involving a higher number of samples compared to those presented here. Overall, the samples included in the map have been chosen basing on the quality of the extracted metagenome and for their geographical distribution in the Mediterranean Sea. Most of them have been collected from strategic areas, widely interested from the production, transformation and transport of oil, hence potentially subjected to a higher risk of pollution. To create the biodiversity map we focus on the prokaryotic communities, analyzing both the bacterial and archaeal 16S rRNA gene using universal primer. Seven out of the eight sites analyzed by pyrosequencing are located in the Mediterranean coastline (Italy, Greece, Morocco, Tunisia, Egypt) while the eighth sample come from the Aqaba Gulf. For each sample, the rarefaction curves indicated a good species sampling coverage. Pyrosequencing results showed that each of the sediment hosted a peculiar bacterial community. Overall, the bacterial communities showed a higher diversity compared to the archaeal communities, which were characterized by higher evenness values, especially in the sites located in the Aqaba Gulf, the Haven shipwreak and the harbour of Messina. The depiction of archaeal communities showed the predominant role of methanogenic communities in the polluted sediments of the sites located along the Italian coastline. In the case of the Aqaba Gulf’s sample the pyrosequencing data on the archaeal community showed that about 88% of the retrieved sequences were unknown, likely being unreliable at the kingdom level. For this reason, we indicated as the most abundant group the genus Nitrosopumilus even if it represent only 10% of the total archaeal sequences. In the case of the bacterial communities, we could obtain the description of the taxonomic groups at the family level. A collaborative manuscript including both the pyrosequencing obtained at UMIL and the metagenomic data obtained by CSIC partner is under preparation at the moment. The whole dataset on the characterization of the bacterial communities in the different polluted sites studied will be included in that publication. The established microbial ecology map of the Mediterranean Sea provides a link between the geography of the Mediterranean Sea and the information on the taxonomic composition of the bacterial and archaeal communities elaborated during the project, showing that it is possible to trace the prokaryotic diversity in the Mediterranean basin.
An additional activity conducted within Task 3.2 was the establishment of databases of protein and nucleotide sequences of different catabolic genes involved in the biodegradation of hydrocarbon compounds, aiming to develop a functional gene array termed BiodegChip. The study was realized at UTUN to develop a DNA microarray focused on the study of catabolic genes of aromatic BTEX pollutants in complex microbial communities. BTEX is an acronym that indicates unsubstituted or alkyl-substituted monoaromatic hydrocarbons including benzene, toluene, ethylbenzene, and o-, m-, and p-xylene. A total of 3558 protein sequences of validly described members of 20 different catabolic protein families were collected from reference pathways. These protein sequences constitute the protein sequence database. The majority of the different catabolic protein families considered in the present study contains a large number of protein sequences, which have to be used independently as seeds for search using BLASTP against 200 non redundant protein and environmental sequence databases (GeneBank, RefSeq, EMBL, DDBJ and PDB). For this reason, for all the protein families containing > 50 protein sequences, a phylogenetic analysis is recommended using ClustalX and MUSCLE conducted in MEGA5. The evolutionary histories of proteins were inferred using the neighbour-joining method and the p-distance model. In the second step, all representative protein sequences from the divergent branches were used to perform TBLASTN searches. In order to design probes for the BiodegChip array, we thereby built a databases of corresponding DNA sequences of the created catabolic protein database, which is used for the physical construction of the microchip detection system for biodegradation genes. All the sequences that constitute the database for the BiodegChip have been included in the Deliverable 3.3.
WP4. Establishment of collections of microbial isolates and metabolites
Different enrichment and culturing techniques were used for establishing different large collections of microbial isolates having different features for potential application in bioremediation. Isolates were obtained using both non selective rich media and mineral media supplemented with different pollutant mixtures as unique carbon source. Sub-collections were created basing on the screening of isolates for specific activities.
Pollutant degrading bacteria were isolated by the different ULIXES partners from several contaminated sites in the Med Sea and the Gulf of Aqaba:
MUCSAT and UMIL isolated from the seashore of El Max district (site 1);
TUC established microbial cultures from the Elefsina Gulf seashore close to oil refinery (site 2) and from the wetlands of Keri in Zakynthos (site 3);
UNIBO established mixed PCB dechlorinating anaerobic cultures from the Venice lagoon (site 4);
UMIL and ETS established microbial cultures from the Ancona harbour (site 5);
IAMC and UMIL cultured from Milazzo harbour (site 6), Augusta bay (site 7) and several surface open sea sites located across the oil tanker routes in the Mediterranean basin (site 17);
UTUN isolated microorganisms from the Bizerte lagoon (site 8) and the Sebkha of Soliman (site 9) from the Oued Hamdoun site close to Monastir and from SidiThabet in Tunisia;
YU and UMIL analyzed samples from Aqaba Gulf polluted seashores (site 10);
UH2C established a collection of isolates from the lagoon of Mar Chica (site 11);
IAMC and UMIL analyzed samples from natural deep-sea hydrocarbon seepage sites (Amsterdam, Kula, Napoli and Olimpi mud volcanoes and the hypersaline anoxic lakes Tyro and Urania, sites 13-16), and from the tanker Haven sunk offshore Genoa (site 12).
The ULIXES isolates database, available to the public at the URL http://www.lab-biotech.com/data/isolates.php includes several collections details.
Four tasks related to the construction of specific sub-collections were planned in WP4 including: (i) collection 1, hydrocarbon degrading microbes (including 3 sub-collections for biofilm and EPS formation, biosurfactant production, and high molecular weight hydrocarbon degradation); (ii) collection 2, halogenated compounds degrading microbes; (iii) collection 3, biopalladium nanoparticles precipitating prokaryotes; and (iv) collection 4, metal biostabilizing prokaryotes.
Task 4.1. Collection 1: Hydrocarbon degrading microbes
To isolate and characterize hydrocarbon (HC) degrading microbes, isolation procedures were mainly based on the use of minimal media supplemented with the targeted pollutant(s). UMIL established a bacteria collection of 235 isolates by enrichment culture using diesel and crude oil as unique carbon sources. Alcanivorax and Marinobacter were shown to be predominant representing respectively 67% and 23% of the whole collection. A sub-collection of 179 Alcanivorax isolates was further characterized by DNA fingerprinting using ITS and BOX-PCR. The genotyping study showed that all the detected genotypes were retrieved exclusively from one site, hence demonstrating the occurrence of a geographic divergence at the strain level. The used approach confirmed the huge biodiversity hosted in the Mediterranean Sea in terms of microbial resources to be exploited for oil remediation. From the contaminated sites 8 and 9, UTUN isolated a collection of 185 hydrocarbonoclatic bacteria using BHMS and MMC media supplemented with crude oil, pyrene, phenanthrene or naphthalene as the sole carbon source. Beside molecular identification (16S rRNA sequencing and ITS-PCR), the collection was characterized in term of biotechnological potential (i.e. biosurfactant production, emulsification activity, HC degradation), which allowed the selection of 16 “efficient” isolates. These selected bacteria were used to setup designed consortia in microcosm experiments and they showed interesting bioremediation potential. Beside several isolates from UMIL and UTUN, TUC constructed several reconstituted and natural consortia and established a collection of biosurfactant producing strains (sub-collection 1B), on which they evaluated biofilm formation, and EPS production (sub-collection 1A). To investigate the latter feature, several enriched and designed consortia were used in different conditions in presence of diverse carbon sources. The results showed that HC biodegradation efficiency, in the presence or absence of biofilm, varied according to the specific used consortium/strain. Within sub-collection 1B, biosurfactant production, characterization and purification were investigated in pure strains and mixed cultures, on which 16S rRNA pyrosequencing community screening was realized. The results showed that biosurfactant production remained constant in low concentration over time and that it was independent from the total culture biomass. Partner YU established a collection of 75 hydrocarbonoclastic isolates from which they selected the most efficient to be applied in WP8 in bioremediation experiments to test the clean-up potential of oil polluted water and sediment samples through microcosm experiments and by in situ-like processes through on-site mesocosms at oil contaminated sites in the Gulf of Aqaba. From the Mar Chica lagoon, shown to be contaminated by heavy metals (site 11), UH2C performed isolation trials, showing that several strains were able to grow in the presence of heavy metals, PAH and chlorinated compounds and, in particular, three isolates were selected for the degradation of anthracene. Finally, sub-collection 1C established by IAMC, includes bacteria selected on sterile mineral medium able to degrade high molecular weight hydrocarbons, such as squalene, pyrene, dibenzothiophene. A total of 40 isolates were obtained from polluted sediments and seawater, tar sample and mesocosms samples, and were assigned mainly to Gammaproteobacteria (Alcanivorax, Cycloclasticus, Marinobacter, Neptunomonas). These strains are of high interest in the frame of the ULIXES project and could be exploited for future bioremediation purposes.
Task 4.2. Collection 2: Halogenated compounds degrading microorganisms
To establish this collection, research efforts were realized mainly by UNIBO, UMIL and UTUN. Samples from contaminated sediments of the Venice lagoon (UNIBO) have been used to develop anaerobic slurry primary microcosms for the enrichment of anaerobic PCB-dechlorinating cultures on which DGGE analysis were performed to follow the evolution of the bacterial community diversity during the dechlorination process. The results of the enrichments indicated that two Chloroflexi phylotypes represent two dehalorespirers having different PCB dechlorination specificities. Using q-PCR, UNIBO researchers showed also that the growth of both phylotypes was associated to PCB dechlorination and that two strains identified as Dehalobium chlorocoercia and an uncultured Chloroflexi sp., were the responsible for dechlorination activities in the different sediments. UMIL focused on the selection of 1,2-dichloroethane (1,2-DCA) degrading cultures by establishing anaerobic microcosms. Molecular analysis performed on the metagenome extracted from the setup microcosms revealed the selection of peculiar bacterial communities from each sediment and confirmed that the enrichment of such peculiar bacterial community was driven by the sediment type used as inocula. Partner UTUN selected dimethoate-degrading microbes from polluted environmental samples, leading to the isolation of 35 strains that were assigned to three phylogenetic groups (Firmicutes, Bacteroidetes and Proteobacteria) and represented potential candidate for dimethoate bioremediation processes.
Task 4.3. Collection 3: Biopalladium nanoparticles precipitating prokaryotes
Thirty facultative anaerobic marine strains and sixteen aerobic strains have been isolated by partner UGENT from contaminated marine sediments collected from different sites (Priolo Gargallo bay and Mar Chica Lagoon). Eight strains showed the ability of concurrent biohydrogen production and Pd precipitation. X-ray diffraction (XRD) and thin section transmission electron microscopy (TEM) analysis indicated the presence of metallic Pd nanoparticles of various sizes (5-20 nm) formed either in the cytoplasm, in the periplasmic space, or extracellularly. Subsequently, the isolates were screened for their ability to generate bio-hydrogen (to activate the Bio-Pd) and to produce bio-surfactants to increase the solubility of pollutants. Promising results were obtained, indicating the selection of microbial resources potentially exploitable for interesting bioremediation applications.
Task 4.4. Collection 4: Metal biostabilizing prokaryotes
From the sediments collected from the Ancona harbour, partner ETS established a collection of metal biostabilizing microbes. For this, five experimental sets, with the addition of organic substrates, were studied in comparison with non-amended controls. The isolation of seventy-two bacteria was performed independently from sediments collected from each of the different microcosm setup. The time course incubations resulted in a significant reduction of total hydrocarbon concentrations in all the experimental systems, coupled with significant changes of metal speciation in sediments. In particular, the fraction of Cu, Pb, Ni and Cr extractable with standard techniques was reduced (on average 13% for Cu, 11% for Pb and 3% for Cr) indicating the potential of these strains to positively influence the mobilization of the investigated heavy metals.
Overall the mixed cultures and the bacteria isolated in the ambit of WP4 constitute a promising resource that was exploited in the ambit of the more applicative WPs of the ULIXES project, namely WP7 and WP8.
WP5. Metabolome analysis of the microbiome associated to selected polluted sites
During the ULIXES project, we completed the following activities within the WP5, which produce a number of outcomes:
• An in silico array was produced and amended in the ULIXES project. The array, which contains a set of 5,315 gene sequences encoding enzymes participating in biodegradation steps, allows by using a simple BLASTP search (score > 45 and E-value < 10e-3) inferring, identifying and quantifying the types and diversity of such genes from any kind of sequence data.
• A total of approximately 310 Mbp high quality sequences have been obtained, by 454 and Illumina Hiseq 2000 sequencing, either from raw or microcosm samples from sediment samples from four major oil refinery polluted Mediterranean Sea sites on the coastlines of Morocco (lagoon of Mar Chica), Tunisia (Bizerte lagoon), Egypt (El-Max site), Greece (Eleysina bay), and Italy (Gulf of Genoa, strait of Messina and Syracuse, and Ancona port) as well as on the coastline of the Red Sea (Gulf of Aqaba along the Jordanian coast). The sequences were obtained from the original samples from the sites as well as from microcosms enriched on specific compounds. All the obtained sequences have been deposited in public databases with the following accession numbers: IDs PRJNA222659, PRJNA222657, PRJNA222660, PRJNA222661, AZIB00000000, AZIC00000000, AZID00000000, AZIE00000000, AZIF00000000, PRJNA222667, PRJNA222664, PRJNA222665, PRJNA222666, PRJNA222663, AZIG00000000, AZIH00000000, AZII00000000, AZIJ00000000, and AZIK00000000. According to these figures, the ULIXES project contributed to provide, to the best of our knowledge, the biggest set of metagenomic sequences from the Mediterranean and the Aqaba Gulf in the Red Sea deposited in databases. Moreover, the overall dataset contributed to increase the knowledge on the genetic diversity and functions of the microbiomes associated to the polluted sites from the Southern side of the Mediterranean Sea, that have been rarely considered in previous metagenomic studies. For samples locations see Figure 2.
• At least 83,000 16S rRNA gene fragments obtained by 454 pyro-sequencing or Illumina Hiseq 2000 sequencing have been identified from sediment samples on the coastlines of the Mediterranean and Red Sea sites (see above). Accordingly, at least 18,435 operational taxonomic units (equivalent to distinct species) were identified. This constitutes, to the best of our knowledge, the biggest set of environmental species within the Mediterranean and the Aqaba Gulf in the Red Sea deposited in databases.
• Meta-genomic (theoretical) and metabolomic (experimental) evidences for the metabolism of 31 distinct pollutant substrates and/or intermediates including alkanes and PAH-like derivatives have been successfully identified in the ULIXES project. A total of 223,416 out of a total of 10,907,240 (or 2.05%) potential protein-coding genes, covering 56 types of major activities, were found as being involved in such processes.
• Three protocols have been developed to: reconstruct biodegradation networks from DNA (1) and 16S rRNA (2) meta-genome sequences, as well as meta-proteomic and meta-metabolomic data (3). Graphical representation of these networks was developed using the igraph package for the R platform and in house algorithms. Additionally, a fourth graphical method was produced by using the Ocean Data View software, in which spatial distributions of percentages of genes encoding enzymes participating in biodegradation steps are plotted as iso-surface distributions. These tools were crucial for inferring putative and active biodegradation reactions in the network, and pathway organizations at the global, organismal and spatial levels.
• A total of 18 uric acid and ammonium-amended microcosms have been produced and analyzed in terms of: (1) community structure; (2) geochemical parameters; (3) presence of uric acid degrading members of the Halomonas genus; (4) the comparison of diversity and distribution of catabolic genes for alkane and aromatic degradation as consequence of the applications of ammonium and uric acid. Results highlighted that Halomonas spp. members may promote biodegradation efficiency by converting uric acid into ammonium which can be readily utilized by marine hydrocarbonoclastic consortia. Additionally, examining the sediment of Ancona port (northern Italy) we demonstrated that while ammonium improved the presence of microbes degrading phthalate, gallate, catechol to 2-hydroxymuconate semialdehyde and toluene (via anaerobic pathway), uric acid improved the presence of dinitrotoluene- and halogenated catechol-degrading microbes.
WP6. Microbial metagenomics and proteomics of the polluted sites
During the ULIXES project, we completed the following activities within the WP6, which produce a number of outcomes, which are highlighted below. To note is that the activities and results within this WP derived from sequences resources and material derived from sediment samples from four major oil refinery polluted Mediterranean Sea sites on the coastlines of Morocco (lagoon of Mar Chica), Tunisia (Bizerte lagoon), Egypt (El-Max site), and Italy (Gulf of Genoa, strait of Messina and Syracuse, and Ancona port) as well as on the coastline of the Red Sea (Gulf of Aqaba along the Jordanian coast), as well as from the polyaromatic hydrocarbon degradation Cycloclasticus sp. ME7.
• A total of 42 metagenomic libraries (28 fosmid libraries and 14 cosmid libraries) containing a total of 1.4 million clones (about 1.25 million in fosmids and about 150,000 in cosmids) have been produced.
• At least, 1,221 out of approx. 1.2 millions clones encoding enzymes of interest have been identified by screening of all 42 libraries.
• 34 fosmid clones have been fully characterized in respect to their substrate profile.
• A total of 306,948 potential protein-coding genes (cut-off of 20 amino acid-long sequences) have been identified within the ULIXES meta-genomic resources. They encode alpha/beta hydrolase family proteins and extradiol dioxygenases.
• At least 173 out of 306,948 genes encoding biotech-relevant enzymes of interest amended in ULIXES project have been cloned and expressed using the Ek/LIC 46 based expression vector in Escherichia coli BL22.
• Apart from activity-based enzyme mining, we have generated five metagenome sequencing datasets (by HiSeq Illumina sequencing) from crude-oil enrichment cultures amended with uric acid or ammonium. Resulting data was mined for genes encoding oxidoreductases and hydrolases with a subset of 50 enzymes that were cloned in E.coli.
• At least 74 biotech-relevant enzymes have successfully expressed and fully characterized with regard to substrate spectra and temperature range. A battery of at least circa 100 different substrates has been used to evaluate the biochemical properties. Characterized enzymes showed elevated optimum temperatures along the temperature gradient of Mediterranean and Red Sea (with lower optimum temperatures in Northern Mediterranean and elevated optimum temperatures in Red Sea).
• A crystal structure has been solved, deposited in the Protein Databank (PDB) and published. The structural co-ordinates reported for Cycloclasticus sp. ME7 CCSP0084 alpha/beta hydrolase appears in the PDB under accession code 4I3F.
• At least six mutant proteins were generated. Mutations were introduced into plasmid p15TV-Lic containing CCSP0084 gene encoding a dual esterase: MCP hydrolase from the α/β hydrolase family from Cycloclasticus sp. ME7. The biochemical analysis of the corresponding mutant variants was undertaken with four distinct substrates and the potential role of those residues in determining substrate profile was determined. Through this analysis, we were able to provide first indications about the residues dictating the evolution between esterases and meta-cleavage product alpha/beta hydrolases, that is, the evolution between a non-degradative and a degradative enzyme.
• At least, a set of 188 gene clusters presumably participating in the production of known secondary metabolite compound classes (poliketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, bata-lactams, butyrolactones, siderophores, melanins and others) have been identified using AntiSMASH (ANTIbiotics & Secondary Metabolite Analysis Shell; http:// antismash.secondarymetabolites.org). To note is the high number of interesting multifunctional genes of polyketide synthases (PKS) or non-ribosomal peptide synthases (NRPS) which are the main responsible for the biosynthesis of industrial cytotoxic compounds (for details see Figure 3).
To note is that within WP6, ULIXES project has contributed to the full characterization of the biggest set of proteins from the Mediterranean and Aqaba Gulf in the Red Sea, which constituted the biggest set of purified protein from a single marine location. In addition, it has delivered the biggest set of expression clone libraries around polluted sites in the Mediterranean and Aqaba Gulf in the Red Sea. Finally, within WP6 ULIXES consortium has demonstrated, for first time, that polluted sites around the Mediterranean and Red Sea may contain the higher number of gene clusters encoding secondary metabolite compound classes, namely, polyketides and non-ribosomal peptides, reported for free-living bacteria in a marine sample.
WP7. Establishing new product/processes for bioremediation
Task 7.1. Using hydrocarbon degradation enzymes and microorganisms to obtain valuable chemicals (fine chemicals and biosurfactant)
The main objective of the ULIXES research within the Task 7.1 was the development the biosynthesis of valuable fine chemicals and new biosurfactants at the laboratory scale by means of the hydrocarbon degrading microbes obtained in WP4. The achievement of this objective implied: (i) production and optimization of in-house protocols that allow investigating the reactivity-profile of enzymes produced in the frame of ULIXES project in order to develop novel and/or optimized biotransformation processes, (ii) the selection of marine bacteria able to produce biosurfactants to be tested in lab scale bioprocesses for the ex-situ bioremediation of marine sediments contaminated by hydrophobic chlorinated contaminants. ULIXES provided the first experimental evidences to demonstrate the biotechnological potential of MCP hydrolases, previously thought to be restricted to biodegradation pathways, has to be reassessed and that C-C meta-cleavage product hydrolases may be useful for important transformations, such as chiral resolutions and lactone hydrolysis. The discovery and success biotech testing of dual esterases - MCP hydrolases from the alpha/beta hydrolase family could be considered as a top achievement in relation to the development of new biocatalysis processes using enzymes previously considered to have low biotech applicability.
Additionally, ULIXES provided a set of at least 31 enzymes for chiral synthon production using the uncommon substrate specificities and enantioselective enzymes. Accordingly, these enzymes can be used for novel biocatalysis processes related to chiral ester resolutions and the concomitant production of pharmaceuticals. Finally, ULIXES has provided a set of at least 40 enzymes that have a substrate preference distinct from other reported alpha/beta hydrolase, as they are catalytically most active in the hydrolysis of poly-aromatics. These enzymes offer a new window for the stereo-assembly of multi-aromatic molecules useful in biology, chemistry and material science, for which enzyme processes have not yet being established. The hydrolases are catalytically active towards PAH esters, which can lead to the potential applicability to generation a unique set of complex aromatic molecules with yet unknown properties. We believe that some of the enzymes may open new opportunities to design a-la-carte enantiomeric forms of complex aromatic molecules, due to the regio- and enantio-selective properties of these enzymes. Such derivatives are known to have industrial relevance for the production of microelectronic components with photolytic properties, printed circuit boards, silk screen printing devices, optical disks, black matrix of colour tubes, dyes, flavouring compounds, plasticizers, perfumes, pesticides, mold growth biomarkers, insect repellent, fibers and films productions, and solid rocket propellants, to cite some reported applications.
Finally, isolates able to produce surface active molecules in different marine mineral media were screened, and they belong to the genera Alkanivorax, Paracoccus, Sulfitobacter, Shewanella, Pseudoalteromonas and Roseovarius. The latter three isolates showed high rate of growth, biosurfactant productivity and stability, and were thus compared in terms of ecotoxicity of the surface active molecule produced towards the marine bacterium Vibrio fisheri. The biosurfactant produced by the Shewanella sp. showed the lowest toxicity and was identified as the most promising biosurfactant for biostimulation applications. Therefore, it was selected for enhancing the bioavailability of hydrophobic chlorinated pollutants in the ex-situ bioremediation of marine sediments in lab-scale bioreactors.
Task 7.2. Development of small scale bioprocesses for the reductive dehalogenation of chlorinated compounds
The main objective of this task was to develop small scale bioprocesses for the reductive dehalogenation of chlorinated compounds at laboratory scale, by the means of the microbial consortia obtained in WP4. The up-scaling of the 1,2-DCA dehalogenating enriched cultures (see Deliverable 4.2) to optimize the conditions for the reductive dechlorination of this pollutant were unsuccessful. To overcome the instability of the established consortia from the marine polluted sites in the framework of ULIXES project, an alternative plan was setup with new dehalogenating enrichment cultures. These consortia were gradually adapted to the salinity value typical of seawater (about 3%). Unfortunately, the process of adaptation to higher salinity values stopped at the first step. In addition, the enriched bacteria did not show 1,2-DCA degradation activities at 0.25% salinity value in the medium even if they were highly efficient in the 1,2-DCA removal at 0% salinity value. Further efforts should be conducted in order to discover promising bacterial consortia involved in the dechlorination of 1,2-DCA in marine ecosystems with the optimization of reductive dechlorination activity against 1,2-DCA.
Even though the 1,2-DCA degrading enriched cultures could not be successfully used for the cleanup of this chlorinated solvent form seawater and sediment, the aim of D7.4 was still achieved. In fact, we achieved the setup of batch and/or continuous anaerobic bioreactors for dehalogenation of chlorinated solvents in marine water and slurry small-scale anaerobic reactors for the dechlorination of PCB mixtures in real sediments of the Venice Lagoon.
The bioremediation process for PCB contaminated sediments was tested in anaerobic shaken reactors and static microcosms supplemented with the selected sediment from the Venice lagoon (sediment E) in WP4. Within WP7, different strategies for the enhancement of PCB dechlorination activities were investigated: i) bioaugmentation of sediment with a marine PCB dechlorinating culture, ii) biostimulation of PCB dechlorinating microbes by providing an electron donor, iii) PCB mobilization by employing biosurfactant, and combinations of all these activities. The most active PCB dechlorinating culture was obtained from marine sediments of the Venice lagoon (i.e. culture from sediment D). The combination of this consortium with a suitable (i.e. lactate) electron donor allowed to achieve the bioaugmentation and biostimulation purposes in PCB dechlorination activity. The biosurfactant produced by the marine isolate XP8 were suitable for increasing the PCB bioavailability. Biostimulation process realized using lactate had no effect on PCB dechlorination by the indigenous microbial community of sediment; on the contrary, the bioaugmentation with the selected PCB dechlorinating culture promoted dechlorination of the PCB, which was further enhanced by biostimulation with lactate. Dechlorination process in the bioaugmented culture supplemented with the selected biosurfactant was partially inhibited. This might be due to some toxic effect of biosurfactant on the dechlorinating microorganisms. The same behaviors were observed in both shaken bioreactors and in static microcosms, where the absence of mixing resulted in lower PCB dechlorination rates and extents. In conclusion, bioaugmentation combined with biostimulation was found to be the most effective strategy to promote PCB reductive dechlorination for ex-situ treatments of PCB contaminated marine sediments.
Task 7.3. Development of novel biopalladium nanoparticles dechlorination processes
Bio-palladium (bio-Pd) nanoparticles combined with a hydrogen donor in order to produce reactive radicals, showed highly catalytically activity in multiple remediation processes, such as the dechlorination of halogenated compounds. The required H2 for activation of bio-Pd catalysts could be provided directly by the microbes, through the fermentative conversion of organic compounds. The general objective of Task 7.3 was to develop bio-Pd nanoparticles able to dehalogenate toxic compounds, such as PCBs and Cl-solvents (TCE), polluting marine environments.
In the framework of WP7, UGent has delivered the following objectives:
(i) Identification and application of 46 indigenous Mediterranean marine isolates for the synthesis of very small, narrow sized palladium nano-catalysts.
(ii) Optimised fermentative production of hydrogen to activate the nano-catalysts, using one of the above microorganisms.
(iii) Complete dechlorination of TCE in synthetic marine water, marine water and sandy marine sediments slurries, using the formed bio-Pd-NPs.
(iv) Enhanced catalytic activity of Bio-Pd nanoparticles for the reductive dehalogenation of TCE, upon modification of the nanoparticle size.
(v) Bio-Pd nanoparticles were able to dechlorinate both PCB20 and Aroclor 1254 PCBs in marine water, where PCBs can be fully dechlorinated to the less harmful biphenyl.
(vi) Activation of bio Pd and further dehalogenation of TCE using either source of H2 (bogenic or exogenous).
(vii) Immobilized catalysts in organic polymers were successfully degraded TCE in marine water and marine sediment slurries.
The newly biogenic nanoparticles developed within the ULIXES WP7 can open a new perspective for future marine nano-based remediation applications.
Task 7.4. Definition of a fate model for metal and metalloids in polluted sediments
The partner EcoTS studied the hydrocarbon biodegradation, mobilization and stabilization of metals through biostimulation experiments. The experimental design included the addition of substrates (sodium acetate and lactose) and mineral nutrients. The biodegradation of hydrocarbons was more efficient after 60 days of incubation, while best results were obtained in presence of acetate and nutrients plus acetate. These treatments resulted in a significant increasing of total bacterial abundance and biomass compared to the control system. The geochemical fractionation of heavy metals in the sediments during the biostimulation experiments was studied, considering the exchangeable and carbonate bound fractions, iron and manganese oxides fraction, organic and sulfide fraction, and the residual fraction. Results indicated a range of different behaviors in terms of mobility/bioavailability of each metal in the sediment matrix. The best performances in terms of immobilization of metals (25% of heavy metals immobilized after 60 days incubation) were achieved with the addition of nutrients and substrates. The rates of mobilization/immobilization of each metal were statistically analyzed for the production of a fate model, as well as to select the best treatment strategies for contaminated sediments. The ecotoxicological assays (Microtox, algal growth inhibition and immobilization of crustaceans), was performed to assess the eco-compatibility of sediment treatments, and suggested a slight reduction of toxicity in systems displaying the best performances of metal immobilization.
The overall results indicated that the biostimulation of autochthonous bacterial community can be an effective strategy for the immobilization of heavy metals in marine sediments. It is noteworthy that specific treatments have to be carefully designed to obtain good performances and re-use options.
WP8. Testing new product/processes by pilot bioremediation treatments in the field
The work package 8 included four different tasks that were carried out by four different partners of the ULIXES consortium, namely YU, MUCSAT, TUC, IAMC.
The overall aim of WP8 was to test selected microbial resources previously selected within the WP4 in a pilot and field scales. Furthermore, UMIL and ETS partners supported the monitoring activity of part of the setup bioremediation processes, investigating respectively the bacterial community structure during the experiments and assessing the eco-toxicity of the treated sediments. Each task was devoted to test microbial resources in the four different sites, all located in industrial areas contaminated by hydrocarbons, chosen as representatives of different latitudes and climatic conditions in the Mediterranean and Red Sea, the latter constituting a reference example of the potential climate situation of the Mediterranean in the next years following the global warming trend.
The WP8 activities were finalized to:
i) Simulate bioremediation models from experimental microcosms, mesocosms and pilot in situ field studies;
ii) Verify the efficiency of the whole approach adopted with ULIXES, i.e. whether the microbial resources retrieved with the microbiological surveys can be effective new products to improve the effectiveness of bioremediation up to the field scale.
Task 8.1. Treatment at the site 1, El-Max district, Alexandria, Egypt
MuCSAT partner performed several preliminary activities aimed the setup of bioremediation treatment of site 1, El-Max district. Five different natural consortia were obtained and characterized from highly polluted stations located at El-Max bay. Moreover, during the consortia growth under laboratory conditions, analysis of the remaining crude oil components were performed in order to choose the most suitable consortium to be applied for the in situ biodegradation experiment. The consortium R, previously obtained from station R, was selected for the in situ biodegradation of crude oil into seawater. A study of different immobilization supporters were performed using three different immobilization material that could be used as bacterial supporter to carry on the bacteria during the in situ biodegradation of crude oil. The results showed good degradation rate: about 99% of Unresolved Complex Mixture (UCM) and 94% of the Total Resolved Peak ratio (TRPs) were removed after 28 days of exposure of the cells to the oil in the seawater using specific devices (D8.4). An experimental trial was performed to study the effectiveness of immobilization of the cells on enhancement of the oil degradation. The results showed that the immobilization of cells generally increased the biodegradation ability, as the residual Total Petroleum Hydrocarbons (TPH) decreased in presence of the immobilized cells compared with the treatment performed using the free cells.
The Plackett-Burman experimental design method was applied to test the effect, in term of oil biodegrading capabilities, of six bacterial isolates used in a consortium. The data showed that the combined use of Pseudomonas plecoglossici and Bacillus aerophilis for the remediation of crude light petroleum pollution enhanced the degradation capability. Future experiments may reveal the optimization of the content of such mixture and formulate it further for in situ bioremediation purposes.
Task 8.2. Treatment at the site 2, the Elefsina Gulf, Attika, Aegean Sea, Greece
TUC partner initially proposed a plan that involved a two-phase experimental design. Phase A consisted of a small scale in situ aeration experiment and an ex situ landfarming mesocosm experiment. Phase B included a larger scale in situ aeration approach, combining the results obtained from phase A. Unfortunately, excavation activities were realized at the target contaminated site and the water depth increased to 90-95 m. Therefore, it was not possible to perform the in situ aeration experiments initially foreseen in the DoW. As a contingency plan, TUC extended the landfarming experiments, doubling the originally planned experimental time (8 weeks instead of the proposed 4) and performed an additional accurate set of chemical (sediment hydrolytic activity), microbiological (total bacterial population monitoring using flow cytometry) and eco-toxicological analyses. The results obtained by TUC for the landfarming ex situ treatment showed that the addition of allochthonous degraders, i.e. allochthonous bioaugmentation, did not enhance biodegradation even though the added degrading consortia had been acclimated in sediment slurry before their application to the sediment and their initial CFUs were 10-fold higher than those of the autochthonous viable bacteria. The degradation patterns of saturated and aromatic compounds, total bacterial population numbers and sediment hydrolytic activity of the bioaugmentated treatments were similar to those of the respective control treatment. On the other hands, biostimulation proved to be necessary, as the above parameters were significantly lower in the treatment. Moreover, the early peaks in total bacterial populations and sediment hydrolytic activity that were observed in the treatment receiving the dead bacterial biomass, indicated that the addition of organic nutrients may accelerate the degradation process. Additional microbiological analyses were performed at UMIL to dissect the composition of the bacterial community in the original sediment and to follow the evolution of the bacterial community structure during the different landfarming treatment experiments. To clarify the role of biostimulation on the autochthonous bacterial communities the sediments have been analysed through Denaturing Gradient Gel Electrophoresis (DGGE), a method that allow both the identification of the inoculated consortia and the study of the overall bacterial community.
Task 8.3. Treatment at the site 10, the Aqaba Gulf, Red Sea, Jordan
Partner YU performed several experiments to enrich consortia obtained from several polluted and unpolluted sites located in the Aqaba Gulf: the marine science station, the phosphate loading berth, the oil terminal and the industrial jetty site. These sites were sampled in the frame of the WP2 activities, aiming to assess the efficiency of different natural consortia and to have a snapshot of the microbial community structure. In the YU laboratory, different experiments were setup using 500 ml flasks, 5000 ml vessels and 12000 ml aquaria, as part of WP4, to enrich the natural consortia and to increase their biomass to be used later as inocula for the large tanks (2000 L). An on-site experiment was held at the shores of the marine science station (MSS) using large tanks (2000 L). The seawater was collected from the MSS site and used as media to support the microbial growth, supplying 0.5% crude oil as the sole carbon source. Chemical and microbiological analyses were carried out during the course of the experiment in order to monitor the progress of microbial growth and asses the crude oil biodegradation, monitoring several parameters for each consortium. The bacterial strains isolated from petroleum polluted sites in the Gulf of Aqaba were predominated by Gammaproteobacteria, which is a common phylum in marine environments. Fifty bacteria isolated from all the sampling sites were positive to the biosurfactant production tests; forty-eight isolates showed the ability to form biofilm and forty-six were able to produce exopolysaccharides. The removal efficiencies of n-alkanes were about 80-90% within 7 days of incubation, where the consortium FW showed the highest degradation activity. The removal efficiency in the presence of individual hydrocarbons phenanthrene, fluoranthene and pyrene by two of the tested consortia reached the highest level after growing the mixed cultures for 16 days. The oxygen concentration had a significant effect on the degradation of hydrocarbons. A correlation could be inferred between the removal of hydrocarbon, the increase in microbial load (CFU) and the CO2 production. Additionally, the samples provided by MUCSAT and TUC for field testing, were enriched and tested in the lab as a first stage. In experiments conducted, there was a clear difference in the n-alkanes removal efficiencies between the MUCSAT and TUC consortia.
Task 8.4. Treatment at the site 12, shipwreck site of the oil tanker Haven, Genoa Gulf, Italy
Partner IAMC-CNR have carried out a series of biostimulation approaches and obtained a better knowledge on the structure and dynamics of indigenous bacterial communities in parallel with the biodegradation processes. The mesocosms experiments were initially considered as preliminary test before the in situ treatment on site 12, corresponding to the oil tanker Haven shipwreck. In this context, sampling activities were performed in the area of the Haven shipwreck where different tar samples were collected for the cultivation of microbial consortia capable of petroleum degradation and for the description of the inhabiting microbial community achieved by molecular methods. However, despite numerous attempts, IAMC-CNR failed to obtain the official permission signed by the Italian Ministry for Environment for the realization of the field experiments on the tanker Haven shipwreck site. The realization of bioremediation experiment would have nevertheless been very challenging and difficult to perform due to significant depth of the shipwreck (about 80 meters) and the difficulties in obtaining the official permission from the local authority, the Italian Naval Authority and the Municipality of Arenzano (Genova), to enter the site. As a contingency plan, IAMC-CNR proposed the treatment of a chronically contaminated and easily accessible site located in the harbour of Messina. Almost 2 tons of contaminated sand sediments were collected and placed in 14,000 L mesocosms. The sediments for the in situ bioremediation study were further polluted with crude oil and the effect of different biostimulation approaches were validated in mesocosms experiments, monitoring in parallel the community structure and dynamics of indigenous bacterial communities during the biodegradation processes. Moreover, in June 2013 Partner IAMC-CNR suggested and included another area for in situ bioremediation treatments, namely the shoreline site located in the proximity to the refinery of Gela (Caltanissetta, Sicily, Italy). The new proposed area represents the most attractive scenario for the realization of in situ experiments. In fact, on 4th June 2013, an oil spill from the pipeline of “Topping1” installation of ENI Refinery of Gela released in the marine environment around 5,000 liters of heavy crude oil. Although quick action were undertaken by the Italian Naval Authority and the ENI S.p.A to combat with the spill, significant part of oil was spilled out, thus contaminating both open sea and the beach. Differently from what previously occurred for the Haven shipwreck site, IAMC-CNR succeeded to obtain the official permission signed by Italian Authorities to monitor the recovery process of the shoreline. Partner IAMC-CNR installed in the contaminated beach of Gela a 10 m2 area for trial field and environmental monitoring. This experimental area, indeed, represented a unique opportunity to work in the area affected by an actual oil spill, where direct application of specific strategies for environmental recovery could be placed, monitored and validated. In this new experimental area partner IAMC-CNR carried out two different strategies for recovery of polluted sediments. In particular, partner IAMC-CNR performed in situ biostimulation (addition of inorganic nutrients) and bioaugmentation (addition of inorganic nutrients plus biomass of indigenous Marinobacter sp. cells previously isolated from the same site).
Potential Impact:
Overall, the ULIXES project had a high environmental added value since it especially focuses on the exploitation of microorganisms that represent a renewable and sustainable resource for the clean-up and bioremediation of the polluted coastal marine environments in the Mediterranean Sea. Bioremediation and enhanced natural attenuation based on microbial products can be, with no doubts, considered as an environmentally friendly process. The approach applied in the ambit of the ULIXES research had the main goal of setting up pollutant- and site-tailored bioremediation processes for remediating marine ecosystems in a sustainable manner, and to develop innovative processes for environmental protection. To pursue this aim a holistic approach, comprising microorganisms cultivation, molecular ecology, OMICs based analyses and development of innovative bioprocesses and field pilot studies, has been applied by the ULIXES partners, in accordance to their specific well established expertise and available facilities. All the activities foreseen in the ULIXES DoW have been performed, leading to the achievement of the foreseen deliverables and the obtainment of most of the expected results. The establishment of a large collection of biodegrading strains and mixed cultures had been finalized (WP4). The ULIXES microbial collection encompasses bacteria of high interest due to their ability to form biofilm, produce biosurfactants and degrade high molecular weight hydrocarbons. Moreover, bacteria able to mobilize different metals, to degrade halogenated compounds and to produce activated bio-palladium nanoparticles have been isolated from the samples collected by the ULIXES partners across the Mediterranean Sea. The ULIXES microbial collection constitutes a massive reservoir of genetic resources that has been organized in a database available to the whole consortium. The collection has been already partially exploited within the project for the setup of bioremediation processes (WPs 7-8), and could be easily consulted by all the partners for further experiments beyond the project end, which can allow the set-up of new biodegrading microbial inocula for the treatment of polluted seawater and sediments. Until now, according to the result of a detailed survey of the scientific literature on marine bioremediation studies, minor attention was devoted by the international scientific community on the study of environmental pollution and safety in the Southern Mediterranean countries. In this contest, the EU project ULIXES represented a novel and tangible effort to integrate the North and South sides of the Mediterranean basin for the setup and improvement of clean up strategies exploiting natural microbiological products and the so called Microbial Resource Management. In the frame of the ULIXES project, the consortium realized the widest metagenomic and 16SrRNA pyrosequencing dataset of the Mediterranean Sea and the Gulf of Aqaba in the Red Sea produced until now (WPs 3-5-6). This dataset encompasses both taxonomic and functional biodiversity data and its sequences will become available to the public as soon as common manuscripts (which involve the whole consortium and are under preparation) will be published. The performed OMICs analyses allowed to get a detailed biodegradation metabolome map of each hydrocarbon-polluted site, correlated with a microbial biodiversity metagenomic map obtained through metagenomics and 16S rRNA pyrosequencing. To achieve a detailed picture of the diversity of the biodegradation potentials in each site, and in particular in the Southern Mediterranean, was one of the key objectives of the ULIXES research. This result boosts the filling of the knowledge gap on the bioremediation potential in a yet overlooked region, the Southern Mediterranean that is one of the major oil-producing areas on Earth, identifying site-specific metabolic routes and genes of potential biotechnological application.
Overall, the results showed that the three-years EU project ULIXES led to the consolidation of a consortium of partners that proficiently cooperated in the planned research activity. The consortium, within the ULIXES project, has published a high number of scientific papers. Moreover, several studies will be finalized after the official end of the project leading to additional results that will provide a high quality contribute to the scientific research on the bioremediation field.
In addition to the scientific achievements obtained within the ULIXES project, the research results had also consequences potentially relevant for social and political aspects of the research, which have to be taken into account when evaluating their implementation. The involvement of scientists from the two sides of the Mediterranean Sea promoted an active exchange of information and knowledge between partners of the different countries. This proactive cooperation resulted also in short visit/training periods of young scientists in the partner’s laboratories, an activity that will be possibly continued beyond the official end of the project, according to the availability of internal funding opportunities of the involved laboratories. The dissemination activities, in addition to the scientific publications, included several activities like the establishment of the project website (www.ulixes.unimi.it) the participation to national and international conferences, the establishment of different databases, the preparation of leaflet, brochure, newsletters, and the organization of a ULIXES final conference (MedRem-2014). MedRem-2014, the final ULIXES conference held in Hammamet, Tunisia on 16-18 January 2014, put together scientists from 26 countries, including the participation of companies and stakeholders operating in the sector of energy and oil and gas extraction and management. The feedback obtained during the conference together with the stakeholder database created under the ULIXES project indicate a high potential for the implementation of the ULIXES results as well as leave the concrete possibility that the project may have potential future influence on the oil management policy in the countries of the Southern Mediterranean, towards more environmental-friendly strategies. Overall, the ULIXES consortium positively evaluated the implementation of the obtained results, which will have an impact primarily at the research level but also on the social, economic and cultural activities by promoting a more conscious behavior in respect of ecosystem protection.
The research activities carried out within the WP7, on the other hand, shed a light on new opportunities to use bacteria as i) nano-engineer to produce nanoparticles that can be applied in the remediation of polluted matrices and ii) key player of dehalogenating processes. These results gave a contribute to the set-up of innovative processes, and can be implemented during the management of polluted sediment and water of both marine and freshwater origin, as reported for example in the following publications of the ULIXES project:
• De Corte S., Hennebel T., De Gusseme B., Verstraete W., Boon N. (2011) Bio-palladium: from metal recovery to catalytic applications. Microbial Biotechnology 5:5-17.
• De Corte S., Hennebel T., Fitts J.P. Sabbe T., Bliznuk V., Verschuere S., van der Lelie D., Verstraete W., Boon, N. (2011). Biosupported bimetallic Pd/Au nanocatalysts for dechlorination of environmental contaminants. Environmental Science & Technology 45:8506-8513.
• De Corte S., Sabbe T., Hennebel T., Vanhaecke L., De Gusseme B., Verstraete W., Boon N. (2012) Doping of biogenic Pd catalysts with Au enables dechlorination of diclofenac at environmental conditions. Water Research 46:2718-2726.
• Hennebel T., De Corte S., Verstraete W., Boon N. (2012) Microbial production and environmental applications of Pd nanoparticles for treatment of halogenated compounds. Current Opinion in Biotechnology 23:1–7.
• Hosseinkhani B, Hennebel T and Boon N. (2014) Biogenic hydrogen production in marine environments for hydrogen driven remediation strategies. New biotechnology (Under revision).
• Hosseinkhani B., Hennebel T., Van Nevel S., Verschuere S., Yakimov M.M. Cappello S., Blaghen M., Boon N. (2014) Biogenic nanopalladium based remediation of chlorinated hydrocarbons in marine environments. Environmental Science &Technology 48:550-557.
• Zanaroli G., Balloi A., Negroni A., Borruso L., Daffonchio D., Fava F. (2012) A Chloroflexi bacterium dechlorinates polychlorinated biphenyls in marine sediments under in situ-like biogeochemical conditions. Journal of Hazardous Materials 209-210:449-457.
• Zanaroli G., Negroni A., Vignola M., Shu H-Y., Fava F. (2012) Enhancement of microbial reductive dechlorination of polychlorinated biphenyls (PCBs) in a marine sediment by nanoscalezerovalent iron (NZVI) particles, Journal of Chemical Technology and Biotechnology 87:1246-1253.
Additional publications acknowledging ULIXES show that the discovered microbial resources (e.g. bacteria, biosurfactant, enzyme) can be implemented in tailored bioremediation techniques for either the in situ or ex situ treatment marine waters and sediments polluted with petroleum hydrocarbons, halogenated compounds and heavy metals, providing input to the discussion about the biostimulation/bioaugmentation effectiveness. These studies, which have been also complemented by synthetic ecology studies to address general principles of the system functionality under invasion/augmentation processes, provide novel perspectives for the realization of field bioremediation treatments. See among others the following publications of the ULIXES project:
• Alcaide M., Tornés J., Stogios P.J. Xu X., Gertler C., Di Leo R., Bargiela R., Lafraya A., Guazzaroni M.E. López-Cortés N., Chernikova T.N. Golyshina O.V. Nechitaylo T.Y. Plumeier I., Pieper D.H. Yakimov M.M. Savchenko A., Golyshin P.N. Ferrer M. (2013) Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/β hydrolase family. Biochemical Journal 454(1):157-66. doi: 10.1042/BJ20130552.
• De Roy K., Marzorati M., Negroni A., Thas O., Balloi A., Fava F., Verstraete W., Daffonchio D., Boon N. (2013) Environmental conditions and community evenness determine the outcome of biological invasion. Nature Communication, 4:1383.
• Cappello S., Genovese M., Della Torre C., Crisari A., Hassanshahian M., Santisi S., Calogero R., Yakimov M.M. (2012) Effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics during assays of oil spill bioremediation: A microcosm study. Marine Pollution Bullettin 64:2820-2828.
• Cappello, S., Antonella C., Hassanshahian M., Genovese M., Santisi S., Yakimov, M.M. (2012) Effect of a bioemulsificant Exopolysaccharide (EPS2003) on abundance and vitality of marine bacteria. Water, Air, & Soil Pollution, 223:3903-3909.
• Amer R., El-Gendy N. Sh., Taha T., Farag S. and Abdel Fattah Y. Biodegardation of crude oil by using indigenous bacterial consortium isolated from contaminated area in Egyptian Mediterranean ecosystem. Environmental Science Process and Impact. (Under revision)
• Mahjoubi M., Jaouani A., Guesmi A., Ben Amor S., Jouini A., Cherif H., Najjari A., Boudabous A., Koubaa N ., Cherif A. (2013) Hydrocarbonoclastic bacteria isolated from petroleum contaminated sites in Tunisia: isolation, identification and characterization of the biotechnological potential. New Biotechnology, 30:723-733.
• Nikolopoulou M., Eickenbusch P., Pasadakis N., Venieri D., Kalogerakis N. (2013) Microcosm evaluation of autochthonous bioaugmentation to combat marine oil spill. New Biotechnology, 30:734-742.
• Nikolopoulou, M., Pasadakis N., Kalogerakis N. (2013) Evaluation of autochthonous bioaugmentation and biostimulation during microcosm-simulated oil spills, Mar. Pollut. Bull. 72:165–173.
• Gertler C., Nather D.J. Cappello S., Gerdts G., Quilliam R.S. Yakimov M.M. Golyshin P.N. (2012) Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study. FEMS Microbiol Ecol 81:520-536.
In the context of treatment application for field bioremediation intervention, a patent authorization has been requested to the Hellenic Industrial Property Organisation and, at the moment, it is under evaluation.
Genomic studies, as those reported below, were also performed during the ULIXES project, providing novel information on the functional capabilities of the bacteria involved in oil hydrocarbons remediation.
• Guazzaroni M.E. Herbst F.A. Lores I., Tamames J., Peláez A.I. López-Cortés N., Alcaide M., Del Pozo M.V. Vieites J.M. von Bergen M., Gallego J.L. Bargiela R., López-López A., Pieper D.H. Rosselló-Móra R., Sánchez J., Seifert J., Ferrer M. (2012) Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation. The ISME Journal 7:122-136.
• Kube M., Chernikova T.N. Al-Ramahi Y., Beloqui A., Lopez-Cortez N., Guazzaroni M.E. Heipieper H.J. Klages S., Kotsyurbenko O.R. Langer I., Nechitaylo T.Y. Lünsdorf H., Fernández M., Juárez S., Ciordia S., Singer A., Kagan O., Egorova O., Alain Petit P., StogiosP., Kim Y., Tchigvintsev A., Flick R., Denaro R., Genovese M., Albar J.P. Reva O.N. Martínez-Gomariz M., Tran H., Ferrer M., Savchenko A., Yakunin A.F. Yakimov M.M. Golyshina O.V. Reinhardt R., Golyshin P.N. (2013) Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispiraantarctica. Nature Communications. 4:2156.
Overall, the consortium positively evaluated the implementation of the results of the project. Moreover, several studies will be finalized after the official end of the project leading to additional results that will provide a high quality contribute to the scientific research on the bioremediation field. These studies will be submitted as publication, which complete list (at the end of the project) is included in the Deliverable 9.7.
List of Websites:
www.ulixes.unimi.it
