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UNIBO activities in ULIXES

UNIBO activities in ULIXES. Fabio Fava Giulio Zanaroli Noura Raddadi Roberta Guerra DICAM, Faculty of Engineering, Alma Mater Studiorum -University of Bologna. Bologna, Italy. Project meeting, Chania, Greece – July 3, 2011. UNIBO involvement in WPs and RTD tasks.

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UNIBO activities in ULIXES

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  1. UNIBO activities in ULIXES Fabio Fava GiulioZanaroli NouraRaddadi Roberta Guerra DICAM, Faculty of Engineering, Alma Mater Studiorum-University of Bologna. Bologna, Italy Project meeting, Chania, Greece – July 3, 2011

  2. UNIBO involvement in WPs and RTD tasks WP 2. Sampling and characterization of the Mediterranean Sea sitesWP 4. Establishment of collections of microbial isolates (and metabolites)WP 7. Establishment of new product/processes for bioremediation Project meeting, Chania, Greece – July 3, 2011

  3. Temporal distribution UNIBO RTD tasks D7.2. August 2013 D7.4. November 2013 Project meeting, Chania, Greece – July 3, 2011

  4. WP 2. Sampling and characterization of the Mediterranean Sea sitesTasks & Activities • Task 2.1: Sampling organization & geochemical characterization of the coastal target sites • Sampling of dredged sediments and water collected from contaminated and non-contaminated locations of the Venice Lagoon • Geochemical analyses of dredged sediment samples for: • (i) concentration of pollutants: (chloro-)hydrocarbons, heavy metals • (ii) total nitrogen and nitrogen species • (iii) total phosphorous and phosphate concentrations • (iv) mineralogical composition • (v) grain size distribution • (vi) total sulphur and sulphur species • (vii) total iron concentration • (viii) total and dissolved organic carbon • (ix) pH, alkalinity, and conductivity • Analysis of water samples for pH, concentration of pollutants detected in the sediment and of the main electron acceptors (such as sulfate, nitrate, nitrite…). Project meeting, Chania, Greece – July 3, 2011

  5. WP2. PCB-contaminatedsediments/water fromVeniceLagoon (a) Di Domenico et al.1998. Organohalogen Comp. 39: 205 Moret et al. 2001. Chemosphere 43:559 Frignani et al.2001. Chemosphere 43: 567 Moret et al. 2005. Marine Pollution Bulletin 50: 167

  6. WP2. PCB-contaminatedsediments/water fromVeniceLagoon (b)

  7. WP 2.Sampling and characterization of the Mediterranean Sea sites. Marine sediment samples from the Venice lagoon (*) All available sediment F has been used Project meeting, Chania, Greece – July 3, 2011

  8. WP 2.Sampling and characterization of the Mediterranean Sea sites. Marine water samples from the Venice lagoon The characterization of selected sediments in terms of content of C, N, P, heavy metals, density, pH is in progress. Samples of sediments obtained from two distinct non contaminated areas are also under characterization. In any case, we expect to receive some new samples of dreadged contaminated sediment s from the “Port Authority” and “Magistrato alle Acque” of Venice. Project meeting, Chania, Greece – July 3, 2011

  9. WP 2. Sampling and characterization of the Mediterranean Sea sitesMilestones & Deliverables Contributionto: MS3) Achievementofcoastalsamples:Obtainmentofdredgedsediment and water samplesfrom the VeniceLagoon site [month 6] D2.1) Report on the geochemicalcharacterizationof the sites: Geochemicalcharacterizationof the sedimentsobtainedfrom the VeniceLagoon [month 20] Project meeting, Chania, Greece – July 3, 2011

  10. WP 4.Establishment of collections of microbial isolates and metabolitesTasks & Activities • Task 4.2. Collection 2. Halogenated compounds degrading prokaryotes • Detection and characterization of native PCB reductive dechlorination processes as well as of microorganisms potentially responsible for them in contaminated sediments of the Venice lagoon. • Setting up of primary slurry microcosms of PCB-contaminated sediments of the Venice lagoon site under in situ like biogeochemical conditions and assessment of the transformation of weathered PCBs via monitoring of: • (i) the concentration and type of PCBs occurring in the sediment • (ii) the concentration of the major native acceptors (SO42-, NO3- ) • iii) the production and composition of biogas • (iv) the structure and composition of the indigenous microbial community (with UNMIL) • Enrichment of the indigenous PCB-transforming microbial populations in sterile slurries inoculated with the most active primary microcosms, via: • (i) supplementation of exogenous PCB mixtures (Aroclors) at high concentrations • (ii) supplementation of electron donors (i.e., H2, lactate, pyruvate, H2 releasing factors) Project meeting, Chania, Greece – July 3, 2011

  11. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Enrichment cultures from sediment “E” Enriched anaerobic cultures were previously obtained from sediment E suspended in its overlaying water and spiked with 5 coplanar PCB congeners (100 mg/kg dry sediment each) for the first 3 culturing steps and with Aroclor 1254 (750 mg/kg dry sediment) at the fourth culturing step. 25% (v/v) 10% (v/v) 10% (v/v) M1C Sed. 25% v/v Coplanar PCBs (500 mg/kg) M2C Sed. 25% v/v Coplanar PCBs (500 mg/kg) M3C Sed. 10% v/v Coplanar PCBs (500 mg/kg) M1AR Sed. 20% v/v Aroclor 1254 (750 mg/kg) Static incubation in the dark at 30°C, in anaerobic conditions: headspace purged with filter-sterilized N2:CO2 (70:30); Autoclave-sterilized microcosms as abiotic controls Periodical sampling: PCB concentration (GC-ECD), Anions concentration (IC-CD), Methane produced (GC-TCD) Project meeting, Chania, Greece – July 3, 2011

  12. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Enrichment cultures from sediment “E” PCB chlorination degree Other microbial activities PCB congener distribution after 35 weeks M1AR CULTURE

  13. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Enrichment cultures from sediment “E” Further enrichment is underway in the presence of Aroclor 1254 (1000 mg/kg dry sediment) M2AR-20 Sed. 20% v/v Aroclor 1254 (1000 mg/kg) M2AR-10 Sed. 10% v/v Aroclor 1254 (1000 mg/kg) 2% (v/v) Inoculum M1AR Sed. 20% v/v Aroclor 1254 (750 mg/kg) M2AR-2 Sed. 2% v/v Aroclor 1254 (1000 mg/kg) Static incubation in the dark at 30°C, in anaerobic conditions: headspace purged with filter-sterilized N2:CO2 (70:30). Autoclave-sterilized microcosms as abiotic controls Periodical sampling: PCB concentration (GC-ECD), Anions concentration (IC-CD), Methane produced (GC-TCD), Methagenomic DNA extraction for molecular fingerprinting analyses (PCR-DGGE). Project meeting, Chania, Greece – July 3, 2011

  14. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Enrichment cultures from sediment “E” PCB chlorination degree Sulfate consumption after 3.5 weeks PCBs distribution after 3.5 weeks No methane has been accumulated under any condition so far

  15. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Cultures from sediment “F” Anaerobic primary cultures from sediment F suspended at 25% (v/v) in its overlaying water and spiked with Aroclor 1242 (500 mg/kg dry sediment) were developed ALC Supplemented with Methanol & Ethanol, 7.4 mM each ACD Supplemented with Formate & Acetate 7.4 mM each Non-amended Static incubation in the dark at 30°C, in anaerobic conditions: headspace purged with filter-sterilized N2:CO2 (70:30); Autoclave-sterilized microcosms as abiotic controls Periodical sampling: PCB concentration (GC-ECD), Anions concentration (IC-CD), Methane produced (GC-TCD) Project meeting, Chania, Greece – July 3, 2011

  16. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Cultures from sediment “F” Non-amended Distribution of PCB congeners after 32 months of incubation ACD Concentration (mmoles/kg dry sed.) ALC Non-amended Sterile ACD ALC PCB

  17. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Cultures from sediment “F” Sulphate-reducing and methanogenic activities ALC Sterile Non-amended ACD ALC ACD Non-amended Project meeting, Chania, Greece – July 3, 2011

  18. WP 4. Establishment of collections of microbial isolates and metabolitesActivities: Cultures from sediment “F” New enrichments were prepared from the primary cultures established with sediment F 3 weeks ago. Sediment was suspended at 20% (v/v) in its filter-sterilized overlaying water and spiked with Aroclor 1254 (1000 mg/kg dry sediment) Non-amended ACD ALC 2% v/v 2% v/v 2% v/v Non-amended-2 ACD-2 ALC-2 Static incubation in the dark at 30°C, in anaerobic conditions: headspace purged with filter-sterilized N2:CO2 (70:30) Incubation started 3 weeks ago. No results are available yet. Project meeting, Chania, Greece – July 3, 2011

  19. WP 4. Establishment of collections of microbial isolates and metabolitesMilestones & Deliverables Contribution to: MS8) Obtainment of mixed cultures: Obtainment of mixed cultures from dredged sediment and water from the Venice Lagoon [month 18] MS12) Selection of microbial cultures for bioremediation: Selection of microbial cultures for bioremediation of PCB contaminated sediments [month 24] D4.2) Establishment of collection 2: Collection of halogenated compounds degrading prokaryotes (Collection of PCBs dechlorinating cultures from the Venice Lagoon) [month 24] Project meeting, Chania, Greece – July 3, 2011

  20. Temporal distribution UNIBO RTD tasks D7.2. August 2013 D7.4. November 2013 Project meeting, Chania, Greece – July 3, 2011

  21. Thankyou!

  22. WP 7. Establishing new product/processes for bioremediationTasks & Activities (1) • Task 7.1. Using hydrocarbon-degrading enzymes and microorganisms to obtain valuable chemicals (fine chemicals, biosurfactants) • Comparison of the 10 most promising biosurfactant-producing isolates obtained in WP4 (by TUC) for their ability to grow and accumulate crude biosurfactants in presence of weathered crude oil in 1L stirred tank bioreactor under batch conditions via: • (i) haemolytic, qualitative drop collapsing, quantitative oil displacement and emulsification activity tests on cultural broths; • (ii) emulsifying activity at different pH, temperatures and salinity degree of the most promising, partially purified (solvent extraction and column fractionation) biosurfactants. • Ecotoxicity assessment of the selected most promising biosurfactants through an ecotoxicological biotest based on the use of the bioluminescent bacterium Vibrio fischeri Project meeting, Chania, Greece – July 3, 2011

  23. WP 7. Establishing new product/processes for bioremediationTasks & Activities (2) • Task 7.2. Development of small scale bioprocesses for the reductive dehalogenation of chlorinated compounds • Contribution to the development of 3L-upflow biofilm reactors packed with Vukopor (or other materials) for the continuous treatment of seawater contaminated with chlorinated solvents from Bizerte lagoon carried out by UNMIL: • (i) Process optimization in terms of organic load, hydraulic residence time and redox potential; • (ii) Process monitoring and assessment through chemical measurements (GC-ECD, IC-CD), ecotoxicity tests (Vibrio fisheri) and molecular microbial ecology techniques (16S rRNA genes and catabolic genes) Project meeting, Chania, Greece – July 3, 2011

  24. WP 7. Establishing new product/processes for bioremediationTasks & Activities (3) • Task 7.2. Development of small scale bioprocesses for the reductive dehalogenation of chlorinated compounds (continued) • Development of 2L-slurry phase anaerobic bioreactors for the batch, ex-situ treatment of actual site PCB-contaminated sediments of Porto Marghera (Venice Lagoon) at ambient temperature: • (i) Process optimization in terms of inoculum type (selected microbial consortia from WP3) and size, types and amounts of supplemented electron donors and biosurfactant types (selected in task 7.1) and amounts; • (ii) Process monitoring and assessment through chemical measurements (GC-ECD, IC-CD), ecotoxicity tests (Vibrio fisheri) and molecular microbial ecology techniques (16S rRNA genes and catabolic genes)(in collaboration with UMIL) Project meeting, Chania, Greece – July 3, 2011

  25. WP 7. Establishing new product/processes for bioremediationMilestones & Deliverables MS15. Optimization of the novel biodegradation processes [month 35] (i) Continuous treatment of seawater contaminated by chlorinated aliphatics in Packed Bed Bioreactors inoculated with the selected isolates/microbial consortia; (ii) Ex-situ batch treatment of PCB-contaminated sediments in slurry phase bioreactors inoculated with the selected microbial consortia. D7.2) Development of new biocatalytic processes [month 32]: biosurfactants production and use for enhancing the bioremediation of PCB contaminated sediments D7.4) Optimization of the conditions for reductive dechlorination processes [month 35] Project meeting, Chania, Greece – July 3, 2011

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