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ULIXES-1 ST Year Meeting Capo Granitola , Mazara del Vallo , Italy January 22-2012»

ULIXES-1 ST Year Meeting Capo Granitola , Mazara del Vallo , Italy January 22-2012». FP7-KBBE-2010-4. Study of multiresistance to heavy metals , chlorinated compounds and some hydrocarbons of bacterial strains isolated from lagoon « Mar Ckika , Morocco ». FP7-KBBE-2010-4.

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ULIXES-1 ST Year Meeting Capo Granitola , Mazara del Vallo , Italy January 22-2012»

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  1. ULIXES-1ST Year MeetingCapo Granitola, MazaradelVallo, ItalyJanuary 22-2012» FP7-KBBE-2010-4

  2. Study of multiresistance to heavymetals, chlorinated compounds and somehydrocarbons of bacterialstrainsisolatedfromlagoon« Mar Ckika, Morocco » FP7-KBBE-2010-4 Presented by: Mr. BLAGHENMohammed PhDstudent: BENZHA Fatima

  3. • The Lagoon of Mar Chika (35°08'52''N, 2°50'53''O) is an area of ​​112 Km2, it is linked to the Mediterranean Sea by an artificial pass which is far from the shore by 7,5km. Mar Chika islocated on the northwest coast of Morocco Mediterranean in front of the Alboran Sea. • The lagoon is isolated from the Mediterranean by a strip of fine sand and dunes (25Km), while it had a communication channel to the sea by the key of Boucana. • Despite Mar Chika is of biological, ecological and landscape interest it is exposed to continuous pollution by the town of Nador, and also from agriculture and industries that discharge pollutants in the lagoon. MediterraneanSea

  4. «The lagoon of Mar Chika» Nador Laggon of Marc Chika Nador The lagoon of Mar Chika

  5. Materiels & Methods Sampling strategy Physicochemical analysis Bacteriological study

  6. • Samplingstrategy Samples (water and sediment) were carried from 15 sites (10 P; 5 NP)

  7. • PhysicochemicalAnalysis • Parametersmesasured in situ: pH, conductivity, temperature, dissolvedoxygen, salinity.

  8. • PhysicochemicalAnalysis Parameters measured in the laboratory : Chemical oxygen demand (COD), Alkaliniy (P.alkalinity, M.alkalinity), Calcium hardness, Total Hardness, Dosage of chloride. J. Rodier, B. Legube, N. Merlet (Juin2009) .L'analyse de l'eau: Eaux naturelles, eaux résiduaires, eau de mer

  9. Parameters measured in the laboratory: Chemicaloxygendemand (COD): is a measure of the capacity of water to consume oxygen during the decomposition of organic matter and the oxidation of inorganic chemicals such as ammonia and nitrite. COD measurements are commonly made on samples of waste waters or of natural waters contaminated by domestic or industrial wastes. . • Alkalinity : isdefined as the measure of the water capacity to neutralizeacids. There are two types of alkalinity : • Phenolphtalein Alkalinity • Methyl Orange Alkalinity Aminot A.; Chausspied M (1983): « Manuel des analyses chimiques en milieu marin » Organic http://science.jrank.org/pages/1388/Chemical-Oxygen-Demand.html#ixzz1Qh0kSt3N

  10. Parameters measured in the laboratory: Calciumhardness of water : is the concentration of Ca2+ in the water and generally represents a significant portion of total hardness. Total Hardness: is an indicator of mineralization of the water. It is the sum of the concentrations of metal cations with the exception of alkali metals and hydrogen ion, and it is mostly proportional to the concentration of calcium and magnesium which sometimes are added to the concentrations of iron, aluminum, manganese and strontium. Aminot A.; Chausspied M (1983): « Manuel des analyses chimiques en milieu marin » Organic http://science.jrank.org/pages/1388/Chemical-Oxygen-Demand.html#ixzz1Qh0kSt3N

  11. • Bacteriologicalstudy • Microbiological analysis is based on research of bacteria considered as indicators of water contamination. • The quality of sampling is an essential step for good analysis. • The sample must be taken in a sterilized bottle in a specific way to prevent accidental contamination or variation in the quality of the bacteria present at sampling.

  12. • Bacteriologicalstudy • 1- Isolation: The Isolates were cultivated on nutrient agar plates and purified for further screening. • 2- Identification: Using the gallery API 20E and API 20NE. • 3- Determination of minimum inhibitory concentrations: (heavy metals, chlorinated compounds and hydrocarbons) by preparing serial dilutions The growth was monitored by measuring the absorbance at 600nm using a Shimadzu UV-1800 spectrophotometer. • 4- Study of the behavior of bacteria resistant to chlorinated compounds: (Pentachlorophenol "PCP") by performing growth curves in the presence of PCP and glucose as a control, then we opted for an GS-MS ( Equipped with a general purpose column, temperature programmed from 80 to 280°C (10°C/min), Gas: helium 99.999% (1,46 ml/min), Injection volume: 1µl, Injector temperature: 270°C).

  13. Results & Discussion

  14. Results of the parametersmeasured in situ

  15. Results of the parametersmeasured in laboratory

  16. Thepollutedsite n° 4

  17. Results of the bacteriological party • The bacterial part revealed, at first, the existence of different bacterial strains that can be distinguished referring to nude eye observation. • The identification of this isolates revealed the presence of different strains, including the staphylococci, obvious referring to the color of the colonies (Orange, Yellow and White), streptococci, Enterobacteriaceae and non-Enterobacteriaceae . White Orange

  18. Identification of strainsisolatedfrom water S.Ficaria P. Aeruginosa A.Salmonicida R.Radiobacter

  19. Identification of strainsisolatedfromsediment P. Luteola Brev. Vesicularis Mannheimia. haemolytica O. Anthropi

  20. Tests of resistance of strainsisolatedfrom water 24h 48h 24h 48h 24h 48h 24h 48h Incubation Time Minimal inhibitory concentration of the tested heavy metals (mM)

  21. Tests of resistance of strainsisolatedfrom water Incubation Time 24h 48h 24h 48h 24h 48h 24h 48h Minimal inhibitory concentration of the tested hydrocarbons and chlorinated compounds (mM)

  22. Tests of resistance of strainsisolatedfromsediment Incubation Time 24h 48h 24h 48h 24h 48h 24h 48h Mannheimia. haemolytica Minimal inhibitory concentration of the tested heavy metals (mM)

  23. Tests of resistance of strainsisolatedfromsediment Incubation Time 24h 48h 24h 48h 24h 48h 24h 48h Mannheimia. haemolytica Minimal inhibitory concentration of the tested hydrocarbons and chlorinated compounds (mM)

  24. Study of the behavior of P.Aeruginosa in a different medium (glucose, PCP and anthracene) • Thus, the results show that the lag phase pass from 8 to 24h, when the carbon source is the PCP. • Concerning anthracene, this phase remains almost the same, in comparison with the glucose.

  25. Fig.3: GC spectrum of the sample standard PCP Results of GC-MS Fig.4: GC spectrum after 24h of incubation Fig.5: GC spectrum after 48h of incubation

  26. GC spectrum of the sample standard PCP MS spectrum of the sample standard PCP

  27. GC spectrum of the sample standard PCP MS spectrum of the sample standard PCP

  28. GC spectrum after 24h of incubation in the presence of Ochrobacter.Anthropi MS spectrum after 24h of incubation in the presence of Ochrobacter.Anthropi

  29. GC spectrum after 24h of incubation in the presence of Seratia.Ficaria MS spectrum after 24h of incubation in the presence of Seratia Ficaria

  30. GC spectrum after 24h of incubation in the presence of Brev.Vesicularis MS spectrum after 24h of incubation in the presence of Brev.Vesicularis

  31. GC spectrum of the sample standard Anthracene Fig.3: MS spectrum of the sample standard Anthracene

  32. GC spectrum after 24h of incubation in the presence of Brev.Vesicularis GC spectrum after 24h of incubation in the presence of Brev.Vesicularis

  33. Heavy metalsRemoval and Analysis

  34. Heavy metals Oligo elements (Fe, Cu, Zn...) Toxic metals (Pb, Hg, Cd...) Used in industry POLLUTION

  35. Mercury: toxic Metal • Source: - Natural - Anthropogenic • Transformation in the environment (CH3)2Hg  CH3Hg+ Hg2+ Hg0

  36. Mercury resistance Mercuric reductase Hg2+ Hg0 NADPH + H+ NADP+

  37. Fluidizedbedbioreactorused

  38. Mercury amounts evolution in both mercurial and oxidizing solutions Cleanup and volatilization rates obtained using fluidized bed bioreactor

  39. NADPH Hg2+ E-FADE-FADH2 Hg0 (Inactive) (Active) Electrochemistry

  40. Conclusions • These identified strains showed a significant minimal inhibitory concentrations concerning heavy metals, hydrocarbons and chlorinated compounds, and proved an interesting potential for biodegradation. • Thus, the obtained results may suggest that the isolated strains could be involved in bioremediation, in a heavily polluted area, and a feature that may prove to be extremely useful for use in the field of water purification. • These poly-resistantfactors, compared to thosereported in the literature, canbe a keyprocess of naturalattenuation, contemplated as an interesting option for variousbioremadiationprocess in view of contaminated sites. -Taoufik J., Zeroual Y., Moutaouakkil A., Moussaid S., Dzairi F.Z., Talbi M., Lee K., Loutfi M., Blaghen M. (2004) Aromatic hydrocarbons removal by immobilized bacteria (Pseudomonas sp., Staphylococcus sp.) in fluidized bed bioreactor. Annals Microbiology. 54: 189-200. - Marianna Czaplicka (2004). Sources and transformations of chlorophenols in the natural environment. Science of the total environment. 322: 21-39.

  41. Thank you for your attention

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