La Russa, Marco (2009) GREEN ALGAE SELECTION USEFUL FOR PHYCOREMEDIATION OF OLIVE-MILL WASTEWATERS AND INCREASE OF THEIR LIPID CONTENT BY GENETIC ENGINEERING. [Tesi di dottorato] (Inedito)

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Abstract

The main aim of this work was the selection and the characterization of algal strains useful for phycoremediation of olive-mill wastewaters (OMW). 100 algal strains from the ACUF collection of the Department of Biology of the University of Naples were screened for their capability to release enzymes with phenol-oxidase activity in the culture broth. The presence of enzymes with phenol-oxidase activity was revealed by ABTS enzymatic assay in 16 of the 100 analysed culture broths. Further enzymatic assays were carried out in order to: 1) evaluate the enzymatic activity of the culture broths in presence of ABTS and different phenolic compounds, as 2,6-DMP and syringaldazine; 2) evaluate the capability of the culture broths to decolourizate the azo dye RBBR. On the base of the results achieved with the enzymatic assays, two algal strains were selected in order to evaluate their capability to grow in, and to degrade the phenolic component of, OMW: Chlamydomonas pitschmannii and Scenedesmus vacuolatus. The two algae were grown for 21 days in OMW diluted with growth medium and the cell concentration and the total phenols amount were measured weakly. S. vacuolatus showed a capability to grow in presence of OMW, and to reduce the phenolic content, higher than C. pitschmannii. The second part of the PhD work was carried out at the Algae Biotechnology & Bioenergy research group of the University of Bielefeld; during this period the research was focused on the characterization of genes encoding for putative enzymes involved into the lipid biosynthetic pathway in the green alga Chlamydomonas reinhardtii. 5 genes encoding for putative diacylglycerol acyltransferases (CrDGAT1-5) were cloned into expression vector pGenD and used for the genetic manipulation of Chlamydomonas in order to obtain mutants able to overexpress these genes. For each gene, 1 to 3 mutants were able to over-express the new CrDGAT construct. The mutant with the highest expression level was selected by quantitative real time PCR analyses for phenotype characterization during the standard growth condition and during nitrogen and sulphur depletion. Number of cells, optical density and total amount of chlorophyll were evaluated in order to verify if the over-expression of these genes could affect the cell phenotype. The total lipid amount was measured by Nile Red fluorescence assay and compared with the wild type. During the normal growth condition no differences were noticed between mutants and wild type strain, demonstrating that the over-expression of these genes doesn’t affect the cell phenotype and suggesting the presence of a second bottleneck in the lipid biosynthetic pathway. When cultivated in nitrogen depletion, all cultures showed the highest lipid accumulation, however without differences between mutants and wild type strains. During the sulphur starvation, mutants D3-18 and D4-66 showed an increase in the lipid accumulation in average 2.0 and 1.8 times higher than the wild type.

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