Carpine, Roberta (2017) Cyanobacteria for PHB production. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Cyanobacteria for PHB production
Creators:
CreatorsEmail
Carpine, Robertaroberta.carpine@gmail.com
Date: 5 April 2017
Number of Pages: 105
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Scuola di dottorato: Biotecnologie
Dottorato: Biotecnologie
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Marzocchella, AntonioUNSPECIFIED
Olivieri, GiuseppeUNSPECIFIED
Hellingwerf, J. KlaasUNSPECIFIED
Date: 5 April 2017
Number of Pages: 105
Uncontrolled Keywords: Cyanobacteria, PHB, bioplastic
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 09 - Ingegneria industriale e dell'informazione > ING-IND/25 - Impianti chimici
Date Deposited: 18 Apr 2017 13:10
Last Modified: 08 Mar 2018 08:44
URI: http://www.fedoa.unina.it/id/eprint/11515
DOI: 10.6093/UNINA/FEDOA/11515

Abstract

The study carried out during the present Ph.D. program aimed at investigating the PHB production process in autotrophic cultures of cyanobacteria. The work was carried out at the Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale of the University of Naples ‘Federico II’ and at Molecular Microbial Physiology Group of the Swammerdam Institute for Life Sciences in Universiteit van Amsterdam. The activities were articulated according to three paths: i) the characterization of PHB production process as regards kinetics and yields using different growth media characterized by different nitrate concentration and screening cyanobacteria strains; ii) the characterization of the PHB production process by cyanobacteria according to kinetic models; iii) the development of genetic engineering approach on cyanobacteria to improve PHB production. 1)Selection of the optimal growth media to produce PHB and screening of cyanobacteria strains: characterization in terms of kinetics and yields. The study was aimed at the assessment of both the kinetics and the yields of the cell growth and PHB produced during the growth on different growth media characterized by different nitrate concentrations: BG11 (optimal nitrate concentration), BG1/2 (half of optimal nitrate concentration), BG1/4 (one fourth of the optimal nitrate concentration) and BG0 (nitrogen-starved conditions). Batch tests were focused on the preliminary characterization of the PHB production process from cyanobacteria with the aim of highlighting the relevant features of the process. Synechocystis PCC6803 was growth as model strains on the different culture media. The investigation was carried out in photobioreactors using CO2 as carbon source. The best media for PHB production was BG1/2 (half of optimal nitrate concentration). Five cyanobacteria strains were screened using BG1/2 medium, to select a high PHB producer: Synechocystis PCC6803, Synechocystis aquatilis, Synechocystis fuscopigmentosa, Synecoccoccus nidulans and Chlorogloeopsis Fritshii. 2)Characterization of the PHB production process: kinetic models. An adequate kinetic model to describe the PHB production process is a key issue to address the conditions to maximize the PHB fraction in the cell. A kinetic model of PHB production by Synechocystis PCC6803 from CO2 was proposed using the biochemical networks simulator COPASI. Data form growth tests under dynamic light system (light/dark cycle) carried out at different initial nitrate concentration were used to assess the kinetic and stoichiometric parameters of the proposed model. Two types of cells were considered in the cultures: growing cells (X), which are able to grow, and PHB producing cells (XPHB). The specific velocity (kT) of XPHB formation was considered dependent from the initial concentration of nitrate. The proposed model includes the nitrate and phosphate utilization rate, the PHB production rate, the cell growth and lysis rate. The model adequately predicted the experimental data, indeed the square correlation coefficient of metabolite concentrations, calculated by comparing experiments and simulations, ranged between 0.81 and 0.99. 3)Genetic modified cyanobacteria strain to improve PHB production. In this study Synechocystis sp. PCC6803 was genetically modified with aim to improve PHB production from CO2. Seven mutant strains were created by single and combined mutations: deletion of phosphotransacetylase (pta); the deletion of acetyl-CoA hydrolase (ach); the overexpression of phosphoketolase (xfpk). The tests were carried out on BG11 medium. The mutant strain that gave best performance on BG11 medium was tested on optimized medium (BG1/2 medium).

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