PRODUÇÃO DE POLIHIDROXIALCANOATOS UTILIZANDO SUBSTRATOS RESIDUAIS

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Feeding strategies for tuning poly (3-hydroxybutyrate-co-4-hydroxybutyrate) monomeric composition and productivity using Burkholderia sacchari

Publication . Raposo, Rodrigo S.; Almeida, M. Catarina M. D. de; Fonseca, M. M. R. da; Cesário, M. Teresa

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-4HB)) co-polymers were produced at bench-scale in fed-batch cultivations by Burkholderia sacchari from glucose (main carbon-source) and gamma-butyrolactone (GBL) as co-substrate. As P(3HB-4HB) properties highly depend on the 4-hydroxybutyrate (4HB) molar fraction, it is advantageous to have a thorough knowledge of the process in order to promote the production of the targeted final product. In this work, polymers with a 4HB molar percentage ranging from 1.5 to 8.4% (mol/mol) were obtained as consequence of a fine tuning of the fed-batch operation conditions, namely regarding the co-substrate feeding rate and its addition time, as GBL is toxic to B. sacchari cells. The best results regarding both the 4HB incorporation (molar%) and the co-polymer productivity (7.1% and 1.1g/(L.h) respectively) were reached when a pulse of GBL (<10g/L) was added early in the accumulation phase followed by a constant GBL addition at a rate similar to that of consumption so that a steady co-substrate concentration in the medium was maintained.

2017-12Journal article

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A Burkholderia sacchari cell factory: production of poly-3-hydroxybutyrate, xylitol and xylonic acid from xylose-rich sugar mixtures

Publication . Raposo, Rodrigo S.; Almeida, M. Catarina M. D. de; Fonseca, M. Manuela da; Cesário, M. Teresa

"Efficient production of poly-3-hydroxybutyrate (P(3HB)) based on glucose-xylose mixtures simulating different types of lignocellulosic hydrolysate (LCH) was addressed using Burkholderia sacchari, a wild strain capable of metabolizing both sugars and producing P(3HB). Carbon catabolite repression was avoided by maintaining glucose concentration below 10g/L. Xylose concentrations above 30g/L were inhibitory for growth and production. In fed-batch cultivations, pulse size and feed addition rate were controlled in order to reach high productivities and efficient sugar consumptions. High xylose uptake and P(3HB) productivity were attained with glucose-rich mixtures (glucose/xylose ratio in the feed=1.5w/w) using high feeding rates, while with xylose-richer feeds (glucose/xylose=0.8w/w), a lower feeding rate is a robust strategy to avoid xylose build-up in the medium. Xylitol production was observed with xylose concentrations in the medium above 30-40g/L. With sugar mixtures featuring even lower glucose/xylose ratios, i.e. xylose-richer feeds (glucose/xylose=0.5), xylonic acid (a second byproduct) was produced. This is the first report of the ability of Burkholderia sacchari to produce both xylitol and xylonic acid."

2017-01Journal article

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Polyhydroxyalkanoates: waste glycerol upgrade into electrospunfibrous scaffolds for stem cells culture

Publication . Canadas, Raphaël F.; Cavalheiro, João M.B.T.; Guerreiro, João D.T.; Almeida, M. Catarina M.D. de; Pollet, Eric; Silva, Cláudia Lobato da; Fonseca, M.M.R. da; Ferreira, Frederico Castelo

"This integrated study shows that waste glycerol can be bio-valorized by the fabrication of electrospun scaffolds for stem cells. Human mesenchymal stem cells (hMSC) provide an interesting model of regenerating cells because of their ability to differentiate into osteo-, chrondro-, adipo- and myogenic lineages. Moreover, hMSC have modulatory properties with potential on treatment of immunologic diseases. Electrospun fiber meshes offer tunable mechanical and physical properties that can mimic the structure of the native extracellular matrix, the natural environment where cells inhabit. Following a biorefinery approach, crude glycerol directly recovered from a biodiesel post-reaction stream was fed as major C source to Cupriavidus necator DSM 545 to produce polyhydroxyalkanoates at polymer titers of 9–25 g/L. Two of the P(3HB-4HB-3HV) terpolymers produced, one containing 11.4% 4HB and 3.5% 3HV and the other containing 35.6% 4HB and 3.4% 3HV, were electrospun into fibers of average diameters of 600 and 1400 nm, respectively. hMSC were cultured for 7 days in both fiber meshes, showing their ability to support stem cell growth at acceptable proliferation levels. Comparative results clearly demonstrate that scaffold topology is critical, with electrospun PHA fibers succeeding on the support of significant cell adhesion and proliferation, where planar PHA films failed."

2014-11Journal article

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Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone

Publication . Cesário, M. Teresa; Raposo, Rodrigo S.; Almeida, M. Catarina M. D. de; Van Keulen, Frederik; Ferreira, Bruno S.; Telo, João P.; Fonseca, M. Manuela R. da

"Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate) both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be excellent carbon sources for P(3HB) production. Using a similar feeding strategy developed for P(3HB) production based on WSH, fedbatch cultures were developed aiming at the production of the copolymer P(3HB-co- 4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gammabutyrolactone (GBL) as precursor of the 4HB units. Fed-batch cultures using glucose as carbon source (control) and GBL were developed to achieve high copolymer productivities and 4HB incorporations. The attained P(3HBco- 4HB) productivity and 4HB molar % were 0.7 g/(L·h) and 4.7 molar %, respectively. The 4HB incorporation was improved to 6.3 and 11.8 molar % by addition of 2 g/L propionic and acetic acid, respectively. When WSH were used as carbon source under the same feeding conditions, the values achieved were 0.5 g/(L·h) and 5.0 molar %, respectively. Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB) using gamma butyrolactone as precursor."

2014-11Journal article

Open access

Efficient P(3HB) extraction from Burkholderia sacchari cells using non-chlorinated solvents

Publication . Rosengart, Alessandro; Cesário, M.Teresa; Almeida, M. Catarina M.D. de; Raposo, Rodrigo S.; Espert, Ana; Díaz de Apodaca, Elena; Fonseca, M. Manuela R. da

"A technique using safer, non-chlorinated organic solvents for the extraction of poly-3-hydroxybutyrate (P(3HB)) from bacterial cells was developed, aiming to attain high recovery yields and purities. Some solvents were selected from the GlaxoSmithKline guide as sustainable industrial solvents and the solubility of P(3HB) calculated using predictive equations from literature. Based on the calculated solubility values, anisole, cyclohexanone and phenetole were tested as extraction solvents and the relevant process variables (extraction temperature, extraction time and mass of cells/solvent volume ratio) were addressed. Polymer recovery yields of 97% and 93% were obtained with anisole and cyclohexanone, respectively, at 120–130 °C using a cell/solvent ratio of 1.5% (w/v). Maximum polymer purities using these experimental conditions were 98% for both solvents. The recovery yield and the polymer purity attained with chloroform (reference solvent) were 96 and 98%, respectively. Higher cell/solvent ratios of 6.0% (w/v) showed slightly lower recovery yields and purities. The average molecular weight and the thermal properties of the polymers extracted with the alternative solvents were fully comparable to those of the polymers obtained by chloroform extraction, demonstrating that the applied conditions did not significantly alter the properties of the extracted P(3HB)."

2015-11Journal article

Open access