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Controlled autolysis facilitates the polyhydroxyalkanoate recovery in Pseudomonas putida KT2440.

Martínez V, García P, García JL, Prieto MA - Microb Biotechnol (2011)

Bottom Line: The development of efficient recovery processes is essential to reduce the cost of polyhydroxyalkanoates (PHAs) production.Our results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope.Conditions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solubility in organic solvents such as ethyl acetate.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, Madrid, Spain.

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Sucrose density step‐gradient ultracentrifugation of P. putida strains. Cells were grown under two steps PHA production conditions, with or without 3MB (5 mM) as inducer (see Experimental procedures). A. Tube 1, P. putida KT2440 wild type grown with 3MB; tube 2, P. putida KTHL grown without 3MB; tube 3, P. putida KTHL grown with 3MB; tube 4, P. putida KTHL grown with 3MB broken by a fourfold passage through a French press. The white band located at the interfaces of tubes 3 and 4 corresponds to the free PHA granules released to the extracellular medium. B. Transmition electronic microscopy views of mcl‐PHA accumulating P. putida KTHL cells. Samples were taken after 24 h growing under the two phases fermentation system in the presence (right) or absence (left) of 3MB inducer.
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f4: Sucrose density step‐gradient ultracentrifugation of P. putida strains. Cells were grown under two steps PHA production conditions, with or without 3MB (5 mM) as inducer (see Experimental procedures). A. Tube 1, P. putida KT2440 wild type grown with 3MB; tube 2, P. putida KTHL grown without 3MB; tube 3, P. putida KTHL grown with 3MB; tube 4, P. putida KTHL grown with 3MB broken by a fourfold passage through a French press. The white band located at the interfaces of tubes 3 and 4 corresponds to the free PHA granules released to the extracellular medium. B. Transmition electronic microscopy views of mcl‐PHA accumulating P. putida KTHL cells. Samples were taken after 24 h growing under the two phases fermentation system in the presence (right) or absence (left) of 3MB inducer.

Mentions: After fermentation, cultures were subjected to sucrose density step‐gradient ultracentrifugation to check the presence of PHA granules in the culture supernatant because of the cell disruption induced in the P. putida KTHL strain (Fig. 4A). This method allows the separation of the PHA granules from the cell fraction (non‐disrupted cells and cell debris) as PHA granules have a specific gravity lower than cell fraction, which sediments at the bottom of the tube (Moldes et al., 2004). In the absence of inducer, PHA granules were not released into the medium and sedimented together with the cell fraction (Fig. 4A, tube 2). However, in the presence of the 3MB inducer, a PHA white band was visible at the sucrose step‐gradient interface, indicating the release of the granules to the extracellular medium (Fig. 4A, tube 3). In the case of the wild‐type strain, extracellular PHA granules were not detected even in the presence of 3MB (Fig. 4A, tube 1). Thus, it was clearly demonstrated that PHA granules were effectively released by the self‐disruption system integrated into the recombinant P. putida KTHL strain, proving the potentiality of this system.


Controlled autolysis facilitates the polyhydroxyalkanoate recovery in Pseudomonas putida KT2440.

Martínez V, García P, García JL, Prieto MA - Microb Biotechnol (2011)

Sucrose density step‐gradient ultracentrifugation of P. putida strains. Cells were grown under two steps PHA production conditions, with or without 3MB (5 mM) as inducer (see Experimental procedures). A. Tube 1, P. putida KT2440 wild type grown with 3MB; tube 2, P. putida KTHL grown without 3MB; tube 3, P. putida KTHL grown with 3MB; tube 4, P. putida KTHL grown with 3MB broken by a fourfold passage through a French press. The white band located at the interfaces of tubes 3 and 4 corresponds to the free PHA granules released to the extracellular medium. B. Transmition electronic microscopy views of mcl‐PHA accumulating P. putida KTHL cells. Samples were taken after 24 h growing under the two phases fermentation system in the presence (right) or absence (left) of 3MB inducer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815265&req=5

f4: Sucrose density step‐gradient ultracentrifugation of P. putida strains. Cells were grown under two steps PHA production conditions, with or without 3MB (5 mM) as inducer (see Experimental procedures). A. Tube 1, P. putida KT2440 wild type grown with 3MB; tube 2, P. putida KTHL grown without 3MB; tube 3, P. putida KTHL grown with 3MB; tube 4, P. putida KTHL grown with 3MB broken by a fourfold passage through a French press. The white band located at the interfaces of tubes 3 and 4 corresponds to the free PHA granules released to the extracellular medium. B. Transmition electronic microscopy views of mcl‐PHA accumulating P. putida KTHL cells. Samples were taken after 24 h growing under the two phases fermentation system in the presence (right) or absence (left) of 3MB inducer.
Mentions: After fermentation, cultures were subjected to sucrose density step‐gradient ultracentrifugation to check the presence of PHA granules in the culture supernatant because of the cell disruption induced in the P. putida KTHL strain (Fig. 4A). This method allows the separation of the PHA granules from the cell fraction (non‐disrupted cells and cell debris) as PHA granules have a specific gravity lower than cell fraction, which sediments at the bottom of the tube (Moldes et al., 2004). In the absence of inducer, PHA granules were not released into the medium and sedimented together with the cell fraction (Fig. 4A, tube 2). However, in the presence of the 3MB inducer, a PHA white band was visible at the sucrose step‐gradient interface, indicating the release of the granules to the extracellular medium (Fig. 4A, tube 3). In the case of the wild‐type strain, extracellular PHA granules were not detected even in the presence of 3MB (Fig. 4A, tube 1). Thus, it was clearly demonstrated that PHA granules were effectively released by the self‐disruption system integrated into the recombinant P. putida KTHL strain, proving the potentiality of this system.

Bottom Line: The development of efficient recovery processes is essential to reduce the cost of polyhydroxyalkanoates (PHAs) production.Our results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope.Conditions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solubility in organic solvents such as ethyl acetate.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, Madrid, Spain.

Show MeSH
Related in: MedlinePlus