Controlled autolysis facilitates the polyhydroxyalkanoate recovery in Pseudomonas putida KT2440.
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.
Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, Madrid, Spain.Show MeSH
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Mentions: Pseudomonas putida BXHL strain was monitored by transmition electronic microscopy (Fig. 9) showing that, similarly to KTHL strain, the addition of 3MB generated deep alterations in the cell envelopes but most of the granules remained inside the cells. Remarkably, when we tested the susceptibility of P. putida BXHL strain grown in rich medium to the effect of membrane disrupting agents such as DOC, SDS and EDTA, we observed that this strain was even more sensitive than P. putida KTHL mutant, suggesting that tolB mutation contributes to alter the stability of cell envelope. As an example, Fig. 7 shows the growth curves of P. putida strains in LB medium with and without inducer and in the presence or absence of 0.075% DOC. It can be observed that induced P. putida BXHL is up to three times more sensitive to the detergent than induced P. putida KTHL. In addition, we compared the PHA granule release in P. putida wild type and BXHL mutant strains after simultaneous incubation with 10 mM EDTA and 0.1% SDS. In this experiment, both strains were cultured using the two steps culture fermentation system with 3MB. After 24 h of incubation in the PHA production conditions media (this is, the end of second step), the cultures were centrifuged and resulting sediments were suspended in distilled water supplemented with 10 mM EDTA and 0.1% SDS. After 8 h of incubation at room temperature, cultures were subjected to sucrose density step‐gradient ultracentrifugation for analysing the presence of PHA granules in the culture supernatant (Fig. 10). It is worth to mention that none viable cell was detected at this condition. P. putida BXHL induced to self‐disruption and incubated with the chemical agents (Fig. 10A, tube 2) showed a much thicker white band when compared with that of the wild‐type strain (Fig. 10A, tube 1). These findings indicated the release of almost all granules to the extracellular medium. To confirm these results, the intracellular PHA content of the cell fraction present in the sediments after the ultracentrifugation step of the 3MB induced P. putida BXHL strain incubated with and without the chemical agents was quantified by GC‐MS (see Experimental procedures). When no chemical agents were added, the PHA content present in the culture sediment was 0.7 g l−1, similar to that found in the total culture (0.86 g l−1). However, PHA was not detected at all in the sediment of cells incubated with 10 mM EDTA and 0.1% SDS, suggesting that this sediment is only composed of cell debris and that most of PHA (93% of CDW) was released to the extracellular medium, as shown in Fig. 10B.
Affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, Madrid, Spain.