Descripción: Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Descripción: The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Descripción: The effect of eliminating D-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP+ ratio was higher than the NADH/NAD+ ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP+. In 60-h fed-batch cultures, K24KL reached 41.9 g · liter-1 biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g · g-1, the highest reported using this substrate. © 2010, American Society for Microbiology.

Descripción: A recombinant E. coli strain (K24K) was constructed and evaluated For poly(3-hydroxybutyrate) (PHB) production from whey and corn steep liquor as main carbon and nitrogen sources. This strain bears the pha biosynthetic genes from Azotobacter sp. strain FA8 expressed from a T5 promoter under the control of the lactose operator. K24K does not produce the lactose repressor, ensuring constitutive expression of genes involved in lactose transport and utilization. PHB was efficiently produced by the recombinant strain grown aerobically in fed-batch cultures in a laboratory scale bioreactor on a semisynthetic medium supplemented with the agroindustrial by-products. After 24 h, cells accumulated PHB to 72.9% of their cell dry weight, reaching a volumetric productivity of 2.13 g PHB per liter per hour. Physical analysis of PHB recovered from the recombinants showed that its molecular weight was similar to that of PHB produced by Azotobacter sp. strain FA8 and higher than that of the polymer from Cupriavidus necator and that its glass transition temperature was approximately 20°C higher than those of PHBs from the natural producer strains. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Descripción: Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible. Copyright © 2007, American Society for Microbiology. All Rights Reserved.