Yarrowia lipolytica lipase production enhanced by increased air pressure

To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l−1 at 1 bar to 533·5 U l−1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high-density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity

Lipase induction in Yarrowia lipolytica for castor oil hydrolysis and its effect on γ-decalactone production

γ-Decalactone is an aromatic compound of industrial interest, resulting from the biotransformation of ricinoleic acid, the major constituent of castor oil. In order to increase the availability of the substrate to the cells for the aroma production, castor oil previously hydrolyzed can be used. This hydrolysis may be promoted by enzymatic action, more specifically by lipases. In this work, the influence upon the aroma production of the lipase produced by Yarrowia lipolytica, a microorganism able to carry out the biotransformation, was studied. In a first approach, lipase induction conditions were analyzed using different Y. lipolytica strains and culture conditions, such as the inoculation mode of the lipase production medium. Lipase production was not affected by the cells centrifugation, so this step was eliminated, reducing the time and phases of the process. Moreover, Y. lipolytica W29 was shown to be the most adequate strain for lipase production. To investigate the importance of castor oil hydrolysis, the pre-addition of an inducer of lipase production (olive oil) to the biotransformation medium was tested. Results showed that the highest aroma production (1,600 mg L−1) was obtained without a lipase inducer. However, the pre-induction of lipase decreased the lag phase for γ-decalactone secretion.The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/28039/2006 and SFRH/BD/63701/2009). They also acknowledge the kind supply of Yarrowia lipolytica IMUFRJ 50862 by Professor Alice Coelho from UFRJ, Brazil, and the supply of Y. lipolytica W29 by Prof. Yves Wache, ENSBANA, Dijon, France

Choice of Steel Material to Avoid Brittle Fracture for Hollow Section Structures

European cold-formed hollow sections in general exhibit better toughness properties than required by EN 10219. However, limits in applying the toughness related rules for the choice of steel material in EN 1993 1 10 to cold-formed hollow sections still constitute barriers to free marketing. By requests from European producers a conservative assessment procedure has been developed which is based on toughness measurements and a concept using effective strains. Due to the cold-forming process such plastic strains may occur in the bent areas of the profiles associated with a noticeable reduction in material toughness. For the degradation of these toughness properties an appropriate temperature shift Delta Tcf has been derived for both circular and rectangular hollow sections. In order to guarantee the proper application of this temperature shift Table 2.1 in EN 1993 1 10 had to be extended to lower temperatures down to -120 °C.JRC.G.5-European laboratory for structural assessmen

Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography

<p>Abstract</p> <p>Background</p> <p>During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells <it>in vitro </it>and <it>in vivo</it>. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.</p> <p>Methods</p> <p>Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.</p> <p>Results</p> <p>Our results show that unconcentrated LV vector stocks with titers in excess of 10⁸transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm²tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 10¹⁰TU were recovered from a single HYPERFlask vessel.</p> <p>Conclusion</p> <p>The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.</p

Influence of fluid dynamic conditions on 1,3-propanediol production from glycerol by Shimwellia blattae: Carbon flux and cell response

The fluid dynamic conditions play a key role in the development and scaling up of bioprocesses. In aerobic cultures, oxygen is an essential substrate for microbial growth, production and culture maintenance; an effective gas-liquid transfer must be achieved. Changes in fluid dynamics due to stirrer speed can affect negatively to the culture, causing hydrodynamic stress (increasing shear stress) or oxidative stress (by an increase of available oxygen in the liquid phase)

Yarrowia lipolytica growth under increased air pressure: influence on enzymes production

Improvement of microbial cell cultures oxygenation can be achieved by the increase of total air pressure, which increases oxygen solubility in the medium. In this work, a pressurized bioreactor was used for Yarrowia lipolytica batch cultivation under increased air pressure from 1 to 6 bar. Cell growth was strongly enhanced by the pressure rise. Fivefold and 3.4-fold increases in the biomass production and in specific growth rate, respectively, were observed under 6 bar. The increase of oxygen availability caused the induction of the antioxidant enzyme superoxide dismutase, which indicates that the defensive mechanisms of the cells against oxidative stress were effective and cells could cope with increased pressure. The pregrowth of Y. lipolytica under increased pressure conditions did not affect the lipase production ability of the cells. Moreover, the extracellular lipase activity increased 96% using a 5-bar air pressure instead of air at 1- bar pressure during the enzyme production phase. Thus, air pressure increase in bioreactors is an effective mean of cell mass and enzyme productivity enhancement in bioprocess based in Y. lipolytica cultures

Behaviour of Composite Beams Made Using High Strength Steel

BRIE