Carmine Landi

A novel process-based model of microbial growth: self-inhibition in Saccharomyces cerevisiae aerobic fed-batch cultures

AbstractBackgroundMicrobial population dynamics in bioreactors depend on both nutrients availability and changes in the growth environment. Research is still ongoing on the optimization of bioreactor yields focusing on the increase of the maximum achievable cell density.ResultsA new process-based model is proposed to describe the aerobic growth of Saccharomyces cerevisiae cultured on glucose as carbon and energy source. The model considers the main metabolic routes of glucose assimilation (fermentation to ethanol and respiration) and the occurrence of inhibition due to the accumulation of both ethanol and other self-produced toxic compounds in the medium. Model simulations reproduced data from classic and new experiments of yeast growth in batch and fed-batch cultures. Model and experimental results showed that the growth decline observed in prolonged fed-batch cultures had to be ascribed to self-produced inhibitory compounds other than ethanol.ConclusionsThe presented results clarify the dynamics of microbial growth under different feeding conditions and highlight the relevance of the negative feedback by self-produced inhibitory compounds on the maximum cell densities achieved in a bioreactor.

Effect of auxotrophies on yeast performance in aerated fed-batch reactor

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae, one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance.

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1β production: optimization, modeling, and physiological aspects

Saccharomyces cerevisiae CEN.PK113-5D, a strain auxotrophic for uracil belonging to the CEN.PK family of the yeast S. cerevisiae, was cultured in aerated fed-batch reactor as such and once transformed to express human interleukin-1β (IL-1β), aiming at obtaining high cell densities and optimizing IL-1β production. Three different exponentially increasing glucose feeding profiles were tested, all of them “in theory” promoting respiratory metabolism to obtain high biomass/product yield. A non-structured non-segregated model was developed to describe the performance of S. cerevisiae CEN.PK113-5D during the fed-batch process and, in particular, its capability to metabolize simultaneously glucose and ethanol which derived from the precedent batch growth. Our study showed that the proliferative capacity of the yeast population declined along the fed-batch run, as shown by the exponentially decreasing specific growth rates on glucose. Further, a shift towards fermentative metabolism occurred. This shift took place earlier the higher was the feed rate and was more pronounced in the case of the recombinant strain. Determination of some physiological markers (acetate production, intracellular ROS accumulation, catalase activity and cell viability) showed that neither poor oxygenation nor oxidative stress was responsible for the decreased specific growth rate, nor for the shift to fermentative metabolism.

Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay.

Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.

Production in fed-batch Reactor of Bacillus subtilis Lipase A immobilized on its own producer Saccharomyces cerevisiae cells

Production in Fed-Batch Reactor of Bacillus subtilis LipaseA Immobilized on its own Producer Saccharomyces cerevisiae Cells Lucia Paciello, Carmine Landi, Jesus Zueco, Palma Parascandola Department of Industrial Engineering, University of Salerno, via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy Department of Microbiology, University of Valencia, Avda Vicente Andres Estelles 46100, Burjassot, Valencia, Spain lpaciello@unisa.it