Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

How Instrument Transformers Influence Power Quality Measurements: A Proposal of Accuracy Verification Tests.

The integration of renewable energy sources on a large scale in the electrical energy distribution systems, as well as the widespread of non-linear loads, has led to a significant increase in power quality (PQ) disturbances. For this reason, PQ monitoring is also becoming a key task in medium voltage (MV) grids. The measurement of PQ at MV levels can only be performed using instrument transformers (ITs) to scale down the level of voltage and current to levels suitable for the input stage of PQ instruments. However, no international standards currently require the verification of the errors introduced by ITs in the measurement of PQ phenomena. Moreover, this issue is only partially addressed in the scientific literature, where papers dealing with specific and limited aspects of the problem can be found. For this reason, this paper aims to comprehensively assess the issue, proposing IT accuracy verification tests for different PQ parameters. First, a set of PQ phenomena relevant for IT testing is chosen, as well as the associated ranges of variation, based on a review of the enforced standards and the scientific literature. For each selected PQ phenomenon, possible performance indices and test waveforms are proposed. Finally, the proposed procedure is validated by applying it to the characterization of two different types of commercial voltage transformers.

A cytofluorimetric analysis of a Saccharomyces cerevisiae population cultured in a fed-batch bioreactor.

The yeast Saccharomyces cerevisiae is a reference model system and one of the widely used microorganisms in many biotechnological processes. In industrial yeast applications, combined strategies aim to maximize biomass/product yield, with the fed-batch culture being one of the most frequently used. Flow cytometry (FCM) is widely applied in biotechnological processes and represents a key methodology to monitor cell population dynamics. We propose here an application of FCM in the analysis of yeast cell cycle along the time course of a typical S. cerevisiae fed-batch culture. We used two different dyes, SYTOX Green and SYBR Green, with the aim to better define each stage of cell cycle during S. cerevisiae fed-batch culture. The results provide novel insights in the use of FCM cell cycle analysis for the real-time monitoring of S. cerevisiae bioprocesses.

Low Power Contactless Voltage Sensor for Low Voltage Power Systems.

Contactless measurements represent the desirable solution in many contexts, where minimal cabling is required or, in general, cabling is not possible. This paper presents a new contactless voltage sensor for low voltage power systems. It is based on a contactless capacitive probe, which surrounds the power cable. It has two concentric electrodes insulated by a shield. A low power analog conditioning circuit evaluates the power line voltage by measuring the current in one of the capacitances of the probe. All the single stages of the circuit have been designed by using low-power rail-to-rail operational amplifiers, supplied at 3.3 V, in order to minimize the power absorption. The sensor has been characterized in various conditions, with sine waves and distorted signals, varying the frequency and the harmonic distortion. The influence of the current, flowing into the power cable, on the voltage measurement has been evaluated too. It shows a good accuracy (lower than 0.3%) from 100 V to 300 V, with a power consumption less than 5 mW.

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

BACKGROUND: Microbial 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. RESULTS: A 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. CONCLUSIONS: The 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.

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1ß 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-1ß (IL-1ß), aiming at obtaining high cell densities and optimizing IL-1ß 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.

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.