The gastrula transition reorganizes replication-origin selection in Caenorhabditis elegans.

Although some features underlying replication-origin activation in metazoan cells have been determined, little is known about their regulation during metazoan development. Using the nascent-strand purification method, here we identified replication origins throughout Caenorhabditis elegans embryonic development and found that the origin repertoire is thoroughly reorganized after gastrulation onset. During the pluripotent embryonic stages (pregastrula), potential cruciform structures and open chromatin are determining factors that establish replication origins. The observed enrichment of replication origins in transcription factor-binding sites and their presence in promoters of highly transcribed genes, particularly operons, suggest that transcriptional activity contributes to replication initiation before gastrulation. After the gastrula transition, when embryonic differentiation programs are set, new origins are selected at enhancers, close to CpG-island-like sequences, and at noncoding genes. Our findings suggest that origin selection coordinates replication initiation with transcriptional programs during metazoan development.

Dielectric monitoring and respirometric activity of a high cell density activated sludge.

A novel method was developed to assess the viability of activated sludge present in a biological wastewater treatment process and signify its distinction from respirometric activity. The respirometric activity and viability of activated sludge at high cell density, such as typically encountered in membrane bioreactors, were investigated in batch and fed-batch systems. The method for measuring the viability of activated sludge was based on the sludge permittivity monitored online by a capacitive sensor. Results from permittivity measurement were compared with usual biological activity measurement through oxygen uptake rate determination. The similar downward trend was observed for both measurements. The respirometric activity and permittivity, respectively, reduced to 50% and 68% of initial value in the fed-batch system and 18% and 27% of initial value for the batch system which was due to quantitative and qualitative changes in the microbial culture in the activated sludge. The novel method allows to made distinction between viable versus dead and inactive versus active microbial cells in the activated sludge system and can be used for better and more efficient control of the biological processes.

Biomass characterization by dielectric monitoring of viability and oxygen uptake rate measurements in a novel membrane bioreactor.

The application of permittivity and oxygen uptake rate (OUR) as biological process control parameters in a wastewater treatment system was evaluated. Experiments were carried out in a novel airlift oxidation ditch membrane bioreactor under different organic loading rates (OLR). Permittivity as representative of activated sludge viability was measured by a capacitive on-line sensor. OUR was also measured as a representative for respirometric activity. Results showed that the biomass concentration increases with OLR and all biomass related measurements and simulators such as MLSS, permittivity, OUR, ASM1 and ASM3 almost follow the same increasing trends. The viability of biomass decreased when the OLR was reduced from 5 to 4 kg COD m(-3)d(-1). During decreasing of OLR, biomass related parameters generally decreased but not in a similar manner. Also, protein concentration in the system during OLR decreasing changed inversely with the activated sludge viability.

Determination of yeast viability during a stress-model alcoholic fermentation using reagent-free microscopy image analysis.

A dedicated microscopy imaging system including automated positioning, focusing, image acquisition, and image analysis was developed to characterize a yeast population with regard to cell morphology. This method was used to monitor a stress-model alcoholic fermentation with Saccharomyces cerevisiae. Combination of dark field and epifluorescence microscopy after propidium iodide staining for membrane integrity showed that cell death went along with important changes in cell morphology, with a cell shrinking, the onset of inhomogeneities in the cytoplasm, and a detachment of the plasma membrane from the cell wall. These modifications were significant enough to enable a trained human operator to make the difference between dead and viable cells. Accordingly, a multivariate data analysis using an artificial neural network was achieved to build a predictive model to infer viability at single-cell level automatically from microscopy images without any staining. Applying this method to in situ microscope images could help to detect abnormal situations during a fermentation course and to prevent cell death by applying adapted corrective actions.

Single-cell analysis of S. cerevisiae growth recovery after a sublethal heat-stress applied during an alcoholic fermentation.

Interest in bioethanol production has experienced a resurgence in the last few years. Poor temperature control in industrial fermentation tanks exposes the yeast cells used for this production to intermittent heat stress which impairs fermentation efficiency. Therefore, there is a need for yeast strains with improved tolerance, able to recover from such temperature variations. Accordingly, this paper reports the development of methods for the characterization of Saccharomyces cerevisiae growth recovery after a sublethal heat stress. Single-cell measurements were carried out in order to detect cell-to-cell variability. Alcoholic batch fermentations were performed on a defined medium in a 2 l instrumented bioreactor. A rapid temperature shift from 33 to 43 °C was applied when ethanol concentration reached 50 g l⁻¹. Samples were collected at different times after the temperature shift. Single cell growth capability, lag-time and initial growth rate were determined by monitoring the growth of a statistically significant number of cells after agar medium plating. The rapid temperature shift resulted in an immediate arrest of growth and triggered a progressive loss of cultivability from 100 to 0.0001% within 8 h. Heat-injured cells were able to recover their growth capability on agar medium after a lag phase. Lag-time was longer and more widely distributed as the time of heat exposure increased. Thus, lag-time distribution gives an insight into strain sensitivity to heat-stress, and could be helpful for the selection of yeast strains of technological interest.

Assessing yeast viability from cell size measurements?

During microbial cell cultures, environmental conditions affect cell physiology and subsequently process efficiency. Physiological changes result in changing cell morphology, such as cell size variations. The aim of this work was to study cell size evolution of a Saccharomyces cerevisiae population exposed to various stresses during alcoholic batch fermentations, and to evaluate the potential use of cell size measurements to infer cell viability. During a reference culture, without perturbation, viability as assessed by propidium iodide staining (PI) remained 100% and mean cell diameter was found to be above 5microm. A rapid temperature shift from 33 to 43 degrees C at 50gl(-1) of ethanol resulted in an immediate arrest of growth and triggered a progressive loss of viability from 100% to 0% and a decrease of mean cell diameter from 5.2 to 3.7microm. Cell size distribution curves obtained with a cell counter showed an increasing subpopulation of significantly smaller cells. At single-cell level, combined microscopy size measurements and PI staining showed that this subpopulation was exclusively composed of dead cells. Similar results were obtained after acetic acid or furfural additions. Accordingly, a multivariate data analysis was achieved to estimate the ratio of dead cells from cell size distributions obtained using the cell counter.

Identification of Saccharomyces cerevisiae strains for alcoholic fermentation by discriminant factorial analysis on electronic nose signals

An electronic nose (E-nose) coupled to gas chromatography was tested to monitor alcoholic fermentation by Saccharomyces cerevisiae ICV-K1 and Saccharomyces cerevisiae T306, two strains well-known for their use in oenology. The biomass and ethanol concentrations and conductance changes were measured during cultivations and allowed to observe the standard growth phases for both yeast strains. The two strains were characterized by a very similar tendency in biomass or ethanol production during the fermentation. E-nose was able to establish a kinetic of the production of aroma compounds production and which was then easy to associate with the fermentation phases. Principal Component Analysis (PCA) showed that the data collected by E-nose during the fermentation mainly contained cultivation course information. Discriminant factorial analysis (DFA) was able to clearly identify differences between the two strains using the four main principal components of PCA as input data. Nevertheless, the electronic nose responses being mainly influenced by cultivation course, a specific data treatment limiting the time influence on data was carried out and permitted to achieve an overall performance of 83.5 percent.