A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: part I--Synthesis protocol.

In this study, a new biosynthetic tracer was developed to characterize the virus retention dynamics of membrane systems. This new tracer is a modified bacteriophage obtained by the grafting of enzymatic probes to an MS2 bacteriophage, one of the smallest non-pathogenic bacteria viruses, with an average diameter of about 30 nm. A protocol for the synthesis and purification of this new tracer was developed in this work. The production of this biosynthetic tracer was first qualitatively shown by a chromatographic characterization and an enzymatic test. The average number of probes grafted per phage was then quantified for three batches of tracers made from the same native phage suspension and the same batch of enzymatic probes. This quantification demonstrated the reproducibility of the synthesis protocol developed.

A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: part II. Biochemical and physicochemical characterizations of the new tracer.

In a previous work, a reproducible procedure to produce a new biosynthetic tracer was developed. This new tracer is an MS2 bacteriophage with enzymatic probes grafted on its surface, which can induce enzymatic activity of the tracer. In this paper, the biochemical and physicochemical characteristics of this new tracer are determined. A protocol was developed to determine the specific enzymatic activity kcat(TRACER) of the tracer, which was found to be 2.93±0.78×10(4) min(-1) on average. Physicochemical characterizations of this new tracer showed that it is representative of viruses and may thus be used as a virus surrogate to assess the virus retention of membrane systems inline. Notably, the mean diameter and molecular weight of the tracer were found to be respectively 64.1±0.3 nm and 12,140±3654 kDa, which are within the size and molecular weight ranges of pathogenic viruses carried by water. The tracer surface was also studied and revealed the considerable porosity of the grafted probe layer, with a mean porosity of 88%, which could explain why the zeta potential of the tracers (-14.34±1.66 mV) was nearly the same as that of the native MS2 phages. Finally, a comparison between filtration of the reference microorganism used for membrane performance assessment (the MS2 phage) and the tracer suspensions showed the same filtration behaviour.

Dynamic microbial response under ethanol stress to monitor Saccharomyces cerevisiae activity in different initial physiological states.

Dynamic Saccharomyces cerevisiae responses to increasing ethanol stresses were investigated to monitor yeast viability and to optimize bioprocess performance when gradients occurred due to the specific configuration of multi-stage bioreactors with cell recycling or of large volume industrial bioreactors inducing chemical heterogeneities. Twelve fed-batch cultures were carried out with initial ethanol concentrations (P(in)) ranging from 5 g l(-1) to 110 g l(-1) with three different inoculums in different physiological states in terms of viability and quantity of ethanol produced (P(o)). For a given initial cell viability of 50%, the time to reach the maximum growth rate and maximum ethanol production rate was dependent on the difference P(in) - P(o). Whatever the initial physiological state, when the initial ethanol concentration P(in) reached 100 g l(-1), the yeasts died. Experimental results showed that the initial physiological state of the yeast was the major parameter to determine, the microorganisms' capacities to adapt and resist environmental changes.

Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway.

Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.

Very high ethanol productivity in an innovative continuous two-stage bioreactor with cell recycle.

The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially capable of giving very high ethanol productivity-was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30 degrees Celsius and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l(-1) and ethanol concentrations of 31 and 65 g l(-1) were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l(-1) in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l(-1) h(-1)) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3 degrees GL) in the two-stage system.

Synergistic temperature and ethanol effect on Saccharomyces cerevisiae dynamic behaviour in ethanol bio-fuel production.

The impact of ethanol and temperature on the dynamic behaviour of Saccharomyces cerevisiae in ethanol biofuel production was studied using an isothermal fed-batch process at five different temperatures. Fermentation parameters and kinetics were quantified. The best performances were found at 30 and 33 degrees C around 120 g l(-1) ethanol produced in 30 h with a slight benefit for growth at 30 degrees C and for ethanol production at 33 degrees C. Glycerol formation, enhanced with increasing temperatures, was coupled with growth for all fermentations; whereas, a decoupling phenomenon occurred at 36 and 39 degrees C pointing out a possible role of glycerol in yeast thermal protection.

Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process.

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions--oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)--on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l(-1)), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l(-1) in 45 h) and average productivity (3.3 vs 2.6 g l(-1) h(-1)) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l(-1). We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l(-1) in 45 h) in order to achieve a highly competitive dynamic process.

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process.

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.