Growth of Schizosaccharomyces pombe on glucose-malate mixtures in continuous cell-recycle cultures. Kinetics of substrate utilization.

The aerobic growth of Schizosaccharomyces pombe on mixtures of glucose and malate was investigated during continuous high cell density cultures with partial cell-recycle using a membrane bioreactor. Determination of the specific metabolic rates relative to substrates and products allowed the capacity of the yeast to metabolize malic acid under both oxidative metabolism (carbon limited cultures) and oxidofermentative metabolism (carbon sufficient cultures) situations to be characterized. Under carbon limiting conditions, the specific rate of malate utilization was dependent on the residual concentration and a limit for a purely oxidative breakdown without ethanol formation was observed for a characteristic ratio between the rates of substrate consumption qM/qG of 1.63 g.g-1. In addition, the mass balance analysis revealed the incorporation of malic acid into biomass. In carbon excess environments, the specific rate of malate utilization was dependent on both the residual malate and the specific rate of glucose consumption indicating that in addition to its conversion into ethanol malate can be respiratively metabolized for qM/qG ratios higher than 0.4 g.g-1.

Characterization of monascidin A from Monascus as citrinin.

Following our investigations on red pigments and monascidin co-production by Monascus species, the antibiotic called monascidin A was characterized as citrinin. Evidence was given by qualitative methods, mass spectra and NMR. Citrinin, a nephrotoxic agent was produced both by Monascus purpureus and Monascus ruber, either in submerged culture of concentrations of 270 and 340 mg/l, respectively, or in solid state culture of concentration of 100 and 300 mg/kg dried matter, respectively. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives from Monascus spp. avoid the occurrence of citrinin.

Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose.

The metabolic and energetic characterization of the growth of Leuconostoc oenos on glucose-citrate or glucose-fructose mixtures enables the potential role of this bacterium in the wine-making process to be ascertained. Moreover, mixotrophic conditions remain a suitable means for improving biomass productivities of malolactic starter cultures. When the malolactic bacterium L. oenos was grown in batch cultures on complex medium at pH 5.0 with glucose-citrate or glucose-fructose mixtures, enhancement of both the specific growth rate and biomass production yields was observed. While growth was possible on fructose as the sole source of energy, citrate alone did not allow subsequent biomass production. The metabolic interactions between the catabolic pathways of the glucose cosubstrates and the heterofermentation of hexoses led to an increased acetate yield as a result of modified NADH oxidation. However, the calculated global coenzyme regeneration showed that the reducing equivalent balance was never equilibrated. The stimulatory effects of these glucose cosubstrates on growth resulted from increased ATP synthesis by substrate-level phosphorylation via acetate kinase. While the energetic efficiency remained close to 10 g of biomass produced per mol of ATP, the increase in the specific growth rate and biomass production yields was directly related to the rate and yield of ATP generation.

Production of single-cell oil from prickly-pear juice fermentation by Cryptococcus curvatus grown in batch culture.

The biomass of Cryptococcus curvatus, an oleaginous yeast, reached 11 g/l and accumulated 46% (w/w) lipid when grown for 35 h in batch culture on diluted (25%) prickly-pear juice. The C:N ratio of the juice was about 50 g/g. The efficiency of substrate conversion was 0.48 g/g for biomass and 0.22 g/g for lipids. The extracted lipids were mainly oleic (18:1) and palmitic (16:0) acids and the quality of pipid approached that of palm oil.

Efficiency of fatty acid synthesis by oleaginous yeasts: Prediction of yield and fatty acid cell content from consumed C/N ratio by a simple method.

In nitrogen-limited media, growth and fatty acid formation by the oleaginous yeast Rhodotorula glutinis, i.e., yield and fatty acid cell content, have been characterized regarding carbon and nitrogen availabilities. It was shown that the formation of fatty acid free biomass was limited by nitrogen availability, whereas the fatty acid production was directly dependent on the consumed C/N ratio. According to these observations, the fraction of substrate consumed for fatty acid synthesis was estimated by using a simple method based on the actual yields, i.e., the mass of carbon source strictly converted into fatty acids and fatty acid free biomass. From these results, relationships were established allowing to predict in a simple and performing manner the maximal attainable fatty acid cell content and yield from the available carbon and nitrogen. These relationships were validated by using experimental data obtained by various authors with different yeast strains, and the proposed method was compared to the energetic and mass balance method previously described.

Carbon and energy balances in cell-recycle cultures of Schizosaccharomyces pombe.

A strain of the fission yeast Schizosaccharomyces pombe was aerobically grown in a cell-recycle fermentor under various operating conditions, i.e., different bleeding rates and various separate feed rates of glucose and basal medium. Carbon and energy balances were analyzed during steady-state culture regimes, allowing growth yields and maintenance coefficients to be determined under glucose-limited and glucose-excess environments. Special attention was given to the metabolic shift from purely oxidative to respirofermentative glucose catabolism resulting from a change in the growth-limiting factor. No maintenance requirements for the carbon source and for energy were observed during glucose-limited culture regimes and oxidative catabolism. Under glucose excess and respirofermentative metabolism, the m(G) coefficient was shown to be growth-linked, whereas the enhancement of the apparent m(e) coefficient observed for increased residual glucose concentrations could be assigned to a decline in the ATP yield.

Estimation of the energetic biomass yield and efficiency of oxidative phosphorylation in cell-recycle cultures of Schizosaccharomyces pombe.

The energetics of growth of the fission yeast Schizosaccharomyces pombe was studied in continuous high-cell concentration cultures using a cell-recycle fermentor. Under non-O2-limited conditions, steady-states were obtained at various specific growth rates (partial cell-recycle) with purely oxidative (glucose limitation) or respiro-fermentative (glucose excess) metabolic behaviour. The stoichiometry of biomass synthesis was established from the elemental composition of the cells and measurements of all the specific metabolic rates, i.e. consumption of glucose and O2 and production of CO2, ethanol and other products. The theoretical yield factor for biomass on glucose was YG,X = 0.85 C-mol.C-mol-1 and maintenance requirements were negligible. Assuming a constant coupling between energy generation and biomass formation for both respirative and respiro-fermentative breakdown of glucose, the biomass yield from ATP (YATP) and the efficiency of oxidative phosphorylation (P/O ratio) could be determined as 9.8 g biomass.mol ATP and 1.28 mol ATP.atom of O2, respectively.

Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures.

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation of glucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass.mol ATP-1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP.mol of glucose-1. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP.mol of glucose-1, or more correctly an energy gain of 0.28 mol of ATP.mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+.mol of malate-1(depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactate -/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate -/H+ symport, though a low-affinity malate- uniport was also implicated. The measured electrical component (delta psi) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.

High-concentration cultivation of Lactococcus cremoris in a cell-recycle reactor.

High-cell concentration cultivation of Lactococcus cremoris, a homofermentative lactic acid producer, in a cell-recycle fermentor is described. Cross-flow filtration allowing continuous removal of the inhibitory metabolite, the influence of dilution rate on growth was investigated in total or partial cell-recycle cultures. The dependence of growth characteristics on operating conditions was identified and quantified using lactose as the carbon source. Growth kinetics could be described by both lactate removal efficiency and nutrient availability. Based on physiological observations, biomass and lactic acid productivities were predicted in partial cell-recycle cultures.

Effects of Partial O(2) Pressure, Partial CO(2) Pressure, and Agitation on Growth Kinetics of Azospirillum lipoferum under Fermentor Conditions.

Azospirillum lipoferum crt1 was grown in batch cultures under standard conditions at 85% saturation of dissolved oxygen (DO) and 30-g/liter glucose concentrations. Kinetic studies revealed nutritional limitations of growth and the presence of an initial lag phase prior to consumption of glucose. The influences of various gaseous environments and shear stress on growth, i.e., various conditions of agitation-aeration, were characterized. Faster growth in the first stages of the culture and shorter duration of the lag phase were observed at DO concentrations of <30% saturation. The possible influences of dissolved CO(2) concentration or shear stress or both were discounted, and we confirmed the detrimental effect of high DO levels (up to 80% saturation) and the favorable influence of low DO concentrations (lower than 30% saturation) on growth. It was concluded that the gaseous environment, i.e., the DO concentration, needs to be considered as an operating parameter and be accurately controlled to ensure optimal growth of Azospirillum cells.

Determination of maintenance coefficients of Saccharomyces cerevisiae cultures with cell recycle by cross-flow membrane filtration.

The fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single-stage process with recycle of the cells via cross-flow micro-filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.

Alkaloid accumulation in Ca-alginate entrapped cells of Catharanthus roseus: Using a limiting growth medium.

The limitation of growth of Catharanthus roseus cells was investigated with a view to their entrapment in a Ca-alginate matrix. An examination of the effects of lowered 2,4-D and phosphate concentrations on cell viability and indole alkaloid biosynthesis enabled a growth limiting and product formation stimulating medium to be designed. Entrapped cells showed a retention of both respiratory activity and biosynthetic capacity over an extended period of time compared with free cells. Evidence is presented which suggests that immobilization in Ca-alginate beads acts to stabilize cells, resulting in enhanced product accumulation.

[Metabolic activity of Candida lipolytica adsorbed to bentonite with hydrophobic chains]. / Activité métabolique de Candida lipolytica adsorbé sur bentonites à chaînes hydrophobes

The respiration of Candida lipolytica on n-tetradecane is decreased in the presence of bentonite. This inhibition is less pronounced by the introduction of hydrophobic chains on the bentonite. Oxygen demand of resting cells varies with the length of hydrophobic chains. In fermentor, addition of adsorbant does not stimulate growth, but assimilation of n-tetradecane is enhanced with certain concentrations of the adsorbant. On glucose, addition of hydrophobic chains on the adsorbant does not change the effects of bentonite on exogenous respiration and substrate assimilation.

Lethal effects of electric current on Escherichia coli.

An attempt has been made to use low-voltage alternating current to kill microorganisms such as Escherichia coli. The bactericidal effect depends on the energy passing through the suspension and on the time during which the cells are left standing in the medium after the treatment. Most of the toxicity is due to an indirect effect developed with unalterable electrodes in the presence of chlorides in the medium. This method might be applied to eliminate pollution of natural waters.