Cultivation of the yeast Candida lipolytica on hydrocarbons. II. One-stage continuous cultivation on gas oil

Continuous cultivation of the yeast Candida lipolytica on gas oil was studied from the viewpoint of biomass production and oil deparaffination. Optimum conditions wore found at the dilution rate D = 0.16–0.19 when biomass productivity 1.7 g/l/hr and yield coefficient. y = 0.92 were achieved. At deparaffination to the same freezing point, more than double the production of biomass and deparaffined oil during a given time unit was achieved in a continuous process than in batch cultivation. Consumption of substrate was followed in both cultivation processes and it was confirmed that individual n-alkanes of gas oil were degraded at various rates and yields. Results proved optimum cultivation conditions to depend on concentration and composition of the paraffinic fraction of gas oil used. To achieve these conditions the continuous process may be controlled by choice; of suitable dilution rate and concentration of gas oil.     

Growth of yeast on n-alkanes. I. Stochastic model

In a system where yeast cells grow on n-alkanes dissolved in oil drops suspended in water, the dispersed oil phase will, in most cases, be fully segregated. This means that each drop has its own history that depends on its degree of saturation with yeast cells. This degree of saturation with yeast cells is determined by a stochastic process depending on adsorption, desorption, and cell production. Although many authors mention segregation as a phenomenon likely to occur, so far this segregation has hardly been taken into account. In this paper the interaction of the population of completely segregated oil drops with the population of yeast cells, which results in growth, is described. The consequences of the model are elucidated by the discussion of some extreme cases. The batch fermentation of hydrocarbons by yeast cell is simulated by means of a Monte Carlo procedure. Adsorption, desorption, and production of yeast cells are considered as chance processes. The history of all individual drops is recorder. The influence of the chance of desorption appears to be much larger than that of the chance of adsorption (at the investigated range). Also the size of the inoculum at the start of the process appears to have a strong influence on the course of fermentation.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary Microsomal membrane fractions of the yeast Candida maltosa were investigated with respect to their ability to catalyse the oxidation of n-alkanes, fatty alcohols and fatty acids. Analysis of intermediates of n-hexadecane oxidation led to the conclusion that monoterminal attack was predominant, whereas diterminal oxidation proceeded as a minor reaction. The oxidation of long-chain primary alcohols to the corresponding aldehydes occurred without addition of nicotinamide adenine dinucleotide (phosphate) [NAD(P)⁺] and was accompanied by stoichiometric oxygen consumption and hydrogen peroxide production, suggesting that an alcohol oxidase instead of an NAD(P)⁺-requiring alcohol dehydrogenase catalysed these reactions. As shown for n-hexadecane, the hydroxylation of palmitic acid was found to be carbon monoxide-dependent, indicating involvement of a cytochrome P-450 system, as in the case of n-alkane hydroxylation.     

Degradation of long-chain n-alkanes by the yeast Lodderomyces elongisporus

Summary Cells of the yeast Lodderomyces elongisporus, precultured on glycerol, were incubated with long-chain n-alkanes. The results whow that monoterminal alkane oxidation is the main pathway of alkane degradation in the investigated yeast. The amount of diterminal activity is negligible, while subterminal degradation did not occur at all.Fatty acids were the first detectable intermediates. Using different n-alkanes, in every case the fatty acids with substrate chain length predominated in the cells. The formation of radioactive fatty acids from (1-¹⁴C)-hexadecane was time-dependent and indicated that desaturation elongation and -oxidation occurred.Extracellularly, the fatty acid pattern was similar, except for the additional presence of fatty acid methyl esters and the prevalence of octadecenoic acid after growth of cells on n-hexadecane.     

Effect of culture conditions on batch growth of Saccharomycopsis lipolytica on olive oil

Summary Batch cultures of Saccharomycopsis lipolytica were grown in minimal medium with olive oil as carbon source. Inocula of glucose-grown cells commenced growth with little lag at rates largely unaffected by variations in the stirring rate or oil concentration. However, growth rates declined when the medium pH was below 7.0. In all cultures, media pH declined with increasing cell concentration. Cell composition during exponential growth was 42% protein and 2% fat. Carbon-limited cells maintained this composition after oil exhaustion but during nitrogen- and oxygen-limited growth, protein content decreased and fat content increased although the protein decrease was only transient with oxygen limitation. Yield coefficients for triglyceride were near unity for all cultures. Free acid concentrations rose rapidly after inoculation. As fermentations progressed, free glycerol appeared and concentrations of di- and monoglycerides passed through maximal values although peak concentrations of di- and monoglycerides persisted for extended times in oxygen- and nitrogen-limited cultures respectively. The fraction of free glycerol consumed was greater in oxygen-limited than in carbon- or nitrogen-limited culture. The basic requirements for growth of yeasts on fatty wastes are discussed with reference to these observations.     

Clutivation of yeast on gas oil

The results achieved by the cultivation of the yeast. Candida lipolytica on gas oil are referred. By using a distillation fraction of gas oil distilling between 180–400°C, containing 10–20% of n-alkanes, the optimal condition for biomass production and deparaffination were estimated for various dilution rates and various amounts of gas oil in the medium. The main factor, which influences the yield coefficient by hydrocarbon fermentation is the polyauxie of the hydrocarbon substrate. The penetration of dispersed hydrocarbons into the yeast cell is demonstrated on electron micrographs and the velocity and reversibility of this process is estimated by using tritium-traced hexadecane.     

Optimization of batch fermentation processes. I. Development of mathematical models for batch penicillin fermentations

Two kinds of mathematical models have been developed for batch penicillin fermentations: (1) general models, based on averaged, nondimensionalized cell and penicillin synthesis curves from plant, scale fermentors and (2) particular models developed from specific sets of experimental data from two sources. Parameter-temperature functions used with the general models were assumed to have general shapes which could apply to many fermentations, i.e., they were based on the familiar temperature response of enzyme-catalyzed reactions. Parameter-temperature functions for the particular models were determined from experimental data for batch runs at various temperatures.     

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica

Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.     

Growth of yeast on n-alkanes. II. Batch experiments

In this paper the results of the Monte Carlo simulations as described in an earlier paper are compared with those of batch experiments. A number of batch experiments were carried out at a low inoculation rate so that only a fraction of the oil drops were inoculated. Under these conditions the effect of the segregation of the oil phase is more clearly demonstrated. Special attention is paid to the preparation of actively growing yeast cells with which the cultures is inoculated. Also a method is developed to estimate the amount of actively growing cells with which the culture is inoculated. The other parameters necessary for the Monte Carlo simulation are measured in separate experiments: the maximum growth rate of the cells, oil drop size, and the drop parameters. Finally the growth curves (measured in the batch experiments) are compared with those calculated with the Monte Carlo procedure. A good agreement is found.     

Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures

The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.     

Degradation of n-alkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8

A bacterial isolate, designated as DQ8, was found capable of degrading diesel, crude oil, n-alkanes and polycyclic aromatic hydrocarbons (PAHs) in petroleum. Strain DQ8 was assigned to the genus Pseudomonas aeruginosa based on biochemical and genetic data. The metabolites identified from n-docosane as substrate suggested that P. aeruginosa DQ8 could oxidize n-alkanes via a terminal oxidation pathway. P. aeruginosa DQ8 could also degrade PAHs of three or four aromatic rings. The metabolites identified from fluorene as substrate suggested that P. aeruginosa DQ8 may degrade fluorene via two pathways. One is monooxygenation at C-9 of fluorene, and the other is initiated by dioxygenation at C-3 and C-4 of fluorene. P. aeruginosa DQ8 should be of great practical significance both in bioremediation of oil-contaminated soils and biotreatment of oil wastewater.     

Gaseous environments modify physiology in the brewing yeast Saccharomyces cerevisiae during batch alcoholic fermentation

Aims:  To investigate the impact of different gaseous atmospheres on different physiological parameters in the brewing yeast Saccharomyces cerevisiae BRAS291 during batch fermentation.
Methods and Results:  Yeasts were cultivated on a defined medium with a continuous sparging of hydrogen, helium and oxygen or without gas, permitting to obtain three values of external redox. High differences were observed concerning viable cell number, size and metabolites produced during the cultures. The ethanol yields were diminished whereas glycerol, succinate, acetoin, acetate and acetaldehyde yields were enhanced significantly. Moreover, we observed major changes in the intracellular NADH/NAD⁺ and GSH/GSSG ratio.
Conclusions:  The use of gas led to drastic changes in the cell size, primary energy metabolism and internal redox balance and E _h . These changes were different depending on the gas applied throughout the culture.
Significance and Impact of the Study:  For the first time, our study describes the influence of various gases on the physiology of the brewing yeast S. cerevisiae . These influences concern mainly yeast growth, cell structure, carbon and redox metabolisms. This work may have important implications in alcohol-related industries, where different strategies are currently developed to control better the production of metabolites with a particular attention to glycerol and ethanol.     

The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures

The kinetics in batch culture of the acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The fastest initial growth and transition from an acid to a solvent metabolism occurs on glucose, with a final 62 g/L glucose conversion. On xylose, an initial slower growth rate and a longer metabolic transition result in higher cellular and acids concentration, thus in a level of fermented sugar limited to 47 g/L. Batch fermentations on mixtures of glucose and xylose show that both sugars can be fermented, with a higher rate for glucose. However, xylose fermentation is inducible and inhibited at glucose level above 15 g/L. Mixtures of glucose and xylose yield the highest amount of fermented sugars, up to 68 g/L, as a result of both a fast metabolic transition on glucose and a strong acid reconsumption on xylose. In all cases, solvent production is triggered at a total acid concentration between 4 and 5 g/L, whereas the final inhibition of the fermentation takes place at a total butanol and acid concentration between 18 and 20 g/L.     

Lipids of Candida lipolytica cultivated in n-alkanes by the industrial method ("Toprina")]

The following lipidic classes are examined in the present study: Total Lipids, Phospholipids, Neutral Lipids, Sterols, and Carotenoid Pigments from dried biomasses of Candida lipolytica grown on n-alkanes by industrial process following the BP technique ("Toprina"). The composition of the lipid classes examined in "Toprina" agree generally with bibliographic data about n-alkanes grown Candida lipolytica in batch cultures.     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

Development and activation of cyanide-resistant respiration in the yeast Yarrowia lipolytica.

Changes in respiratory activity and in the contents of adenine nucleotides (ATP, ADP, AMP) were studied in cells of the yeast Yarrowia lipolytica during the development of cyanide-resistant respiration. The transition of the yeast from the logarithmic to the stationary growth phase due to exhaustion of glucose was associated with decreased endogenous respiration and with the activation of a cyanide-resistant oxidase. Cyanide activated cell respiration during the stationary growth phase. The cyanide-resistant respiration was inhibited by benzohydroxamic acid (BHA), an inhibitor of the alternative oxidase. In the absence of cyanide, BHA had no effect on the cells which had the cyanide-resistant oxidase. This indicates that the cells do not use the alternative pathway in vivo. The decreased endogenous respiration of the cells was accompanied by decreased contents of adenine nucleotides. Addition of cyanide resulted in a sharp decrease in the content of ATP, in a twofold increase in the content of ADP, and in a fivefold increase in the content of AMP. In the absence of cyanide, BHA had virtually no effect on the contents of adenine nucleotides. The decreased rate of oxygen consumption during the transition of the cells to the stationary growth phase was caused by the decreased activity of the main cytochrome-containing respiratory chain (2,4-dinitrophenol (DNP) stimulated respiration). The alternative oxidase was synthesized in the cell but was inactive. Cyanide stimulated respiration due to activation of the alternative oxidase via the AMP produced. The decrease in the cell content of ATP is suggested to be a factor inducing the synthesis of the alternative oxidase.