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 the yeast Torulopsis candida on n-alkanes

Yeast speciesTorulopsis candida was isolated from sea water and grew well on n-alkanes. The results are indicated below, and further studies will determine the economical value of utilizing this species in the industrial production of single-cell protein.This study was carried out at the Istituto di Microbiologia Agraria (DirectorProf. O. Verona), University of Pisa, Italy, during a scholarship offered by the foreign Office of the Italian Government.     

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 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.     

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.     

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.     

Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

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.     

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.     

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.     

Cultivation of the yeastCandida lipolytica on hydrocarbon

The ability of the yeastCandida lipolytica 4-1 to oxidize and utilize various pure aliphatic hydrocarbons occurring in gas oil was studied. It was found that the given strain ofCandida lipolytica oxidized n-alkanes without adaptation, starting with heptane, and utilized them for growth, starting with nonane. Isoalkanes with a single methyl group in the side chain were also oxidized and utilized for growth, but less than the corresponding n-alkanes. The site of the methyl group in the isoalkane chain influences its conversion to biomass. Branched chains at both ends of the isoalkane molecule prevent its utilization for growth ofCandida lipolytica. 1-olefines are also oxidized and utilized for growth, though less than the corresponding n-paraffins. Alkylaromatic hydrocarbons are oxidized from amylbenzene up to decylbenzene, which is utilized only slightly for growth of the yeast.     

Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagenesis

Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 X 10(-7) and 5 X 10(-6) mutants per cell. But one class of glucosamine resistant mutants (GAMRA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed. UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m2 was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.     

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.     

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.     

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.