Vladimir Marić

Cell lipids of the Candida lipolytica yeast grown on methanol

Candida lipolytica yeast, grown on 1% methanol as the only carbon and energy source, synthesized 4.9% of dry cell mass as lipids, 52.3% of which were polar lipids. Polar lipids consisted mainly of phospholipids and sphingolipids as their minor components. The total long-chain bases content has been found to account only for 0.7% of the polar lipids. The long-chain bases composition determined by thin-layer and gas chromatography shows a preponderance of trihydroxy bases and a small amount of dihydroxy bases. The striking finding was the high content of 19-phytosphingosine (90.8% of total long-chain bases). Fatty acid (FA) composition of polar lipids was characterized by the relatively high concentration of unsaturated fatty acids (66.4% of total FA) and by the predominance of fatty acids with 16 carbon atoms (85.0% of total FA).

Isolation and chemical composition of the ceramide of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Ceramide was quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Fatty acids and long-chain bases, obtained after acid methanolysis, were analyzed by gas-liquid chromatography. Fatty acid composition was characterized by predominance of fatty acids shorter than 20 carbon atoms (72.2% of total fatty acids) and by high concentration of fatty acids with 16 carbon atoms (43.3% of total fatty acids). The dominant fatty acids were h16:0 and 18:1. The major fatty acid with more than 20 carbon atoms was h24:0. Hydroxylated fatty acids quantitatively represented almost half of the total fatty acids. The ceramide contained an unusual long-chain base composition. Besides the well-known trihydroxy bases, 18- and 20-phytosphingosine, which is a common characteristic for yeast sphingolipids, a high proportion of more polar bases than the trihydroxy ones, was found. The structures of 4,5-dihydroxyicosasphinanine (1,3,4,5-tetrahydroxy-2-aminooctadecane) and 4,5-dihydroxyicosasphinganine (1,3,4,5-tetrahydroxy-2-aminoeicosane) were tentatively assigned to these bases. Their chromatographic properties were identical to those of synthetic bases.

Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast

The plasma membrane and mitochondria of bottom fermenting brewer’s yeast obtained as a by-product of industrial beer production were isolated and the lipid fraction was analyzed. The phospholipid content accounted for 78 mg/g protein in the plasma membrane and 59 mg/g protein in the mitochondria. Major phospholipids in both preparations were phosphatidylinositol, phosphatidylcholine and phosphatidyl-ethanolamine but their proportions differed significantly. In the plasma membrane phosphatidy linositol, and in the mitochondria phosphatidylcholine were present in the highest concentration (37 and 30 %, respectively). The main classes of neutral lipids (triacylglycerols, ergosterol, squalene and steryl esters) were twice more abundant in the plasma membrane than in the mitochondria (61 and 33 mg/g protein, respectively). A characteristic of the neutral lipid composition of both organelles was the low content of ergosterol (12 and 7 mg/g protein, respectively) and a high content of squalene (25 and 22 mg/g protein). The main feature of the fatty acid composition of both organelles was the preponderance of saturated fatty acids (78 and 79 %, respectively), among which palmitic acid was the principal one. The most expressed characteristics of lipid fractions of the analyzed plasma membranes and mitochondria, high concentration of squalene and preponderance of saturated fatty acids are the consequences of anaerobic growth conditions. The lack of oxygen had possibly the strongest effect on the lipid composition of the plasma membranes and mitochondria of bottom fermenting brewer’s yeast.

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C19 phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine.

Mathematical modeling of mixing in a horizontal rotating tubular bioreactor: "Spiral flow" model

A horizontal rotating tubular bioreactor (HRTB) was designed as a combination of a “thin-layer bioreactor” and a “biodisc” reactor whose interior was divided by O-ring shaped partition walls. For the investigation of mixing in HRTB the temperature step method was applied. Temperature changes in the bioreactor were monitored by six Pt-100 sensors (t90 response time 0.08 s and resolution 0.002 °C) which were connected with an interface unit and a personal computer. In this work a modified “tank in series” concept was used to establish a mathematical model. The heat balance of the model compartments was established according to the physical model and the “spiral flow” pattern. Numerical integration was done by the Runge-Kutta-Fehlberg method. The mathematical mixing model called “spiral flow” model contained four adjustable parameters (N1, Ni, Fcrand Fp) and five parameters which characterized the plant and experimental conditions. The “spiral flow” model was capable to describe the mixing in HRTB properly, and its applicability was much better than with the “simple flow” model, presented earlier.

Microbial acetate oxidation in horizontal rotating tubular bioreactor.

The aim of this work was to investigate the possibility of conducting a continuous aerobic bioprocess in a horizontal rotating tubular bioreactor (HRTB). Aerobic oxidation of acetate by the action of a mixed microbial culture was chosen as a model process. The microbial culture was not only grown in a suspension but also in the form of a biofilm on the interior surface of HRTB. Efficiency of the bioprocess was monitored by determination of the acetate concentration and chemical oxygen demand (COD). While acetate inlet concentration and feeding rate influenced efficiency of acetate oxidation, the bioreactor rotation speed did not influence the bioprocess dynamics significantly. Gradients of acetate concentration and pH along HRTB were more pronounced at lower feeding rates. Volumetric load of acetate was proved to be the most significant parameter. High volumetric loads (above 2 g acetate l−1 h−1) gave poor acetate oxidation efficiency (only 17 to 50%). When the volumetric load was in the range of 0.60–1.75 g acetate l−1 h−1, acetate oxidation efficiency was 50–75%. At lower volumetric loads (0.14–0.58 g acetate l−1 h−1), complete acetate consumption was achieved. On the basis of the obtained results, it can be concluded that HRTB is suitable for conducting aerobic continuous bioprocesses.

Natural zeolite clinoptilolite increases the concentrations of sphingoid bases in the yeast Yarrowia lipolytica

In the present paper, we studied the effect of natural zeolite clinoptilolite on sphingolipid metabolism in the yeast Yarrowia lipolytica. We also investigated if zeolite addition had any impact on cell shape and size, as well as on the pH alterations during the culture growth. High performance liquid chromatography analysis of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from Y. lipolytica showed that their concentrations markedly rose upon the zeolite addition. The largest increase among the identified molecular species of sphingoid bases was seen in C18 phytosphingosine, whose levels rose 6.2‐fold and 22.3‐fold after culturing cells for 24 and 36 hours respectively in the presence of finely ground zeolite. pH measurements of the culture medium showed a similarity between pH profiles of control and zeolite‐supplemented cells, suggesting that ion‐exchange capacity was not probably responsible for the observed change in sphingolipid metabolism. Scanning electron microscopy revealed that zeolite affected cell size and shape. Y. lipolytica cells grown in the absence of zeolite were oval‐shaped with an average cell size of 0.7–2.7 μm, whereas when cultured with zeolite, they were round‐shaped and larger, having an average cell size of 1.3–2.9 μm.

Fermentative bioconversion in a horizontal rotating tubular bioreactor

Abstract The performance of a horizontal rotating tubular bioreactor (HRTB) was investigated with a biological system under non-sterile conditions. A spontaneously developed microbial culture was cultivated in a simple glucose/yeast extract medium. A fermentative bioconversion was examined by different combinations of process parameters (bioreactor rotation speed 5–30 min −1 and medium inflow rate 1–10 l h −1 ). Bioconversion dynamics in HRTB was monitored by withdrawing the samples from five positions along the bioreactor. Investigation in HRTB showed a rapid and an efficient glucose conversion into different products of metabolism. Glucose consumption rate along the HRTB depended on medium inflow rate, while bioreactor rotation speed did not have a significant influence. Complete glucose conversion in HRTB was observed at inflow rates of up to 6.5 l h −1 . The pH gradient along the HRTB was detected at higher medium inflow rates (6.5 and 10 l h −1 ), but did not significantly influence substrate conversion efficiency. A discussion of its potential use and a comparison of HRTB with other bioreactors are also presented.

Effect of Growth Phase on the Content and Composition of Ceramides of the Hydrocarbon-assimilating Yeast Candida lipolytica

Candida lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Ceramides were quantitatively isolated from total lipids of exponential and stationary phase cells by a combination of column chromatography and preparative high-performance thin-layer chromatography. After acid methanolysis their composition was analyzed by gas-liquid chromatography. The ceramide content of the exponential phase cells was two times higher than the one of the stationary phase cells. The composition of long-chain base moiety of ceramides did not change significantly during the growth. In both growth phases 19-phytosphingosine was the major long-chain base. However, the fatty acid composition of ceramides changed greatly during the growth. In the exponential growth phase, ceramides contained predominantly fatty acids greater than 20 carbon atoms, while fatty acids shorter than 20 atoms predominated in ceramides of the stationary phase, 16:0 being the main one. In the exponential growth phase fatty acid moiety of ceramides was characterized by unusually high degree of unsaturation and relatively high proportion of odd-numbered fatty acids. However, the proportion of both, unsaturated and odd-numbered fatty acid decreased significantly in ceramides of the stationary phase. The unexpected finding was the absence of fatty acid hydroxylation of ceramides in the exponential phase cells and unusually low degree of hydroxylation in the stationary phase.

Enzymic hydrolysis of protein during barley spent grains mashing

Abstract High nitrogen barley syrup (HNBS) was obtained by mashing barley spent grains with neutral proteinase. The total nitrogen to carbohydrate ratio in the HNBS was three times higher than in typical barley syrup. In order to enhance the liberation of free amino acids, the spent grains were hydrolysed simultaneously with proteinase and barley peptidase extract; the α-amino to total nitrogen ratio increased from 11.4 to 14.6%. During mashing with peptidase extract, the protein solubilization rate was lower than during mashing with neutral proteinase only. Data analysis by means of the modified Foster-Niemann equation suggested that endopeptidase inhibitors, possibly present in the peptidase extract, caused by decrease of protein solubilization rate. The isolation of the bacterial proteinase inhibitors from peptidase extract indicates that they affected the protein solubilization process.