Fatty Acid and Sterol Composition of Mucor genevensis in Relation to Dimorphism and Anaerobic Growth

Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains γ-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of γ-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of Mucor circinelloides var. lusitanicus INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of gamma-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Lipid composition of the mycelium of the fungus Mucor hiemalis cultivated with trehalose, triacylglycerols, and itraconazole

We investigated the growth and cell lipid composition of the fungus Mucor hiemalis VKMF-1431 cultivated under aerobic conditions in the presence of the morphogenetic agents itraconazole, exogenous triacylglycerols, and trehalose. The sporangiospores of a 6-day culture were used as inocula. Under these conditions, the fungus produced mycelium; nevertheless, solitary yeastlike cells also developed on the glucose-containing medium and in the presence of itraconazole and sterilized triacylglycerols (sTAGs). No yeastlike growth occurred in the system with trehalose and with unsterilized (native) TAGs (nTAGs). With trehalose and nTAGs in the cultivation medium, the ratio between PEA and PC, the two main types of membrane lipids, was low. This testified to a relatively high PC percentage and, accordingly, a stable structure and a highly functional state of the membranes. Moreover, if the development of the fungus occurred exclusively as mycelium formation, the level of polyunsaturated fatty acids (??-linolenic and arachidonic acid) increased in the presence of trehalose and that of linoleic acid increased in the presence of nTAGs. These results may suggest that unsaturated fatty acids and membrane lipids are related to the cell wall formation and the implementation of morphogenetic programs in mucorous fungi.     

Development of Respiration and Mitochondria in Mucor genevensis After Anaerobic Growth: Absence of Glucose Repression

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO(4) fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.     

The effect of lipid content on the elemental composition and energy capacity of yeast biomass

Oleaginous yeasts (18 strains) were grown in ethanol media under various cultivation conditions (33 biomass samples). It was found that lipid and lipid-free fractions of dry biomass have elemental composition and biomass reductivity very close to values which can be considered as biological constants. The energy content of dry biomass strongly depended on the total lipid content. When the lipid content was 64%, the energy value of dry biomass reached 73% of diesel oil; therefore, oleaginous microorganisms can be a promising source of biodiesel fuel. The approach used in this work makes it possible to determine the energy value of biomass by its elemental composition without application of laborious and expensive calorimetric measurements of combustion heats.     

The effect of initial bioenergetic state on the cryostability of yeast cells

The cryostability of Saccharomyces cerevisiae cells decreased when they were cultivated under anaerobic conditions in a liquid growth medium YEPD as compared to the culture grown under aerobic conditions. The effect of cultivation conditions on the different cryostability of S. cerevisiae cells is discussed. The initial state of their bioenergetics was shown to influence the cryostability of yeast cells.     

Metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobacter sphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2–3 times lower in the cells of the M variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa ₃, acting as cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient at the cytochrome aa ₃ level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.     

Impact of morphogenetic effectors on the growth pattern and the lipid composition of the mycelium and the yeastlike cells of the fungus Mucor hiemalis

We investigated the growth and the cell lipid composition of the mycelium and of the yeast-like form of Mucor hiemalis VKMF-1431 obtained under aerobic conditions by treatment with the morphogenetic agents itraconazole, exogenous triacylglycerols (TAGs), and trehalose. The sporangiospores of a 20-day culture were inoculated on the medium with glucose. Under these conditions, the fungus produced both mycelium and yeast-like cells. It was established that, upon the germination of old (20-day) sporangiospores, the fungus predominantly used the mycelium development strategy in the presence of trehalose and TAGs. It was characterized by a low ratio between the two bulk membrane lipids (PEA/PC) and increased levels of PC and polyunsaturated fatty acids (FA). Compared to the mycelium, the yeast cell morphotype obtained on the medium with glucose was distinguished by an elevated PEA/PC ratio, lowered TAG, free sterol (FS) and esterified sterol (ES) levels, a decreased ES/FS ratio that correlated with the reserve sterol pool size, and a lowered content of unsaturated fatty acids (the linoleic and the ??-linolenic acid). These peculiarities of the lipid composition of yeastlike cells correlated with the intensity of yeastlike growth. Light and electron microscopy revealed differences between the above cell morphotypes. With itraconazole, yeast-like cells were characterized by the destruction of the endoplasmic reticulum membranes and formation of a large number of vacuoles. The suggestion was confirmed that the state/age of inoculum sporangiospores exerts an influence on the capacity for dimorphism in mucorous fungi such as M. hiemalis. The data obtained testify to an involvement of lipids in the process of adaptation to environmental factors and to their regulatory role in morphogenetic processes associated with the formation of alternative morphotypes of the mucorous fungus.     

Comparative physiological study of the wild type and the small colony variant of Pseudomonas aeruginosa 20265 under controlled growth conditions

Small-colony variants (SCVs) of Pseudomonas aeruginosa are often found in chronically infected airways of patients suffering from cystic fibrosis. These slow-growing morphological variants have been associated with persistent and antibiotic-resistant infections. Nevertheless, the behavior of SCVs under varied availability of O₂ and iron, two key variables relevant to the lung environment of CF patients and pathogenicity of P. aeruginosa, has not been systematically studied so far. In this work, the effects of O₂ and iron were comparatively studied for a CF P. aeruginosa wild type (WT) strain and its SCV phenotype in a real-time controlled cultivation system. Significant differences in the behavior of these strains were observed and quantified. In general, SCV exhibited a higher fitness than the WT toward aerobic conditions. Under iron rich condition, and despite less release of total extracellular proteins, absence of flagellin and lower siderophore production, the SCV cells grown at fully aerobic conditions showed a higher specific growth rate and a significantly higher cytotoxicity in comparison with the WT cells. The strains behaved also differently towards iron limitation. The phenomena of limited O₂ transfer from the gas to the liquid phase and enhancement of formation of virulence factors under conditions of iron limitation were much more profound in the SCV culture than in the WT culture. These results have important implications for better understanding the pathogenicity of P. aeruginosa and its small-colony variants.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

THE BIOGENESIS OF MITOCHONDRIA : III. The Lipid Composition of Aerobically and Anaerobically Grown Saccharomyces cerevisiae as Related to the Membrane Systems of the Cells

The growth conditions known to influence the occurrence of mitochondrial profiles and other cell membrane systems in anaerobic cells of S. cerevisiae have been examined, and the effect of the several growth media on the lipid composition of the organism has been determined. The anaerobic cell type containing neither detectable mitochondrial profiles nor the large cell vacuole may be obtained by the culture of the organism on growth-limiting levels of the lipids, ergosterol, and unsaturated fatty acids. Under these conditions, the organism has a high content of short-chain saturated fatty acids (10:0, 12:0), phosphatidyl choline, and squalene, compared with aerobically grown cells, and it is especially low in phosphatidyl ethanolamine and the glycerol phosphatides (phosphatidyl glycerol + cardiolipin). The high levels of unsaturated fatty acids normally found in the phospholipids of the aerobic cells are largely replaced by the short-chain saturated acids, even though the phospholipid fraction contains virtually all of the small amounts of unsaturated fatty acid present in the anaerobic cells. Such anaerobic cells may contain as little as 0.12 mg of ergosterol per g dry weight of cells while the aerobic cells contain about 6 mg of ergosterol per g dry weight. Anaerobic cell types containing mitochondrial profiles can be obtained by the culture of the organism in the presence of excess quantities of ergosterol and unsaturated fatty acids. Such cells have increased levels of total phospholipid, ergosterol, and unsaturated fatty acids, although these compounds do not reach the levels found in aerobic cells. The level of ergosterol in anaerobic cells is markedly influenced by the nature of the carbohydrate in the medium; those cells grown on galactose media supplemented with ergosterol and unsaturated fatty acids have well defined mitochondrial profiles and an ergosterol content (2 mg per g dry weight of cells) three times that of equivalent glucose-grown cells which have poorly defined organelle profiles. Anaerobic cells which are low in ergosterol synthesize increased amounts of squalene.     

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.     

Optimization of medium composition for improving biomass production of Lactobacillus plantarum Pi06 using the Taguchi array design and the Box-Behnken method

An immune-enhancing strain, Lactobacillus plantarum Pi06, isolated from a healthy infant was used for biomass production following optimization of the medium in shake-flask culture. Preliminary studies showed that commercial MRS medium and cultivation under static conditions generated higher biomass production than four other tested media with or without a shaking condition. The selected medium composition, consisting of glucose, yeast extract, soy peptone, ammonium citrate, and corn steep liquor, was further optimized using a systematic method that integrated the Taguchi array design and the Box-Behnken method. The response effects of these factors were first investigated using Taguchi design under an L ₁₆ (4⁵) array. The suggested medium composition, derived from Statistica 7.1 using the Taguchi design, was applied to cultivate cells and a biomass of 7.16 g dry cell weight (DCW)/L was obtained. Response surface methodology based on the Box-Behnken method for the three response variables of glucose, yeast extract, and corn steep liquor was then used to further increase the biomass level to 8.94 g DCW/L. The resulting optimum medium consisted of 35 g/L glucose, 35 g/L yeast extract, and 40 mL/L corn steep liquor. Compared with the initial medium, the biomass yield was improved from 4.31 to 8.94 g DCW/L, an enhancement of approximately 107%.     

Lipid accumulation inRhodotorula glutinis on sugar cane molasses in single-stage continuous culture

Microbial lipids produced byRhodotorula glutinis grown in continuous culture with molasses under nitrogen-limiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h^–1. The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.     

Transport of glucose intoSaccharomyces cerevisiae RXII

Evidence is submitted that glucose is found in a chemically unaltered form in cells ofSaccharomyces cerevisiae RXII incubated with glucose, if the culture is grown under aerobic conditions at 30°C and is in the logarithmic phase of growth at the moment of harvesting. Under these conditions, the course of formation of a glucose steady state can be studied under aerobic and anaerobic incubation conditions. The steady state glucose concentration in the cells is the linear function of the glucose concentration in the medium.