Lipid Accumulation Process Research Articles

Lipids of oleaginous yeasts. Part II: Technology and potential applications

AbstractThe process of lipid accumulation in the oleaginous yeasts cultivated in various fermentation configurations when either sugars and related compounds or hydrophobic substances are used as substrates is presented and kinetic models describing both de novo and ex novo lipid accumulation are analyzed. Technological aspects related with single cell oil (SCO) produced by oleaginous yeasts are depicted. The influence of culture parameters upon lipid production process is presented. Lipid production has been studied in batch, fed‐batch, and continuous cultivation systems using yeasts belonging to the species Lipomyces starkeyi, Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, and Yarrowia lipolytica. The potentiality of yeasts to produce SCO as starting material of 2nd generation biodiesel is indicated and discussed. Of significant importance is also the utilization of yeast lipids as substitutes of high added value exotic fats (e.g., cocoa butter). Lipid produced by the various yeasts presents, in general, similar composition with that of common vegetable oils being composed of unsaturated fatty acids, whereas cocoa butter is principally composed of saturated fatty acids, consequently the various strategies that are followed in order to increase the cellular saturated fatty acid content of the yeast lipid are presented and comprehensively discussed.

Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients

Two-stage fermentation process was used for lipid production by Lipomyces starkeyi AS 2.1560 in glucose solution without auxiliary nutrients. In the first stage, cells were cultivated in a nutrient-rich medium for propagation. In the second stage, cells were resuspended in glucose solution to achieve high cellular lipid contents. The effects of the inocula age, cell density and initial glucose concentration on lipid production were briefly studied. When high cell density fermentation was performed in a 7-L stirred-tank bioreactor for 40 h using non-sterile glucose solution as carbon source, the biomass, lipid and lipid content reached 104.6 g/L, 67.9 g/L and 64.9%, respectively. More significantly, lipid productivity reached 2.0 g/L h during the initial 16 h-period and 1.6 g/L h for the entire culture. Our results demonstrated that cell propagation and lipid accumulation processes can be spatially separated, allowing further optimization to improve both processes. The two-stage fermentation method should have a great potential to develop more efficient processes to convert renewable materials into biofuel and related products.

Gestational diabetes: emerging concepts in pathophysiology.

Gestational diabetes affects 3 to 5% of pregnancies in the United Kingdom, contributing to significant maternal and fetal morbidity. Understanding the pathophysiology is important as it guides diagnostic screening and treatment. The insulin resistance of normal pregnancy facilitates provision of metabolic substrates to the fetus and is multifactorial in origin. Recent identification of hepatic and skeletal muscle lipid deposition in Type 2 diabetics, demonstrated by novel magnetic resonance spectroscopy techniques, is likely to be the underlying cause of pathological insulin resistance. Similar mechanisms almost certainly underlie gestational diabetes, although further studies are required to prove this. Women who develop gestational diabetes have demonstrable insulin resistance prior to pregnancy that is part of a chronic process of lipid accumulation ultimately leading to type 2 diabetes later in life. The importance of lifestyle advice and dietary modification and the rationale behind the use of metformin are thus explained.

Commercial sugars as substrates for lipid accumulation in Cunninghamella echinulata and Mortierella isabellina fungi

AbstractCunninghamella echinulata and Mortierella isabellina, fungi capable of accumulating single cell oil (SCO) containing γ‐linolenic acid (GLA), were cultivated on sugar‐based media, at initial substrate concentration 60 g/L. It was demonstrated that the carbon source plays an essential role on lipid accumulation process. Especially, on glucose, C. echinulata produced 3.9 g/L lipids containing 19.5% GLA, whereas M. isabellina achieved 9.9 g/L of lipid, containing 3.5% GLA. Growth on fructose was also satisfactory for both microorganisms. Although M. isabellina failed to grow adequately on saccharose, C. echinulata presented appreciable biomass production of 17.6 g/L, containing 18% of fat. Extra‐cellular invertase activity was also detected in the case of C. echinulata cultures, achieving 0.5 U/mL. Both molds presented satisfactory growth on molasses, with significant amounts of SCO and GLA produced, accompanied by non‐negligible substrate decolorization. Scaling‐up in a 3‐L bioreactor of M. isabellina with glucose as a substrate resulted in 17.5 g/L of biomass, containing 12.7 g/L of lipid. Finally, fractionation of cellular lipid for both strains showed that neutral lipids represented about 90–92% w/w upon total accumulated lipids, whilst fractions of glycolipids plus sphingolipids and phospholipids were 6–7 and 3–4%, respectively.

Comparative proteomic analysis of Rhodosporidium toruloides during lipid accumulation

Intracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an '-omics' level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen-limited medium and performed a comparative and semi-quantitative proteomic analysis of cell samples obtained thereafter by a 2D-LC-MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi-quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering.

Organic nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accumulation inCunninghamella echinulata

To investigate the effect of organic nitrogen on lipogenesis during growth of Cunninghamella echinulata on tomato waste hydrolysate (TWH) media. Cunninghamella echinulata grown on a TWH medium rapidly took up glucose and produced large amounts of lipids. However, when some quantities of the organic nitrogen were removed from TWH (by acid followed by alkaline precipitation of proteins) the uptake of glucose was dramatically reduced and large quantities of fungal biomass having low lipid content were produced. Nevertheless, when glycerol was used as carbon source instead of glucose, the uptake rate as well as the biomass production and the lipid accumulation processes were unaffected by the TWH organic nitrogen removal. Finally, when the fungus was grown on a glucose supplemented TWH medium that contained no assimilable organic nitrogen (after further precipitation of proteins with methanol), the produced biomass contained non-negligible quantities of lipids, although glucose uptake remained low. Lipid analysis showed that the produced lipids comprised mainly of neutral lipids, which were preferentially consumed during lipid turnover. Lipid production on the original TWH medium having glucose as carbon source was 0.48 g of lipid per gram of dry biomass, corresponding to 8.7 g of lipid per litre of growth medium. The produced lipids contained 11.7%gamma-linolenic acid (GLA), hence the GLA yield was more than 1 g l(-1). Organic nitrogen compounds found in TWH favour glucose (but not glycerol) uptake and lipid accumulation in C. echinulata. Agro-industrial wastes containing organic nitrogen, such as tomato waste, are produced in vast amounts causing severe environmental problems. These wastes could be used as fermentation feedstock to produce microbial lipids.

Glycerol-3-Phosphate dehydrogenase (E.C.1.1.1.8) is expressed in cultured chicken embryonic adipofibroblasts and upregulated by embryonic chicken serum

Chicken embryonic adipofibroblasts (CEA) accumulate intracytoplasmic lipids when cultured in medium containing chicken serum (CS), but not in medium with fetal bovine serum (FBS). To characterize this process of lipid accumulation, we evaluated the expression of the enzyme glycerol-3-phosphate dehydrogenase (E.C.1.1.1.8) (GPDH), first in chicken tissues and then in CEA cultured under diverse conditions. GPDH activity in adipose depots from 4-wk-old broiler chickens was similar or higher than that shown by liver, the main organ for fatty acid synthesis in chickens, while skeletal muscle had the lowest levels of GPDH. In vitro, GPDH activity increased in CEA cultured in the presence of CS but not in medium with FBS, paralleling the lipid accumulation by these cells. Both lipid accumulation and GPDH activity were further increased in CEA cultured in the presence of embryonic CS. Our results show that GPDH is highly expressed in avian tissues related to lipid metabolism and therefore can be a reliable marker for avian adipogenesis, and suggest that ECS is an optimum source for the purification of avian adipogenic factors.

Normal and Oxidized Low Density Lipoproteins Accumulate Deep in Physiologically Thickened Intima of Human Coronary Arteries

Diffuse intimal thickening (DIT) that develops as a physiologic adaptation in the arterial wall has been implicated to have a predilection for atherosclerosis. We histologically investigated the lipid accumulation process in the human coronary DIT by focusing on the localization of normal and oxidized low-density lipoproteins (LDLs). Immunohistochemistry for apolipoprotein B 100 (a major apolipoprotein of LDL) and 8-iso-prostaglandin F2α (an oxidative product in LDL) showed substantial accumulation of oxidized relative to normal LDLs in the deep layers of DIT (52/139 segments). Subendothelial deposition of normal rather than oxidized LDLs, known as an early event of fatty streak formation, was less frequently found (13/139 segments). In contrast with fibrofatty lesions, lipid accumulation localized deep in DIT was characterized by fine lipid droplets scattered in the preserved tissue and by its association with neither macrophage accumulation nor apoptosis in the constituent cells. On the other hand, the deep intimal location of lipid accumulation clearly coincided with increased type I and type III collagen and elastic fibers but rarely with sulfated proteoglycans including decorin, which were all strongly expressed in advanced lesions. This lipid accumulation was found only in sites with DIT of more than 200 μm, occasionally extending to the inner media and involving neovessel formation around it. The presence of deep intimal lipid accumulation was associated with reduced endothelium-dependent relaxation to substance P in isolated coronary rings. These results suggest that normal and oxidized LDLs accumulate preferably in the nutritional border zone of established DIT involving local extracellular matrix alterations but independently of inflammatory or apoptotic processes. This may contribute to the functional and morphologic abnormalities seen in human coronary atherogenesis that progresses slowly with age.

Impact of algal diets and emulsions on the fatty acid composition and content of selected tissues of adult broodstock of the Chilean scallop Argopecten pupuratus (Lamarck, 1819)

The impact of dietary fatty acids on the fatty acid composition and content in different organs (digestive gland, adductor muscle and male and female gonad) of Argopecten purpuratus was evaluated during the process of gonad maturation under controlled laboratory conditions. A mixed algal diet of Isochrysis galbana (clone T-Iso), Pavlova lutheri, Chaetoceros gracilis and Tetraselmis suecica was either fed alone (Al) or supplemented with 50% lipids (expressed as % of the algal dry weight (DW)) using an emulsion rich in 22:6 n−3 (Al+EmDHA) or an emulsion rich in 20:5 n−3 (Al+EmEPA). Results showed the algal diet having the greatest impact on the fatty acid composition (mainly an increase of 18:1 n−9, 18:2 n−6; 18:3 n−3 and a decrease of 20:5 n−3) of the digestive gland and female gonad, moderate in the male gonad while the fatty acid composition of the adductor muscle was hardly affected. Our data indicated that 16:1 n−7 and 18:1 n−9 were partially elongated to 18:1 n−7 and 20:1 n−9, respectively. The supplementation of the algal diet with EmDHA or EmEPA (which contained 45% and 19% 22:6 n−3, respectively) increased significantly the concentration of 22:6 n−3 (mg g −1 DW) in the digestive gland, female gonad and male gonad with 78%, 57% and 51%, respectively, for (Al+EmDHA)-fed scallops and 18%, 33% and 19%, respectively, for (Al+EmEPA)-fed scallops, while the 22:6 n−3 concentration in the adductor muscle was not affected. Broodstock conditioning was associated with a significant increase of the lipid content in the female and to a lesser extent in the male gonad. The significant increase (as compared to the initial sample) of the 22:6 n−3 concentration in the digestive gland, female gonad and eggs of A. purpuratus fed 22:6 n−3-rich lipid illustrated that recently ingested fatty acids did significantly contribute to the lipid accumulation process in the maturing female gonad. The similarities between the fatty acid composition of the female gonad and digestive gland suggest that lipids were transferred from the lipid-rich digestive gland to the female gonad.

Production of gamma-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel.

A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio. The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but gamma-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process. When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased. The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163. C. echinulata was also able to grow on orange peel. The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation. Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted. The maximum GLA production was 1.2-1.5 mg/g of fermented peel, calculated on a dry weight basis. After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.

Relationship between the lipid composition of the Anadyr capelin Mallotus villosus and its physiological state

The lipid composition of muscle tissue in the capelin Mallotus villosus from Anadyr population was studied in connection with its physiological state, location, and season of fishery. The content of lipids in sexually mature Anadyr capelin in July–October rose from 8.5 to 11.8%, which is slower than the process of lipid accumulation in the capelin of the related Pribylov population. A predominance was confirmed of the total fatty acids 20 : 1 over the total acids 22 : 1 (the difference was about 15% ) in the lipids of the mature fattening capelin, as well as predominance of the total content of these two monoenoic acids in males compared with females. In the Anadyr capelin, this difference can be characterized as follows: Σ (20 : 1 + 22 : 1)males / Σ (20 : 1 + 22 : 1) females = 1.3. It is obvious that processes of accumulation of monoenoic fatty acids in reserve lipids proceed under strong metabolic control that differs in males and females.

Dependence of the lipid composition of Spirulina platensis (Nordst.) Geitl. on the mode of energy existence of cultures: from photoautotrophy to photoheterotrophy

Dependence of the lipid composition of the blue-green alga Spirulina platensis (Nordst.) Geitl. on the mode of energy existence of cultures was studied. It has been shown that under conditions providing the possibility of alga's transfer to photoheterotrophy, the culture growth rate essentially increased, which was accompanied by some inhibition of the lipid accumulation process. It has been found that the content of monogalactosyldiacylglycerol (MGDG) was twice as low, that of sulphoquinovosyldiacylglycerol (SQDG) was thrice as low, while the content of phosphatidyl-glycerol (PG) was more than 1.7 times as high as in the control. Under conditions favorable for photoheterotrophy, the values of DGDG (digalactosyldiacylglycerol) ratios have increased. The revealed modifications in the composition of polar lipids evidence for a structural reorganization of the membrane apparatus in S. platensis due to changes in carbon nutrition conditions. Such structural reconstructions may provide some correction of the lipid matrix flowability and the sufficient stabilization level of proteins in electron-transport chains, pigments, and enzymes.

Acute coronary syndromes: unstable angina and non-Q-wave myocardial infarction.

Ischemic heart disease includes a wide spectrum of conditions, ranging from silent ischemia and exertion-induced angina, through unstable angina, to acute MI. Unstable angina occupies the center of this spectrum, causing disability and risk greater than that of chronic stable angina but less than that of acute MI1 (Fig 1⇓). Although non–Q-wave MI for many years was considered prognostically similar to unstable angina, recent longitudinal studies indicate that it is similar to Q-wave infarction2 3 (Fig 2⇓). Figure 1. Cumulative 6-month mortality from ischemic heart disease. Diagnosis on admission to hospital (n=21 761; 1985 to 1992). From Duke Cardiovascular Database. Reproduced with permission from Reference 1. Figure 2. Top, Cumulative 1-year combined death or MI among patients with Q-wave and non–Q-wave MI treated with fibrinolysis. Reproduced with permission from Reference 2. Bottom, Risk of subsequent cardiac events in stable convalescing patients after first non–Q-wave and Q-wave MI. Reproduced with permission from Reference 3. The concept of unstable angina has emerged from observations of frequent symptoms preceding acute MI, followed by prospective documentation that unstable symptoms frequently culminated in acute MI. The syndrome was rapidly accepted as a well-defined clinical entity as specific clinical manifestations, pathophysiological mechanisms, laboratory findings, and treatment became better characterized. Unstable angina is currently one of the leading causes of hospital admission for CAD, and non–Q-wave MI accounts for >30% of admissions for acute MI.1 4 Yet, the diagnosis of unstable angina remains clinical, based on symptom recognition. The physician caring for patients with unstable angina is in a privileged position of recognizing rapidly evolving CAD and being able to intervene to prevent irreversible left ventricular damage and progression of CAD. Unstable angina is classically described as a heterogeneous disease, referring to a wide spectrum of clinical manifestations from stable angina to MI, of disease …

Regulatory Studies on Citrate Synthase in Candida 107, an Oleaginous Yeast

The kinetic properties of citrate synthase from Candida 107 were examined. Adenine nucleotides were inhibitory to enzyme activity, the order of efficacy being ATP > ADP > AMP. Inhibition by ATP was competitive with respect to acetyl-CoA and mixed with respect to oxaloacetate. Combinations of adenine nucleotides giving simulated energy charge values were also inhibitory, though the total adenine nucleotide concentration was of greater significance than the relative proportions of each in determining the degree of inhibition. When Mg2+was added at a concentration sufficient to saturate the adenine nucleotides, the inhibition was almost entirely relieved. The apparent absence of any rigorous control of citrate synthase by adenine nucleotides in oleaginous micro-organisms is consistent with previous observations that the flow of carbon through the glycolytic and pentose phosphate pathways to pyruvate and thence to citrate should be uninterrupted during the process of lipid accumulation.

Growth and metabolism of Ricinus communis endosperm in tissue culture

Endosperm tissue from both developing and germinating castor oil seeds (Ricinus communis L.) was grown as a callus in tissue culture. Callus growths were established from endosperm explants (without embryo) at all stages of seed development except from the quiescent unimbibed seed. Best tissue culture growth was observed with endosperm tissue obtained from seeds that had been germinated for 2 days.The lipid reserves diminished in all endosperm tissue which had been established on culture from germinating seeds but the rate of breakdown was much slower in cultures from 0-day germinated seeds. Glyoxylate cycle activity, as tested by acetate-1- and -2-14C incorporation, was not evident in long-term cultures.The process of lipid accumulation found in normal developing endosperm (24 to 36 days after fertilization) was not retained in culture but was replaced by a process resulting in the rapid loss of the already accumulated oil reserves.As far as could be determined the in vivo biochemical capabilities of the castor bean endosperm were not retained for a lengthy period in the tissue culture conditions used.