Lipids of filamentous fungi as a material for producing biodiesel fuel

Species of various filamentous fungus taxa were tested for ability to produce lipids suitable as a material for manufacturing biodiesel. The mucoralean fungus Cunninghamella japonica was found to be a promising lipid producer. The inexpensive medium for its growth developed in this study contained ammonium nitrate as a nitrogen source. With its use, up to 16 g/l biomass and over 7 g/l lipids was obtained. The fungal lipids were dominated by oleic acid. It constituted 50% of total fatty acids. The iodine index of the lipid fraction was 86.61. The heat of combustion of the lipids, 37.13 MJ/kg, was close to the value for rapeseed oil.     

Lipophorin density variation during oogenesis in Rhodnius prolixus

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations of lipophorins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophoin of different densities. Arch. Insect Biochem. Physiol. 35:301-313, 1997.© 1997 Wiley-Liss, Inc.     

Synthesis of biologically active lipids by fungus Mucor lusitanicus 306D grown on media with various composition

Growth and lipogenesis of fungus Mucor lusitanicus 306 D producing gamma-linolenic acid was studied under various regimes of nitrogen and carbon nutrition. Media containing food industry wastes such as maize extract, molasses, and protein hydrolysate were used. Content of gamma-linolenic acid was higher when using carbohydrates such as glucose and molasses as carbon sources and urea as a nitrogen source. At high glucose concentration (100 g/l), fed batch cultivation provided high content of gamma-linolenic acid in lipids (1 g/l). After extraction of lipids, fungus biomass contained 42% proteins with all essential amino acids. Defatted biomass was shown to be effectively assimilated by minks.     

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies: use of fluorescent lipids to study lipid-body formation in an oleaginous fungus

Fluorescent phosphatidic acid and phosphatidylcholine were used to characterize lipid-transport pathways into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Several characteristics of the lipid transport such as temperature dependence and ATP dependence were evaluated. The transport depicted by these fluorescent lipids was consistent with metabolism of radiolabelled lipids, indicating that fluorescent lipids are useful to study lipid-body formation in this fungus. The results dissect lipid transport of phosphatidic acid and phosphatidylcholine into lipid bodies and reveal regulatory steps for lipid-body formation in this fungus.     

Lipid accumulation in Trichoderma species

Abstract Two filamentous fungi, Trichoderma harzianum and Trichoderma viride , were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride . In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.     

Activity of NAD-dependent isocitrate dehydrogenase, isocitrate lyase, and malate dehydrogenase in Mucor circinelloides var. lusitanicus INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of gamma-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of gamma-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Spectrofluorimetric method for the estimation of total lipids in Eremothecium ashbyii fungal filaments using Nile blue and avoiding interference of autofluorescent riboflavin

A rapid, simple and sensitive spectrofluorimetric technique was developed for monitoring total lipids in hyphae of the riboflavin-overproducing fungus Eremothecium ashbyii using the fluorescent probe Nile blue in an aqueous system, avoiding the interference due to autofluorescent riboflavin. The existing methodologies for lipid estimation are tedious, requiring large biomass, solvent extraction and gravimetry. E. ashbyii is a hemiascomycete fungus which accumulates lipids in its mycelia prior to flavinogenesis. This study defines the conditions (wavelength selection and sensitivity) for the spectrofluorimetric quantification of lipids in situ in the macerated mycelia of this fungus in the presence of intracellular autofluorescent riboflavin without the need to extract the lipids from the mycelia. The fluorescent intensity was linear with the lipid concentration of the mycelia (by gravimetry) under three different growth conditions using glucose, olive oil and sunflower oil as carbon sources. This spectrofluorimetic method of lipid estimation can be applied to other fungi and microorganisms.     

Lipid metabolism of Pleurotus sajor caju

The biosynthesis of lipids in the mycelium and sporophore of Pleurotus sajor caju was studied. Whereas in the mycelium the biosynthesis of lipids was directly primarily towards storage (e.g. tri-acylglycerols), in the sporophore it was directed towards structural components (e.g. sterols). The incorporation of ¹⁴C precursors into non-polar and polar lipid fractions was generally similar for ¹⁴C acetate, ¹⁴C palmitate, ¹⁴C oleate and ¹⁴C linoleate in the case of mycelium and sporophore. It appears that linoleic acid was utilised as a source of acetate for lipid biosynthesis in the sporophore. A significantly higher incorporation of label was seen in sporophore sterol than in mycelial sterol. Malate dehydrogenase activity increased in the mycelium grown in the presence of lipids. Lipase of P. sajor caju was inducive. The growth of P. sajor caju was enhanced by increased lipid utilisation. The implications of these results on commercial cultivation of this fungus are discussed.     

Production of 5,8,11-eicosatrienoic acid by a Δ5 and Δ6 desaturation activity-enhanced mutant derived from a Δ12 desaturation activity-defective mutant of Mortierella alpina 1S-4

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3omega9) was attained with a mutant fungus, Mortierella alpina JT-180, derived from delta12 desaturation activity-defective and delta6 desaturation activity-enhanced M. alpina M209-7. Production of 20:3omega9 by JT-180 was 1.4 times greater than that of the parent strain M209-7. This is thought to be due to its enhanced Delta5 desaturation activity, which was 3.3 times higher than that of M209-7. In both strains, 78.5-80.4% of the total lipids comprised triacylglycerol (TG), and 76.6-79.0% of 20:3omega9 was present in TG. Comparing the fatty acid compositions among various lipid species, the highest percentages (24.1-37.6%) of 20:3omega9 in total lipids were found in phosphatidylcholine. For optimization of 20:3omega9 production by JT-180, a glucose concentration of 4% in the culture medium and shifting of the growth temperature from 28 degrees C to 20 degrees C on the 2nd day were shown to be effective. Under optimal conditions, 20:3omega9 production by JT-180 reached 1.92 g/l culture medium in a 10-l jar fermentor (corresponding to 81.5 mg/g dry mycelia and 18.3% of total fatty acids), which is greater than that reported previously from M209-7 (1.65 g/l).     

Differential channelling of liver lipids in relation to susceptibility to hepatic steatosis in two species of ducks

In the human, hepatic steatosis can be associated with an imbalance between synthesis, secretion and storage of hepatic lipids, and exhibits a genetic susceptibility. The effect of overfeeding on hepatic lipid channelling was investigated in two genotypes of ducks that differ in their susceptibility to fatty liver, i.e. the common duck, Anas platyrhynchos, and the Muscovy duck, Cairina moschata. Before overfeeeding, the Muscovy duck exhibited a lower subcutaneous adiposity and a higher muscular development, whereas hepatic composition was similar in both genotypes (>5% lipids and triglycerides accounting for 6–10% lipids). In the plasma lipoprotein profile, HDL predominated (5.5–7.8 g/l) over VLDL (0.09–0.25 g/l) and LDL (0.65–1.06 g/l). All lipid and lipoprotein concentrations were lower in the Muscovy duck. In response to overfeeding, the Muscovy duck exhibited a higher degree of hepatic steatosis (62 vs. 50% lipids), and a lower increase in adiposity and in the concentration of plasma triglycerides (6-fold vs. 10-fold) and VLDL (23-fold vs. 34-fold). Thus, certain genotypes may be more responsive to the dietary induction of fatty liver because of a less efficient channelling of hepatic lipids towards secretion into plasma and adipose storage, and the duck may represent a suitable model in which to study the development of hepatic steatosis and its pathogenesis.     

Activity of NAD-dependent isocitrate dehydrogenase,isocitrate lyase,and malate dehydrogenase in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of γ-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of γ-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Biomass and lipid production of heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> by using biodiesel-derived crude glycerol

Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode, the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microalgal lipid production may be expected.     

Biosynthesis of fructo-oligosaccharides by<Emphasis Type="Italic"> Sporotrichum thermophile</Emphasis> during submerged batch cultivation in high sucrose media

Biosynthesis of fructo-oligosaccharides (FOS) was observed during growth of the thermophilic fungus Sporotrichum thermophile on media containing high sucrose concentrations. Submerged batch cultivation with the optimum initial sucrose concentration of 250 g/l allowed the production of 12.5 g FOS/l. The FOS mixture obtained was composed of three sugars, which were isolated by size-exclusion chromatography. They were characterized by acid hydrolysis and HPLC as 1-kestose, 6-kestose and neokestose. The mechanism of osmotic adaptation of S. thermophile was investigated and sugars and amino acids were found to be the predominant compatible solutes. The fungus accumulated glutamic acid, arginine, alanine, leucine and lysine, in order to balance the outer osmotic pressure. Fatty acid analysis of the membrane lipids showed a relatively high percentage of unsaturated lipids, which is known to be associated with high membrane fluidity.     

Morphological and spectrometric analyses of lipids accumulation in a novel oleaginous microalga, Eustigmatos cf. polyphem (Eustigmatophyceae)

Eustigmatos cf. polyphem is a yellow-green unicellular edaphic microalga belonging to the eustigmatophyte. The characteristics of lipids accumulation of E. cf. polyphem grown in a bubble cylindrical photobioreactor under nitrogen-limited conditions was dissected by morphological and spectrometric analyses. Total lipids accumulation rate increased rapidly at early growth phase, with the emergence of many small lipid bodies. Afterwards, lipid bodies became abundant and enlarged primarily because of the progressive accumulation of lipids and the fusion of nearby lipid bodies. Maximum total lipids and neutral lipids content reached up to 60.59 % and 53.08 % of cell dry weight, accompanied with a biomass dry weight 7.9 g/l. E. cf. polyphem is thus referred to as an oleaginous microalga for biodiesel production due to its high biomass and considerable production of oils.     

The lipid composition of serum lipoproteins in the harbour seal, Phoca vitulina

The density profile of serum lipoproteins and their lipid composition was studied in 12 adult, female harbour seals. The animals were sampled after an approximate 20 hr fast. The density profile of lipoproteins showed that the harbour seals displayed a distinct VLDL (density less than 1.006 g/ml) and HDL band (density about 1.125 g/ml), but no clear LDL band. There was a rather diffuse population of lipoproteins in the density range of 1.019-1.100 g/ml. Mean serum total cholesterol concentration was 5.7 mmol/l; about 60% of this cholesterol was located in the HDL fraction (density greater than 1.063 g/ml). The fasted seals were found to carry 4% of serum total lipids in chylomicrons. These lipoproteins consisted of 51% of triaclyglycerols (on the basis of total chylomicron lipids). The LDL (defined as heparin-manganese precipitable lipoproteins in VLDL and chylomicron-deficient serum) contained 49% of cholesterol and 43% of phospholipids (on the basis of total LDL lipids). The HDL (defined as heparin-manganese soluble lipoproteins in VLDL and chylomicron-deficient serum) contained 36% of cholesterol and 58% of phospholipids (on the basis of total HDL lipids).     

利用平板反应器大量培养高产油绿藻——尖状栅藻的生长和油脂积累规律

以新近分离的淡水绿藻--尖状栅藻(Scenedesmus acuminatus)为研究对象,将改良的BG-11培养基中的初始NaNO₃浓度降低为6.0mmol/L和3.6mmol/L,利用新设计的内置拉筋平板式光生物反应器对尖状栅藻(S. acuminatus)进行大量培养。测定不同时相的生物量、总脂含量、脂组分含量及脂肪酸组成和含量,分析尖状栅藻(S. acuminatus)大量培养时的生长和油脂积累规律。当初始NaNO₃浓度为6mmol/L时其最高生物量(6.27g/L)明显高于初始NaNO₃浓度为3.6mmol/L时的生物量(5.30g/L);而最高的总脂含量在初始NaNO₃浓度为3.6mmol/L时获得为干重的56.6%,高于初始NaNO₃浓度为6mmol/L时的总脂含量(51.6%)。总脂经硅胶柱层析分级后得到三种类型的脂组分:中性脂、糖脂和磷脂,随着培养时间的延长中性脂含量逐渐增加,培养至18d后,中性脂的含量分别达到总脂的 90.9%(6 mmol/L NaNO₃)和 92.0%(3.6 mmol/L NaNO₃)及干重的 47.5%(6.0 mmol/L NaNO₃)和 51.4%(3.6 mmol/L NaNO₃)。主要脂肪酸组成为棕榈酸、棕榈油酸、硬脂酸、油酸、亚麻油酸和亚麻酸,这六种脂肪酸在不同时相的含量变化范围分别为89.92%~96.18%(占总脂肪酸)和12.5%~50.7%(占细胞干重)。总脂、中性脂及总脂肪酸单位体积产率分别为:0.18 g/L/d,0.16 g/L/d和0.15 g/L/d(6.0 mmol/L NaNO₃)及0.16 g/L/d,0.15 g/L/d和0.15 g/L/d(3.6 mmol/L NaNO₃)。研究结果表明,尖状栅藻(S. acuminatus)是一株易于规模化培养、脂肪酸组成适合于生物柴油生产的高产油微藻。     

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.     

Lipid composition and effect of amphotericin B on yeast cells of Paracoccidioides brasiliensis

Yeast cells of Paracoccidioides brasiliensis strain SN, were obtained for analysis of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. Such analysis were made on cells cultured in the presence or absence of amphotericin B and on non proliferating cell suspensions exposed to the antibiotic. Marked alterations in lipid contents were observed in this different conditions. The major alterations were the reduction of total lipids, sterols, and palmitoleic acid in both, proliferating and non proliferating antibiotic exposed cells. The effect of amphotericin B was evaluated also in terms of viability and release of intracellular substances, at different times of exposure. The minimal inhibitory concentration (MIC) determined for that strain of this fungus was 0.2 g/mL.     

Pyrene fluorescence probing of unsaturated lipids in Phytophthora infestans zoospores.

Pyrene rapidly penetrates into isolated zoospores of phytopathogenic fungus Phytophthora infestans localizing predominantly in lipid bodies. An analysis of steady-state monomer and excimer fluorescence spectra, as well as of vibronic structure has suggested a considerable part of the fluorescent probe to be located in a lipid environment. Pyrene partition into hydrophilic phase was observed at its high concentrations. Catalytic hydrogenation of unsaturated lipids in zoospores in situ reduced excimer production. The kinetics of changes of pyrene excimerization suggest that hydrogenation affects both the surface and the intrinsic lipids of the zoospores. The usefulness of pyrene as a fluorescent probe for unsaturated lipids in membranes and lipid bodies of intact cells, and the possible role of eicosapolyunsaturated fatty acids in induction of immune response in potato plants are discussed.     

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

Aims: To investigate the effect of organic nitrogen on lipogenesis during growth of Cunninghamella echinulata on tomato waste hydrolysate (TWH) media. Methods and Results: 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%γ-linolenic acid (GLA), hence the GLA yield was more than 1 g l⁻¹. Conclusions: Organic nitrogen compounds found in TWH favour glucose (but not glycerol) uptake and lipid accumulation in C. echinulata. Significance and Impact of the Study: 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.