Amino acids as carbon,energy and nitrogen sources for Penicillium camembertii

Three groups of amino acids were previously characterized on their ability to be assimilated as carbon source by Penicillium camembertii. In view of a deeper understanding of their metabolic behaviour, growth of P. camembertii on glucose, the limiting substrate, and an amino acid was examined in batch culture. Amino acids from the first group (Cys, His, Lys, Met, Trp and Val) are convenient nitrogen sources, but cannot be assimilated as carbon sources. However, they are also dissimilated, namely used for energy supply by oxidation into CO₂, during stationary phase, after glucose depletion, as shown for lysine; and the corresponding nitrogen was released as ammonium. Growth exhibited diauxic behaviour for the second group of amino acids (Arg, Leu), since they can be assimilated as carbon sources, in addition to their assimilation as nitrogen sources, but only after glucose depletion, as shown for arginine. A clear differentiation between the assimilated and the dissimilated carbon was demonstrated for the third group of amino acids (Ala, Asp, Glu, Gly, Pro, Ser, Thr); it was shown that the carbon from glutamic acid was assimilated, while the carbon from glucose was dissimilated. Copyright © 2006 Society of Chemical Industry     

Carbon assimilation and dissimilation during growth of Geotrichum candidum on amino acids and glucose

BACKGROUND: This work examines the metabolic behaviour of amino acids during Geotrichum candidum growth, in the presence of a primary carbon source like glucose. Amino acids were characterized based on their carbon assimilation and dissimilation by G. candidum, in the presence of glucose as the limiting substrate. RESULTS: The first group (Cys, His, Phe, Thr and Trp) was only used as nitrogen sources by G. candidum, with glucose being the carbon and energy source. Glucose repression was shown for the rest of the amino acids, since only after glucose depletion amino acids from the second group (Gly, Lys, Met, Val) were dissimilated for energy supply by oxidation into CO₂, while those from the third group (Ala, Arg, Asp, Glu, Leu, Pro and Ser) were assimilated as carbon sources (and additionally used as nitrogen sources), leading to a diauxic growth. CONCLUSION: This energy‐saving response was not previously shown for the second fungus involved in ripening of soft white cheese—P. camembertii—leading to simultaneous use of some amino acids and glucose as carbon and energy sources, and hence lower growth rates than those recorded during G. candidum growth. Copyright © 2007 Society of Chemical Industry     

An unstructured model for the analysis of substrate consumption and product release in relation to biosynthesis and cell maintenance during batch cultures of Geotrichum candidum and Penicillium camembertii

An unstructured model has been developed to predict microbial growth based on carbon or nitrogen substrate consumption, ammonia or carbon dioxide production and proton transfer. The model has been validated for batch cultures of Geotrichum candidum and Penicillium camembertii growing on peptones and peptones + lactate based media. The contributions of the considered kinetics to biosynthesis and cellular maintenance can be deduced from this model. The nitrogen source (peptones) was mainly utilized in biosynthesis: for P camembertii growing on peptones, 86% of the metabolized peptones. G candidum metabolized peptones preferentially to lactate as a carbon source, resulting in lactate utilization by a maintenance mechanism during the stationary state. In contrast, P camembertii, which metabolized fewer amino acids as a carbon source, utilized lactate mainly for biosynthesis (83% of the consumed lactate). Most (up to 71%) of the ammonia released was produced by deamination of amino acids utilized as both carbon and nitrogen sources by growth‐associated metabolism. With peptones, proton transfer resulted from ammonia release, most likely as a result of the growth‐associated mechanism, as supported experimentally (55–58% of the released ammonia for both microorganisms). The contribution of lactate to proton transfer resulted in 76% of protons exchanged by a growth‐associated mechanism during P camembertii growth. For total carbon dioxide production, the contributions of the energy supplies for biosynthesis and cell maintenance were similar; except during P camembertii growth in the presence of lactate (65% of growth‐associated CO₂ production). © 2002 Society of Chemical Industry     

Analysis of batch submerged cultivations of Geotrichum candidum growing in lactate with either glutamate or lysine

Amino acid and lactate assimilation by Geotrichum candidum were examined in submerged culture. G candidum growth on lysine and lactate clearly showed that lactate can be assimilated as a carbon source, in addition to an energy source. Lysine was chosen since G candidum did not grow on lysine as the sole carbon and nitrogen source. On the contrary, when a convenient carbon source amino acid (glutamate) was used, lactate appeared to have no effect on G candidum growth; it was only assimilated at a low rate during the stationary state for energy supply, in agreement with previous observations recorded during growth on peptones. The assimilation of glutamate, the more convenient substrate for G candidum growth, required less energy than lysine assimilation, indicated by the lower level of CO₂ released. Copyright © 2004 Society of Chemical Industry     

Diffusion of glutamic acid in relation to growth of Geotrichum candidum and Penicillium camembertii at the surface of a solid medium

Growth of pure cultures of Geotrichum candidum or Penicillium camembertii at the surface of a solid medium was studied. Consumption of nutrients by the microorganisms growing at the surface of the gel induced their diffusion from the core to the rind. Particular attention was paid to the diffusion of glutamic acid, the nitrogen source, in relation to the growth of the microorganism at the surface. The growth kinetic has been described using the Verlhust model. The diffusion coefficient of glutamic acid in sterile culture medium was measured and found to be 0.55 cm² day^?1. With this coefficient and assuming that the glutamic acid consumption was partially linked to growth, the experimental diffusion gradients have been fitted. Good agreement was found between experimental data and the diffusion/reaction model. The glutamic acid diffusion cannot be assumed to limit growth, since noticeable amounts of this substrate always remained at the upper surface of the gel. Copyright © 2004 Society of Chemical Industry     

Nutrient Composition of Larval Nectar Secretions from Three Species of Myrmecophilous Butterflies

A comparative chemical analysis of the larval nectar secretions and hemolymph from three unspecifically and facultatively ant-attended lycaenid species (Polyommatus coridon, P. icarus, and Zizeeria knysna) was performed by using high-performance liquid chromatography techniques. Sucrose was the main sugar component in all three species. In half of the samples of P. coridon, it was accompanied by glucose, whereas other sugars occurred only rarely. In P. icarus and Z. knysna, melezitose was the second-most important component, followed by fructose and glucose. Total sugar contents were 43.6 ± 14.8 g/l (mean ± SD) for P. coridon, 74.2 g/l for P. icarus, and 68.3 ± 22.6 g/l for Z. knysna. Up to 14 different identified amino acids were found in P. coridon nectar, with a total content of 9.7 ± 3.4 g/l. Leucine was always the major component (contributing 50% of overall amino acid content). Other important amino acids were tyrosine, proline, arginine, and phenylalanine. P. icarus nectar contained up to six amino acids with a total content of 1.2 g/l, dominated by tyrosine and phenylalanine. Z. knysna nectar contained alanine and proline, with only 0.3 ± 0.17 g/l total content. In the hemolymph of all species, up to 16 different amino acids occurred relatively regularly, with histidine dominating, followed by serine and proline. The amino acid pattern in hemolymph was considerably different from that of the nectar secretions. Larval diet weakly influenced P. coridon nectar sugars, and with a semisynthetic diet, a more homogeneous amino acid pattern was detected. Comparison with reports from other lycaenid species shows that secretions rich in amino acids are related to intimate, often obligate ant associations, whereas facultative, unspecific myrmecophiles rely on carbohydrates.     

Effects of changes in the major carbon source on the fatty acids ofEuglena gracilis

Euglena gracilis was cultured under both heterotrophic and phototrophic growth conditions using ethanol, glucose or CO₂ as the major carbon source. Total fatty acid analyses indicated that ethanol produced more highly unsaturated acids than did glucose under both growth conditions. Growth in the light on CO₂ yielded a very high content of 18∶3, 16∶3 and 16∶4 (33%), compared to ethanol (11%) or glucose (10%). These two preformed carbon sources enhanced the content of the C₂₀ and C₂₂ polyenes compared to CO₂, and growth in the dark on to CO₂, and growth in the dark on ethanol caused a further increase in these polyenes. Growth in the dark on glucose caused only a slight increase of the C₂₀ and C₂₂ polyenes compared to growth in the light on this carbon source. When the fatty acid patterns of the two dark-grown heterotrophs were compared, two observations were quite evident. First, there was a two-fold increase in the saturated acids in the cells grown on glucose. This was largely due to myristic acid. Second, the C₂₀ and C₂₂ polyenes were almost twice as concentrated in the cells grown on ethanol.     

Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth

Twenty microalgal strains were investigated in photoautotrophic flask cultures for their potential for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production. The highest EPA proportion (% of total fatty acids) was produced by Monodus subterraneus UTEX 151 (34.2%), followed by Chlorella minutissima UTEX 2341 (31.3%) and Phaeodactylum tricornutum UTEX 642 (21.4%). The highest DHA proportion (% of total fatty acids) was obtained in Crypthecodinium cohnii UTEX L1649 (19.9%), followed by Amphidinium carterae UTEX LB 1002 (17.0%) and Thraustochytrium aureum ATCC 28211 (16.1%). Among the 20 strains screened, the EPA yield was high in M. subterraneus UTEX 151 (96.3 mg/L), P. tricornutum UTEX 642 (43.4 mg/L), Chl. minutissima UTEX 2341 (36.7 mg/L), and Por. cruentum UTEX 161 (17.9 mg/L) owing to their relatively high biomass concentrations. The DHA yield was high in C. cohnii UTEX L1649 (19.5 mg/L) and A. carterae UTEX LB 1002 (8.6 mg/L). Heterotrophic growth of these 20 microalgae was also tested on two different carbon sources, acetate and glucose. All microalgae except Nannochloropsis oculata UTEX LB 2164 showed growth on glucose (5 g/L) under heterotrophic conditions. Twelve of them could grow heterotrophically when acetate (1 g/L) was used as their sole carbon and energy source.     

Formulation and optimization of two culture media for the production of tumour necrosis factor-β in Escherichia coli

Two culture media were designed and optimized by statistical techniques for the growth of Escherichia coli K12 HB101 (pCG402) which expressed human tumour necrosis factor-β (TNF-β). Common compounds, such as MgSO₄ and KH₂PO₄, were used to provide the bacteria with the necessary elements for biomass synthesis. For compounds not required for biomass synthesis, such as thiamine, or compounds used for both biomass synthesis and as an energy source, such as glucose, yield coefficients were used. In formulating the composition of the nutrients, carbon was chosen as the limiting substrate in both media. In the complex medium (CM), casein hydrolysate was selected to supply the two auxotrophic amino acids, proline and leucine. The optimal ratio of glucose to casein hydrolysate was determined to be 1:0·6 by using a centre composite design experiment. In the defined medium (DM), the concentrations of the three carbon sources, glucose, proline and leucine were based on their respective yield coefficients (Y_{biomass/glucose}: 0·5, Y_{biomass/proline}: 7, Y_{biomass/leucine}: 13·5). Shake flask experiments based on a fractional design were used to confirm that the two media were glucose limiting. Bacterial growth was improved in CM whereas DM gave higher TNF-β expression. A 2-dm³ fed-batch fermentation using CM was performed and a dry biomass concentration of 20 g dm^?3 was obtained with the expression of soluble TNF-β being 20 mg g^?1 dry biomass. With this fed-batch system, a high biomass concentration and high expression of TNF-β were achieved concomitantly.     

Free Fatty Acids Reduction in Waste Cooking Oil by Rhodosporidium toruloides and Simultaneous Carotenoids,Lipids, and PAL Enzyme Production in a Two-Phase Culture System

Waste cooking oil (WCO) is a problematic waste product that contains free fatty acids (FFAs), preventing it from being valorized easily as biodiesel and poses an environmental hazard if incorrectly disposed. The use of WCO as a carbon source for Rhodosporidium toruloides (R. toruloides) using a two-phase culture system is developed. The normal growth of R. toruloides when cultured in WCO (OD₆₀₀ 52) reveals its ability to use a hydrophobic substrate as the carbon source compared to glucose (OD₆₀₀ 51.9). Interestingly, the extracellular lipase activity when R. toruloides is grown on WCO is 14.4 U mL⁻¹ compared to when grown on glucose (2.4 U mL⁻¹). Additionally, FFA levels in WCO are reduced from 2% to 0.2% at end of fermentation, suggesting that R. toruloides can consume FFA. Furthermore, higher yield of beneficial products: β-carotene (4.57 µg mL⁻¹), torularhodin (4.2 µg mL⁻¹), fatty acids (1 mg mL⁻¹), and phenylalanine ammonia-lyase (PAL) enzyme (0.12 µmol mg⁻¹) are produced when WCO is the carbon source, compared to glucose (4.1 µg mL⁻¹ β-carotene, 3.0 µg mL⁻¹ torularhodin, 1 mg mL⁻¹ of fatty acids, and 0.096 µmol mg⁻¹ PAL enzyme). This is a first study that shows R. toruloides can grow on hydrophobic carbon source.     

磷源对产甘油假丝酵母发酵生产甘油的影响及动力学分析

甘油是一种重要的轻化工原料,广泛应用于化妆品、牙膏、烟草、香精、水性油墨、印染纺织、涂料、合成树脂、皮革、造纸、制药、食品和国防等各个领域的1700多种产品．在我国,由于受合成洗涤剂的冲击,肥皂行业日渐萎缩,甘油产量大幅下降．因此,利用耐高渗酵母发酵生产甘油的产业化前景看好．     

The effect of amino acids and amino acid analogues on growth of an obligate methanotroph,Methylosinus trichosporium OB3b

The sensitivity of the obligate methanotroph, Methylosinus trichosporium OB3b to a number of amino acid analogues and amino acids has been examined. Sulphaguanidine (5 μg ml^?1) norleucine (100 μg ml^?1) m-fluorophenylalanine (50 μg ml^?1), p-fluorophenylalanine (50 μg ml^?1) and S2-aminoethyl-C-cysteine (1000 μg ml^?1) inhibited growth. Proline, threonine, methionine and lysine (2–4 μg ml^?1) also inhibited growth, but arginine and leucine at similar concentrations had no effect. Attempts were made to isolate amino acid analogue resistant mutants, which would be expected to overproduce amino acids. Two approaches were made, selection of spontaneous and induced mutants in plate culture, and selection of a spontaneous mutant during growth in continuous culture under selective conditions, but no mutants were obtained. Possible reasons for failure to obtain such mutants are discussed.     

Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients

BACKGROUND: Protein stability of therapeutic monoclonal antibodies during processing and final storage is imperative for the commercial success of the product. Amino acid addition is one of the options for stabilization of proteins that can be employed during the manufacturing process and storage. RESULTS: Use of arginine in the elution buffer during Protein A purification and subsequent neutralization doubled the yield of antibody compared with the original glycine‐based elution buffer. The role of amino acids in stabilizing monoclonal antibody liquid formulations was then studied using differential scanning calorimetry. Increase in the unfolding transition temperatures (T_m) of immunoglobulin G (IgG) was measured after supplementing a glycine buffer with a range of amino acids. The basic amino acids histidine, lysine, and arginine stabilized all three domains of IgG. The neutral amino acids serine and alanine provided less stabilization; glutamine, proline, and the acidic amino acids provided negligible stabilization. CONCLUSION: The positive charge on the side‐chain of histidine, lysine, and arginine appears to be the most important factor affecting the IgG stabilization. It is probable that the mechanism for IgG stabilisation is the same for all of the basic amino acids and that it binds transiently to IgG side chains, altering water populations in the solvation shell, making unfolding and aggregation less energetically favourable. Copyright © 2011 Society of Chemical Industry     

An Isorhamnetin Rhamnoglycoside Serves as a Costimulant for Sugars and Amino Acids in Feeding Responses of Adult Western Corn Rootworms (<Emphasis Type="Italic">Diabrotica virgifera virgifera</Emphasis>) to Corn (<Emphasis Type="Italic">Zea mays</Emphasis>) Pollen

Adult beetles of Diabrotica virgifera virgifera LeConte (western corn rootworm) feed on pollen of Zea mays L. (corn) and other plant species. To identify D. virgifera feeding stimulants, beetle responses to mixtures of known and novel phagostimulants, presented at their naturally occurring concentrations in maize pollen, were compared to individual component responses applying the amount occurring in 0.2 mg of pollen per cellulose feeding disk. On a molar basis, three major sugars (fructose, glucose, and sucrose) were more prevalent in corn pollen buffer extract (CPE) than free amino acids. Western corn rootworm feeding was stimulated by the three sugars (28% disk consumption) and, to an even greater extent, by a mixture of 21 free amino acids (41% disk consumption). However, the combination of three sugars and 21 amino acids elicited a level of D. virgifera feeding (41% disk consumption) similar to that of the 21 amino acids alone. A novel maize pollen phagostimulant was purified from CPE by using solid-phase extraction followed by RP-HPLC. Based on its mass fragment pattern, two UV maxima (254 and 359 nm), and previous isolation from maize pollen, this phagostimulant is tentatively identified as isorhamnetin 3-O-neohesperidoside. This compound interacted additively with the mixture of three sugars and 21 amino acids, to produce 77% of the phagostimulation level of CPE. Therefore, a possible stimulatory mechanism for D. virgifera feeding on corn pollen has been elucidated.     

Unstructured kinetic model for reuterin and 1,3‐propanediol production by Lactobacillus reuteri from glycerol/glucose cofermentation

BACKGROUND: Lactobacillus reuteri is unable to grow on glycerol as sole carbon and energy source hence, glycerol is used as an alternative hydrogen acceptor during growth on available carbohydrates. Thus, glycerol is converted to reuterin and 1,3‐propanediol (1,3‐PDL), both products with interesting industrial applications. These compounds are commonly produced by using resting cells in two‐step fermentation processes. RESULTS: The glycerol/glucose cofermentation by L. reuteri yields reuterin and 1,3‐PDL at a glycerol concentration higher than 100 mmol L⁻¹. An increase of glycerol concentration from 200 to 400 mmol L⁻¹ showed no additional stimulatory effect on ethanol and acetate production but consistently reduced the lactate concentration. It was also found that reuterin concentration reached a maximum value and subsequently decreased due to its conversion to 1,3‐PDL. An unstructured kinetic model was proposed to describe simultaneously microbial growth, substrates consumption and products formation. A multi‐response nonlinear regression analysis based on Marquardt algorithm combined with a Runge‐Kutta integration method was used to obtain the values of the fitting parameters. CONCLUSIONS: The optimum concentration of glycerol for maximum reuterin and 1,3‐PDL production was 200 mmol L⁻¹. The complete process was satisfactorily described by the kinetic model proposed. Copyright © 2008 Society of Chemical Industry     

碳源对土曲霉中洛伐他汀生物合成的影响

通过摇瓶培养考察碳源种类及其初始水平对土曲霉次级代谢物洛伐他汀生物合成的影响。蔗糖和葡萄糖等快速利用碳源产洛伐他汀量较低,且易积累其直接前体monacolin J;淀粉和甘油等缓慢利用碳源有利于细胞生长和产物合成,其monacolin J积累相对较少,甘油为碳源时,可获得最高产物量(937.5±12.3)mg/L,乳糖不利于细胞生长导致较低的产物量。以淀粉为碳源时,其初始水平的增加可促进洛伐他汀的合成,碳质量分数为2%时对底物利用率较高,4%时最高产物量可达(875.4±23.1)mg/L,monacolin J则在2%—3%时积累较多,同时产生较多的2-甲基丁酸。     

Selected Ectomycorrhizal Fungi of Black Spruce (Picea mariana) can Detoxify Phenolic Compounds of Kalmia angustifolia

Allelopathy has been implicated as a factor contributing toward failure of black spruce (Picea mariana) regeneration in Kalmia angustifolia-dominated sites in eastern Canada. Several phenolic acids of Kalmia origin inhibit primary root growth of black spruce. We tested the hypothesis that some well-adapted conifer ectomycorrhizae can degrade and detoxify water-soluble phenolic compounds produced by Kalmia and use the degraded products as a carbon source to stimulate growth. We found that hyphal growth of Paxillus involutus, a common ectomycorrhizal fungus of black spruce, was stimulated by water leachates of Kalmia leaf and litter. An equimolar mixture of three phenolic acids (ferulic, o-coumaric, and o-hydroxyphenylacetic acid), commonly found in Kalmia, had no negative effects on fungal growth at 1 mM concentration. The o-hydroxyphenylacetic (o-HPA) acid, which is known to be toxic to black spruce, was found to stimulate the growth of Laccaria laccata, L. bicolor, and P. involutus (isolates 211804 and 196554) by 38.4, 29.3, 25.0, and 18.9%, respectively, at 1 mM. Pure ferulic, o-coumaric, and o-HPA acids were degraded by 100, 98, and 79.5%, respectively, within 10 d in the presence of P. involutus 211804. However, L. laccata could not tolerate high concentrations of the Kalmia leachates. P. involutus and L. bicolor used o-HPA acid as a carbon source when cultured in noncarbon nutrient medium. The 0.5 and 0.2 mM o-HPA acid inhibited the root growth of black spruce. However, after solutions had been exposed to a culture of P. involutus, they had no significant effect on seedling growth of black spruce. We concluded that some ectomycorrhizal fungi, such as P. involutus and L. bicolor, are able to degrade Kalmia phenolics. Our findings point to a mechanism by which ectomycorrhizal species can control species interactions in higher plants by changing the rhizosphere chemistry.     

Effect of glucose:xylose ratio on xylose reductase and xylitol dehydrogenase activities from Candida guilliermondii in sugarcane bagasse hydrolysate

The influence of glucose on xylose reductase (XR) and xylitol dehydrogenase (XDH) enzyme activity was evaluated from sugarcane bagasse hydrolysate fermentations with different glucose:xylose ratios (1:25, 1:12, 1:5 and 1:2.5) by employing an inoculum of Candida guilliermondii grown in media containing glucose, a mixture of glucose and xylose, or only xylose as carbon sources. According to the results, the glucose:xylose ratio affected positively this bioconversion and a correlation was not observed between the favourable conditions for xylitol production and the XR and XDH activities. Also, the results were influenced not only by the glucose:xylose ratio in the fermentation medium, but also by the carbon source employed in the growth medium of the inoculum. The optimum condition for xylitol production by C. guilliermondii in sugarcane bagasse hemicellulosic hydrolysate should use hydrolysate with a 1:5 glucose:xylose ratio and inoculum grown in medium containing xylose as the only carbon source. Copyright © 2006 Society of Chemical Industry     

Mathematical model of growth and polyhydroxybutyrate production using microbial fermentation of Bacillus subtilis

Poly-3-hydroxybutyrate (PHB) has been extensively utilized as a biodegradable plastic. The value of substrate inhibition constant (K_I) was also established in shake flask cultures by varying the initial glucose concentration (20–160?g/L) in growth media. Excess carbon source adversely affected the growth of Bacillus subtilis cultures. The production kinetics of PHB was studied using batch fermentation strategy for B. subtilis culture. Batch cultivation was also performed in a 5-L stirred tank bioreactor to obtain a Biomass and PHB yield of 5.25 and 1.6?g/L, respectively. The kinetic data of biomass, PHB production, and substrate consumption was used to estimate the optimized values of the growth and product formation kinetic parameters. Optimal values of kinetic parameters (µ_m value of 0.325, K_S value of 10.53?g/L for glucose, Y value of 0.183?g/g of glucose, K_I value of 105?g/L, m value of 0.12?g/(g h), k₁ of 0.36?g/g, and k₂ value was 0.12?g/(g h)) and the initial values of biomass, substrate and PHB concentration (X?=?0.14?g/L, S?=?9.99?g/L, and P?=?0.13?g/L) were utilized to obtain a modified mathematical model for PHB production. Statistical validity of the mathematical model simulations were measured using F-test. The F-test showed that the statistical validity of the model was more than 95% when compared with experimentally obtained values. This model also predicted that the production of PHB using B. subtilis cultures is mainly growth associated.     

Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source

Crypthecodinium cohnii, a marine alga used for the commercial production of docosahexaenoic acid (DHA), was cultivated in medium containing sodium acetate as principal carbon source; the pH was maintained at a constant value by addition of acetic acid, which also provided an additional carbon source in a controlled manner. The accumulation of lipid by C. cohnii in this pH-auxostat culture was significantly greater than previously reported for batch cultures using glucose as principal carbon source. Of six strains tested in pH-auxostat cultures, C. cohnii ATCC 30772 was the best, with the cells reaching 20 to 30 g dry weight per liter after 98 to 144 h and containing in excess of 40% (w/w) total lipid, with DHA representing approximately half of the total fatty acids in the triacylglycerol fraction. A productivity of 36 mg DHAL⁻¹ h⁻¹ was achieved during cultivation for 98 h using a 5% (vol/vol) inoculum, and DHA production was in excess of 3 g per liter of culture. Most of the DHA was present in neutral lipids.