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.     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

Glucose-containing polymer rings enable fed-batch operation in microtiter plates with parallel online measurement of scattered light,fluorescence, dissolved oxygen tension,and pH

Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.     

Xylitol production in repeated fed batch cultivation

The ability of Candida parapsilosis to produce xylitol was tested using successive substrate supplies, and the importance of the amount of viable cells in enhancing the conversion rate was demonstrated. The suitability of this yeast for the production of xylitol was investigated in repeated fed-batch cultivation, using pure xylose or mixtures of xylose and glucose. The use of this process increased productivity by about 40% compared with simple batch cultivation without loss of yield of product on substrate. The presence of glucose in the culture medium seemed to stimulate the specific growth rate, but had no influence over other fermentative parameters.     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain

By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.     

Culture medium containing glucose and glycerol as a mixed carbon source improves ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine production by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M-Z18

To improve the efficiency of ε-poly-l-lysine (ε-PL) production by Streptomyces sp. M-Z18, batch and fed-batch fermentations with glucose and glycerol (co-fermentations) were performed. The batch fermentations showed that the initial ratio of glucose to glycerol plays an important role in glucose/glycerol co-fermentation. The optimal glucose/glycerol weight ratio was 30/30; this resulted in a maximum ε-PL productivity of 5.26 g/L/d. Glucose and glycerol were consumed synergistically during the co-fermentation process, and the length of time during which the substrate was exhausted was significantly shortened compared with the single carbon source fermentation. Under optimized conditions, fed-batch fermentations with glucose and glycerol as a mixed carbon source achieved maximum ε-PL concentration and productivity values of 35.14 g/L and 4.85 g/L/d, respectively. These values were respectively 1.43- and 1.39-, and 1.17- and 1.16-folds higher than those obtained from fermentations with glucose and glycerol as single carbon sources. The present study is the first to suggest that glucose/glycerol co-fermentation may be an efficient strategy for ε-PL production by Streptomyces sp. M-Z18.     

Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production. The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.     

Enhanced cellulase production using Solka Floc in a fed-batch fermentation

Summary Use of a fed-batch mode of cultivation of T. reesei has permitted high concentrations of substrate to be consumed. This has resulted in the production of high titre cellulase preparations around 30 FPU/ml at high volumetric productivities (177 IU/L.hr).Perhaps the most obvious area for major improvement in the process of cellulose utilization is the production of cellulase enzyme for hydrolysis of wood and agricultural residues. It has been estimated that some 50% of the cost of producing glucose from cellulosic material is attributable to enzyme production alone (Perez, et al., 1980). Improvements in the area would therefore have a dramatic impact, and are of paramount importance if economical hydrolysis processes are to be realized. The first major thrust in the area has been the development of improved mutant strains of T. reesei, free from catabolite repression and capable of constitutive cellulase production (Montenecourt and Eveleigh, 1977; Gallo, 1982).While this effort continues to develop further high yielding mutants, improvement must also come from developments in fermentation techniques. A major advance is the use of fed-batch cultivation, which provides a means of avoiding the agitation and aeration difficulties, as well as repression effects encountered with high substrate concentration batch fermentation. This report briefly compares batch and fed-batch operation over a range of substrate concentrations.     

双碳源单细胞蛋白高密度培养

单细胞蛋白(SCP)培养具有增殖速度快、原料来源广、蛋白质含量高等优点,正在成为重要的蛋白质来源之一。生产SCP的原料包括烷烃、低碳醇类及碳水化合物。由碳水化合物,尤其是由各种农副产品加工的废料、造纸工业废水及木质纤维素水解产物等,生产SCP属于废弃资源的综合利用,具有重要的经济和社会意义。这类原料中常含有多种碳源,例如,在木质纤维水解液中,有以木糖为主的五碳糖,也有以葡萄糖为主的六碳糖,木糖与葡萄糖之比约为1:2。这样,木糖的利用就成了一个关键问题。葡萄糖对木糖代谢的抑制作用随葡萄糖比例的降低而减轻;Slinger和Bothast研究了用混合糖培养Candida shehetane的过程,当木糖与葡萄糖的比例为3:1及1:1时,葡萄糖的利用速度分别比木糖高1.6倍及3.4倍;在低还原势时,酵母产酒精速率降低而细胞增长速率加快,可以达到较高细胞浓度。     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.     

High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.     

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

The enzyme controlled substrate delivery cultivation technology EnBase(?) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.     

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

汉逊德巴利酵母发酵葡萄糖生产D-阿拉伯糖醇

从378株耐高渗酵母中,筛选到1株由葡萄糖发酵高产D-阿拉伯糖醇的酵母。通过生理生化和分子生物学的鉴定,证实该菌株为Debaryomyces hansenii,保藏编号CICIM Y 0504。研究该酵母摇瓶发酵的主要影响因素,确定其摇瓶发酵条件为:葡萄糖200 g/L,酵母膏10 g/L,初始pH值3,装液量20 mL/250 mL,温度30℃。在此条件下发酵120 h,D-阿拉伯糖醇浓度达90.37 g/L,转化率45.18%。在15 L发酵罐对该酵母进行扩大培养,结果表明,初始葡萄糖浓度200 g/L的分批发酵产D-阿拉伯糖醇64.07 g/L,转化率33.94%;葡萄糖浓度控制在30～50 g/L的分批补料发酵产D-阿拉伯糖醇125 g/L,转化率37.5%。研究结果对葡萄糖发酵生产D-阿拉伯糖醇工业化的实现具有重要启示。     

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.     

The secretome of Pichia pastoris in fed-batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time

The quantitative changes of the secretome of recombinant Pichia pastoris (Komagataella phaffii) CBS7435 over the time-course of methanol- or glucose-limited fed-batch cultures were investigated by LC-ESI-MS/MS to define the carbon source-specific secretomes under controlled bioreactor conditions. In both set-ups, no indication for elevated cell lysis was found. The quantitative data revealed that intact and viable P. pastoris cells secrete only a low number of endogenous proteins (in total 51), even during high cell density cultivation. Interestingly, no marked differences in the functional composition of the P. pastoris secretome between methanol- and glucose-grown cultures were observed with only few proteins being specifically affected by the carbon source. The ‘core secretome’ of 22 proteins present in all analysed carbon sources (glycerol, glucose and methanol) consists mainly of cell wall proteins. The quantitative analysis additionally revealed that most secretome proteins were already present after the batch phase, and depletion rather than accumulation occurred during the fed-batch processes. Among the changes over cultivation time, the depletion of both the extracellularly detected chaperones and the only two identified proteases (Pep4 and Yps1-1) during the methanol- or glucose-feed phase appear as most prominent.     

Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells.

Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.     

Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates

ABSTRACT: BACKGROUND: Microbial lipids are a potential source of bio- or renewable diesel and the red yeast Rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. METHODS: R. toruloides was grown in batch and fed-batch cultures in 0.5 l bioreactors at pH 4 in chemically defined, nitrogen restricted (C/N 40 to 100) media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. Lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by GC. RESULTS: R. toruloides was grown on glucose, xylose, arabinose or mixtures of these carbohydrates in batch and fed-batch, nitrogen restricted conditions. Lipid production was most efficient with glucose (up to 25 g lipid L1, 48 to 75% lipid in the biomass, at up to 0.21 g lipid L1h1) as the sole carbon source, but high lipid concentrations were also produced from xylose (36 to 45% lipid in biomass). Lipid production was low (15-19% lipid in biomass) with arabinose as sole carbon source and was lower than expected (30% lipid in biomass) when glucose, xylose and arabinose were provided simultaneously. The presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. High cell densities were obtained in both batch (37 g L1, with 49% lipid in the biomass) and fed-batch (35 to 47 g L1, with 50 to 75% lipid in the biomass) cultures. The highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. However, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply. CONCLUSIONS: Lipid production in R. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. Although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. Constant feeding was better than intermittent feeding. The feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of C5 sugars did.