Modeling lipid accumulation in oleaginous fungi in chemostat cultures: I. Development and validation of a chemostat model for Umbelopsis isabellina

Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures of U. isabellina grown on mineral media with glucose and NH₄ ⁺. The model describes the results of chemostat cultures well for D > 0.04 h⁻¹, but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures.     

Modeling growth,lipid accumulation and lipid turnover in submerged batch cultures of <Emphasis Type="Italic">Umbelopsis isabellina</Emphasis>

The production of lipids by oleaginous yeast and fungi becomes more important because these lipids can be used for biodiesel production. To understand the process of lipid production better, we developed a model for growth, lipid production and lipid turnover in submerged batch fermentation. This model describes three subsequent phases: exponential growth when both a C-source and an N-source are available, carbohydrate and lipid production when the N-source is exhausted and turnover of accumulated lipids when the C-source is exhausted. The model was validated with submerged batch cultures of the fungus Umbelopsis isabellina (formerly known as Mortierella isabellina) with two different initial C/N-ratios. Comparison with chemostat cultures with the same strain showed a significant difference in lipid production: in batch cultures, the initial specific lipid production rate was almost four times higher than in chemostat cultures but it decreased exponentially in time, while the maximum specific lipid production rate in chemostat cultures was independent of residence time. This indicates that different mechanisms for lipid production are active in batch and chemostat cultures. The model could also describe data for submerged batch cultures from literature well.     

Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures

Summary Cryptococcus albidus var. Albidus CBS 4517 was grown in nitrogen-limited and in carbon-limited chemostat cultures. The effect of growth rate and limiting nutrient on lipid accumulation and fatty acid composition was investigated.The maximum lipid content in the biomass was, in both cultivation systems, observed at the lowest dilution rate (growth rate) tested. At this dilution rate, D=0.31 h^-1, cells from the nitrogen-limited culture contained 41% (w/w) lipid and cells from the carbon-limited culture 37%. These results indicate the ability of C. albidus, unlike other oleaginous yeasts, to accumulate lipid also in carbon-limited chemostats.The yield of lipid from carbon source was about the same at D=0.031 h^-1 in nitrogen-limited (Y _L/S=0.16 g/g) as in carbon-limited (Y _L/S=0.17 g/g) cultures and decreased with increasing growth rates. In the nitrogen-limited culture, the lipid productivity was about constant at low growth rates (0.031–0.056 h^-1) and a slight decrease was observed at D=0.08 h^-1, while the specific lipid productivity, q _L, increased to 27.5 mg/g per hour. In the carbon-limited culture, however, lipid productivity increased with increasing growth rates and reached its maximum value near _max, whereas q _L was about constant at 20 mg/g per hour.The fatty acid composition was influenced by the specific growth rate in nitrogen-limited as well as in carbon-limited cultures, although the changes were more pronounced during carbonlimitation. A decrease in the degree of unsaturation (/mole) was also observed with increasing lipid content in the cells.     

Production of the macrolide antibiotic tylosin in batch and chemostat cultures

The production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase–idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (q_tylosin) was found to be a function of the growth rate. The maximum value of (q_tylosin) was observed when D = 0.017 hr^?1. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr^?1, the maximum value of q_tylosin was 0.71 mg tylosin/g dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW-hr. This value of q_tylosin was 40% greater than the maximum q_tylosin observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of q_tylosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of q_tylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased the uptake of phosphate decreased the values of q_tylosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of q_tylosin of 1.1 mg/g/hr being obtained at a growth rate of 0.03 hr^?1. Batch fermentations with this medium did not follow a trophophase–idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.     

Mathematical modelling of lipid production by oleaginous yeasts in continuous cultures

A mathematical model was constructed to describe the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production by oleaginous yeasts. To test this model and to determine some relevant model parameters, the oleaginous yeast Apiotrichum curvatum ATCC 20509 was grown in continuous cultures at various C/N-ratios and dilution rates. It appeared that when nitrogen is limiting for the formation of biomass, the remaining glucose can be converted to storage carbohydrate and storage lipid. No clear dependence of carbohydrate yield on the C/N-ratio could be demonstrated, but lipid yield increased gradually with increasing C/N-ratios.The maximal dilution rate for lipid producing yeast cells appeared to be optimal at relatively low C/N-ratios. It can be concluded that the experimental results fitted well with the mathematical model. By using this model, lipid yield and lipid production rate can be calculated at any C/N-ratio of the growth medium and optimum operation conditions can be predicted for the production of microbial lipids.     

Use of chemostat data for modelling extracellular-inulinase production by Kluyveromyces marxianus in a high-cell-density fed-batch process

Production of extracellular inulinase by low-cell-density (2 kg dry weight·m⁻³) sucrose-limited chemostat cultures of Kluyveromyces marxianus obeyed saturated kinetics at dilution rates ranging from 0.02 to 0.5 h⁻¹. A non-structured Monod-type equation, describing the relation between specific growth rate and specific extracellular-inulinase production rate, was used to fit experimental data. THis equation was subsequently incorporated in a model for the production of biomass and extracellular inulinase in a high-cell-density (> 100 kg dry weight·m⁻³) fed-batchculture of K. marxianus grown on sucrose. The model adequately described biomass production in the fed-batch culture. However, the production of extracellular inulinase in the fed-batch process was slightly higher than predicted by the model. This observation may be related to differences in growth conditions between in the chemostat and fed-batch cultures.     

A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms.

The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor. The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependent isocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, the metabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomes arrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase which converts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate. Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.     

Enthalpy content as a function of lipid accumulation in Rhodotorula glutinis

Microcalorimetry has been demonstrated to be a suitable on-line method for monitoring the lipid production phase of oleaginous yeasts. The choice of lipid extraction method for the oil accumulated by oleaginous yeasts is highly important both for accuracy when quantifying the lipid level and determining the fatty acid composition. The energy content of Rhodotorula glutinis increased from 23.0 kJ/g to 30.6 kJ/g dry biomass during the lipid-accumulating phase and was directly correlated to the analysed level of lipids, when an alkaline hydrolysis extraction method was used. Consequently, bomb-calorimetric measurements of the energy content were shown to be an indirect method of quantifying the lipid content in oleaginous yeasts. The fatty acid composition remained rather constant during the batch growth of Rh. glutinis with approximately 70% unsaturated C18 fatty acids. The high energy content as well as the fatty acid composition of Rh. glutinis makes this yeast a better candidate for use as aquaculture feed compared with the commonly used Saccharomyces cerevisiae.     

Isolation of a yeast-lyzing Arthrobacter species and the production of the lytic enzyme complex in batch and continuous-flow fermentors

A gram-negative bacterium strongly lytic toward living cells of the food yeast Saccharomyces fragilis was isolated by continuous-flow enrichment from compost. The organism was identified as a species of Arthrobacter. The extracellular lytic enzyme complex produced by this bacterium contained β-1,3-glucanase, mannan mannohydrolase, and proteolytic activities. The polysaccharases were inducible by whole yeast cells. In chemostat cultures on chemically defined media, synthesis of the polysaccharases was very slight and only detectable at dilution rates below 0.02 hr^?1. Enzyme production in defined media was not solely dependent on growth rate but also was influenced by the growth limiting substrate and the culture history. The production of individual depolymerases and of the lytic activity was studied in batch and chemostat cultures containing yeast as the limiting substrate. The maximum specific growth rate of the Arthrobacter under these conditions was 0.22 hr^?1. β-1,3-Glucanase and proteolytic activities were synthesized by exponentially growing bacteria but maximum lytic titers did not develop until the specific growth rate was declining, at which time mannan mannohydrolase syntheses was induced. In yeast limited chemostats polysaccharase syntheses were greatest at the lowest dilution rates examined, namely 0.02 hr^?1. Further optimization of enzyme production was achieved by feeding the Arthrobacter culture to a second-stage chemostat. A comparison of lytic enzyme productivities in batch and chemostat cultures has been made.     

Cytosolic NADPH balancing in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.     

Glucose uptake of Cytophaga johnsonae studied in batch and chemostat culture

The influence of different physiological states on the glucose uptake and mineralization by Cytophaga johnsonae, a freshwater isolate, was examined in batch and chemostat cultures. At different growth rates under glucose limitation in chemostat cultures, different uptake patterns for ¹⁴C labeled glucose were observed. In batch culture and at high growth rates the glucose uptake potential showed a higher maximum velocity and a much lower substrate affinity than at lower growth rates. These findings and the results of short-term labeling patterns could be explained by two different glucose uptake mechanisms which enable the strain to grow efficiently both at high and low substrate concentrations. Substrate specificity studies showed that a structural change of the C-2 atom of the glucose molecule was tolerated by both systems. The consequences of these results for the ecophysiological classification of the Cytophaga group and for the operation of continuous cultures are discussed.     

Prediction of lipid accumulation-degradation in oleaginous micro-organisms growing on vegetable oils

Oleaginous micro-organisms (yeasts, moulds), in culture media having the carbon source as limited factor, degrade reserve lipids and produce new biomass, after the onset of carbon exhaustion from the medium. In this paper the process of lipid accumulation-degradation in oleaginous micro-organisms, growing on a vegatable oil was simulated. The model was integrated with 4 different methods and the parameters were optimised with the least squares method. It was found that the degradation of endocellular carbon pool is a very slow process characterised, however, by a good yield in fat-free biomass. Low values of the specific growth rates of the fat-free microbial mass, both from consumption of extra cellular and endocellular carbon pools, favourite the production of microbial lipid. The maximum of the specific rate of lipid accumulation is positively affected by the low values of the specific growth rate of the fat-free microbial mass from consumption of extra cellular carbon pool, but remained unaffected by the specific growth rate of the fat-free microbial mass from consumption of endocellular carbon pool. On the other hand, lipid production and specific rate of lipid accumulation are positively influenced by the high values of the specific rate of storage lipid formation. In conclusion, this numerical model can be used in the laboratory as pilot for planing further experimental work.     

The effect of growth conditions on production and excretion of extracellular antigens by three ascomycetous yeasts

Ascomycetous yeasts produce extracellular antigens that are almost specific for the species. The antigen production by Hansenula wickerhamii and Stephanoascus ciferrii was independent of the carbon source and was proportional to the final cell density of the cultures. The same was true of chemostat cultures of Stephanoascus ciferrii, irrespective of the dilution rate and whether glucose or ammonia was the limiting nutrient. In cultures of Saccharomyces cerevisiae, however, antigen excretion mainly took place in the late exponential growth phase. Large amounts of antigen were extracted from the cell wall of Saccharomyces cerevisiae. A small amount was detected in the cytoplasm.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Growth yield, maintenance requirements, and lipid formation in the oleaginous yeast Apiotrichum curvatum

For guiding and improving the efficiency of the production of lipid, complete insight into the flow of carbon and energy during growth and product formation is necessary. Therefore, data have been collected to determine various important growth parameters for the oleaginous yeast Apiotrichum curvatum. Chemostat experiments at specific growth rates, ranging from 0.05 to 0.15 h(-1) and recycling experiments with 100% biomass retention, with growth rates decreasing from 0.10 to 0.004 h(-1), demonstrated that maintenance requirements of A. curvatum are very low, compared to maintenance requirements described for other yeasts as Saccharomyces cerevisiae and Candida parapsilosis.It also appeared that growth and lipid production are proportional to substrate consumption when specific growth rates are higher than approximately 0.02 h(-1), but that lipid production stops at growth rates below this value. The practical consequences of these data are that fed batch cultures, which are often applied in fermentation industry, can only be useful with lipid producing yeasts when the growth rate in the process is carefully monitored to ensure specific growth rates higher than 0.02 h(-1). Dilution of the culture, partial recycling and/or a continuously increasing nutrient feed are solutions for the expected problems at low growth rates.     

Yield and maintenance relations of yeast growth in the chemostat at superoptimal temperatures

A model is proposed that accounts for the decreases in yield which occur in chemostat cultures of mesophilic yeasts at superoptimal growth temperatures. Two yield depressing effects were identified, one due to increased maintenance requirements by the viable fraction of the population, the other due to energy substrate dissipation by the nonviable fraction. The two effects are functions of the dilution rate, as is the fraction of nonviable cells. Experimental results were obtained on the yield, maintenance, and dissipation of energy substrate in a glucose-limited chemostat culture of a respiration-deficient mutant of Saccharomyces cerevisiae at 39°C. The rates of glucose utilization for maintenance and for dissipation constituted, respectively, 33–28% and 15–9% of the total glucose utilization rate over the range of dilution rates tested (0.038–0.064 hr^?1), while the yield varied over this range from 0.066–0.085 g of biomass (dry wt) per gram of glucose.     

Molar growth yields,respiration and cytochrome profiles of Beneckea natriegens when grown under carbon limitation in a chemostat

The effect of growth rate on the physiology of Beneckea natriegens was studied in chemostat culture. The molar growth yields (Y) from glucose and oxygen, the specific rates of oxygen (q _{O 2}) and glucose (q _glc) consumption and the specific rate of CO₂ production (q _{CO 2}) were linearly dependent on the growth rate over the dilution rate 0.17 h^-1 to 0.60 h^-1. Further increase in the dilution rate resulted in a decrease in growth yield and respiration rate and these changes were coincident with increases in the specific rate of glucose utilisation and of acetate production. The affinity of Beneckea natriegens for glucose was similar when measured either directly in chemostat culture or in a closed oxygen electrode system using harvested bacteria. The total content of cytochromes decreased with increasing growth rate. However, the quantity of CO-binding cytochromes remained independent of growth rate and correlated with the potential respiration rate.     

Enhanced production of lactococcin 972 in chemostat cultures

Lactococcus lactis subsp. lactis IPLA972 is a wild lactococcal strain suitable as a single starter in the manufacture of dairy products. This strain synthesizes lactococcin 972 (Lcn972), a unique bacteriocin that blocks septum formation. In this work, we report on the conditions to optimize biomass and Lcn972 production. In batch cultures, pH 6.8 was found to be optimum for bacteriocin synthesis and both glucose and lactose supported Lcn972 production. The inhibitory activity improved up to eight-fold with increasing carbohydrate concentration. In chemostat cultures, steady states were achieved even at dilution rates higher than _max, due to the strong wall growth. Lcn972 behaved as a true primary metabolite, as it was maximally produced when the cells were actively growing. Bacteriocin yields were improved up to ten-fold in chemostat cultures compared with those achieved in batch.