Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

A mathematical model of growth and alkaloid production in the submerged culture of Claviceps purpurea

A mathematical model of the batch cultivation of Claviceps purpurea was formulated. The main attention was devoted to the effect of exocellular and intracellular phosphate on the growth of the mycelium and production of clavine alkaloid under experimental conditions without limitation by carbon and nitrogen sources. The method of nonlinear regression was used ot predict the optional technological regime of the phosphate addition in the batch culture at different time intervals of additions.     

Growth of Escherichia coli in phosphate limited cultures : Measurements and modelling

The batch cultivation ofEscherichia coli limited by inorganic phosphate shows an exponential growth phase followed by a linear phase when the phosphate in the medium is exhausted. Data analysis and modelling of batch cultivation with different initial concentrations of the limiting factor give information about an exchangeable phosphate store, with RNA as the main component, and a non-exchangeable store.     

Membrane Inlet Mass Spectrometry for the on‐line Measurement of Metabolic Fluxes: Case Lysine‐Production by Corynebacterium glutamicum

A miniaturized reactor system with on‐line measurement of respiration rates by membrane inlet mass spectrometry was applied for the on‐line metabolic flux analysis at different phases of a 1.2 L batch culture of lysine‐producing Corynebacterium glutamicum. For this purpose, cells taken from the batch culture were transferred into the 12 mL mini reactor, and incubated for 15 min with [1‐¹⁸O]glucose. Quantification of oxygen uptake rate and CO₂ mass isotopomer production rates in combination with a simple metabolic model allowed the estimation of the flux partitioning ratio between the pentose phosphate pathway and glycolysis during the process. The relative flux into the pentose pathway increased during growth, and reached maxima at 11 and 17 h cultivation time coinciding with maxima of the differential lysine yield. The developed system is a promising tool for determination of metabolic flux dynamics in industrially relevant batch and fed‐batch cultures.     

Chlorella pyrenoidosa 7-11-05 in batch and in homogeneous continuous culture under autotrophic conditions

The growth ofChlorella pyrenoidosa 7-11-05 in batch and in continuous culture using two types of cultivation vessels was compared. The high values of specific growth rate obtained in batch deep stirred tank culture containing small amount of inoculum are not easily attainable in continuous culture with regard to the high number of autospores formed throughout the ontogenetic cycle ofChlorella. The obtained surface and volume productivities prove that in both types of cultivation vessels a high growth intensity can be achieved. The deep tank perfectly mixed cultivation may become of significance in the future.     

Bakers' yeast cultivation on by-products and wastes of potato and wheat starch production on a laboratory and pilot-plant scale

Saccharomyces cerevisiae was cultivated in a 10 litre laboratory stirred tank, an 80 litre laboratory airlift tower loop and a 4 m³ pilot-plant airlift tower loop reactor using by-products and wastes of potato and wheat starch production in batch and continuous cultures. Potato protein liquor, potato liquor retentate, potato liquor residue, and wheat process water were used as nutrients and glucose from the enzymatic conversion of potato starch as energy source. Besides the performance of the cultivation (cell concentrations, specific growth rates in the first (glucose) and second (ethanol) growth phases, productivities, yield coefficients), the qualities of the effluents (concentrations of phosphate, dissolved organic carbon, dissolved organic nitrogen, and chemical oxygen demand) were also determined in the different reactors as functions of the operational parameters. The optimal conditions were evaluated with regard to cultivation performance and effluent quality. These performances do not vary with the scale of the reactors. The performance of continuous cultures is considerably better than that of batch cultures.     

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.     

Growth behavior and protease production by an icelandicthermus sp. isolate in batch and continuous culture

AThermus strain, producing an extracellular protease, was isolated in a hot spring in Iceland. The main growth characteristics of this isolate were studied with different cultivation vessels and different cultivation techniques. A clear and striking dependence of the growth behavior on the cultivation technique was apparent. Higher maximum yield of biomass, higher productivity of biomass, and higher maximum growth rate were found in continuous cultivations compared with batch cultivations. The substrate utilization and the yield of biomass of this strain were much higher than reported for several otherThermus strains. Reproducibility of kinetic data seemed not to depend on the type of cultivation vessel, on the basis of the types of vessels tested, and instability of the population was not observed during cultivations. Production of extracellular protease in our cultivations was apparently growth associated in batch culture, and the specific rate of production in continuous culture was dependent of the dilution rate, implying that certain kinds of regulatory mechanism(s) might be involved.     

Requirements of Pseudomonas aeruginosaDissociants in Glucose, Nitrate, and Phosphate. Limiting Concentrations of the Nutrients during Batch Cultivation

Requirements of R-, S-, and M-dissociants of Pseudomonas aeruginosaK-2 strain in glucose, nitrate, and phosphate, as well as the error of determination, have been calculated from the data of 250 experiments on their batch cultivation on the media with the various initial content of these resources. The resource requirements of R- and S-dissociants appear indistinguishable within the error limits. The requirements of M-dissociant significantly differ from those of R- and S-forms; however, due to specific growth of the M-form, the confidence intervals of the measured values can overlap with those of R-space and S-forms. The obtained requirement values allow an evaluation of the studied resources concentration limiting the culture growth.     

Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.     

Continuous culture with complete cell recycle to obtain high cell densities in product inhibited cultures; cultivation ofStreptococcus lactis for production of superoxide dismutase

Summary A cultivation system is described for cultivatingStreptococcus lactis in continuous culture with complete cell recycle. The aim was to obtain high cell densities for the production of su-peroxide dismutase (SOD) while avoiding the growth inhibiting effects of the lactic acid produced. This type of cultivation was performed both at constant and at increasing dilution rates. Comparisons made include those between cell mass productivity and SOD productivity in recycling cultivations and batch cultivations. In the recycling cultivation at increasing dilution rates a cell mass of 19 g/1 was obtained after 22 h of cultivation and the SOD productivity was 43.10³ U/1.h which is four times higher than for batch cultivations. The effect of recyclingS. lactis was also considered and no damage of the microorganisms was observed.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

Polymyxin biosynthesis by Bacillus polymyxa during growth limitation by the nutritional sources

The synthesis of the antibiotic polymyxin M was studied under the conditions of batch and continuous cultivation of Bacillus polymyxa var. Ross whose growth was limited with glucose, phosphate and ammonium nitrogen. Polymyxin M was synthesized when the culture growth decelerated as a result of its limitation with the above compounds. Different amounts of the antibiotic were synthesized depending on the type of a limiting factor. The highest productiveness was found in the case of glucose limitation. The optimal conditions for polymyxin M synthesis were established under the conditions of one-step continuous cultivation.     

Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.     

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

Enhanced chitosan production and modeling hyphal growth of Mucor rouxii interpreting the dependence of chitosan yields on processing and cultivation time

Chitosan is a major structural component of fungal cell walls and has diverse medical and other applications. However, cost‐effective culture and extraction methods for fungi need to be developed. Therefore, Mucor rouxii was grown on YPG‐media in both submerged batch and semi‐continuous cultures. Chitosan was extracted from the mycelia to explore strategies to enhance yields and production rates. As observed in earlier studies, M. rouxii is able to adapt to shear stress when cultured semi‐continuously. Modeling the hyphal growth of batch experiments shows that the mycelia were ruptured by shear forces within a short cultivation time shown by a decreased hyphal length. However, an increasing chitosan content was observed with an increasing cultivation period in semi‐continuous cultures, which is an indication for the adaption to shear stress. Semi‐continuous culture resulted in the highest contents of extractable chitosan. The results and models of hyphal growth, including tip extension and branching, suggest that repeated batch cultures may be optimal for chitosan production.     

PHOSPHATE AND SILICATE GROWTH AND UPTAKE KINETICS OF THE DIATOMS ASTERIONELLA FORMOSA AND CYCLOTELLA MENEGHINIANA IN BATCH AND SEMICONTINUOUS CULTURE1

Information on the nutrient kinetics of Asterionella formosa Hass. and Cyclotella meneghiniana Kutz. under either phosphate or silicate limitation was obtained for use in a Monod model and in a variable internal stores model of growth. Short-term batch culture growth experiments were fit to the Monod model and long-term semicontinuous culture experiments and short-term uptake experiments were fit to the variable internal stores model. Mathematical analysis indicates that the parameters of the 2 models may be expressed in terms of each other at steady state. The qualitative results of both batch and steady state culture methods agree. For limiting phosphate experiments. A. formosa is better able to grow at low PO₄-P concentrations than C. meneghiniana, as shown by its lower K for PO₄-P limited growth. The k_Q of A. formosa compared to C. meneghiniana found in long-term semicontinuous culture indicates that A. formosa is almost an order of magnitude more efficient at using internal phosphate for growth. The qualitative results under silicate-limited growth of C. meneghiniana is less than that of A. formosa. The k_Q from semicontinuous culture experiments indicates that C. meneghiniana is the more efficient at using internal silicate for growth. Nutrient uptake experiments showed more variability from a Michaelis-Menten relationship than short-term growth experiments. There were no significant differences between the 2 species in half saturation constants for either phosphate or silicate uptake. We observed a marked dependence of the coefficient of luxury consumption (R) of phosphate on the steady state growth rate. A. formosa has a higher R than C. meneghiniana.     

Maximum glucoamylase production by a temperature-sensitive mutant of Saccharomyces cerevisiae in batch culture

In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (P_i) transporter of S. cerevisiae and is derepressed by P_i starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80 ^ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed, but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching time, t_s. Received 18 September 1997/ Accepted in revised form 07 January 1998