Lactic acid productivity of a continuous culture of Lactobacillus delbreuckii

Summary Maximum volumetric productivities of biomass (1.40 gl^–1h^–1) and lactic acid (8.93 gl^–1h^–1) for a continuous culture ofLactobacillus delbreuckii occurred between dilution rates 0.35h^–1 and 0.40h^–1. All major nutrients were in excess in these cultures. Glucose utilisation was complete at dilution rates of 0.1h^–1 and lower. Product and biomass yields were constant in the dilution rate range studied (0.05h^–1 to 0.50h^–1).     

Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis

Lactose of sweet whey permeate was converted into sodium lactate byLactobacillus helveticus. To increase the, productivity of the lactic acid fermentation and to reduce the amounts of effluents, the bioreactor was coupled with an ultrafiltration module and an electrodialysis unit. Without the electrodialyzer, with total cell recycling and at a dilution rate of 0.88 h^–1, a cellular concentration of 64 gl^–1 and a productivity of 22 gl^–1 h^–1 were obtained. When the electrodialysis unit is coupled, the outlet concentration of lactate was stabilized at 85±5 gl^–1.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

n-3 Polyunsaturated fatty acid productivity of the marine microalgaIsochrysis galbana. Growth conditions and phenotypic selection

Two set of isolates were obtained in an isolation/selection programme to select eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) rich strains ofIsochrysis galbana. EPA content was improved up to an average of 5.3% (d.wt) for the second set of isolates. On the other hand, with the aeration rate, pH and irradiance kept at low levels, the growth rate was slow and EPA synthesis was enhanced, but productivity increased when growth rates were maximum. A model relating steady-state dilution rates in chemostat cultures ofI. galbana to internal average irradiance is proposed. The greatest productivities were obtained between 0.0295 h^–1 and 0.0355 h^–1 with 300 mg m^–3 h^–1 for EPA and 130 mg m^–3 h^–1 for DHA.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

Microbial production of propionic acid and vitamin B12 using molasses or sugar

With a cell concentration of 125 g dry biomass 1^–1 and a dilution rate of 0.1 h^–1,Propionibacterium acidipropionici produces 30 g propionic acid 1^–1 from sugar with a productivity of 3 g 1^–1 h^–1. The yield of propionic acid is approx. 0.36–0.45 g propionic acid g^–1 sucrose and is independent of the dilution rate and cell concentration. Acetic acid is an unwanted by-product in the production of propionic acid. The concentration of acetic acid only increases slightly when the cell concentration is increased. A two-stage fermentation process was developed for the conversion of sugar or molasses of various types to propionic acid and vitamin B₁₂. By fermentation of blackstrap molasses (from sugar beet and sugar cane) in the first fermentation stage 17.7 g propionic acid 1^–1 with a yield of 0.5 g propionic acid g^–1 carbohydrate was produced with a dilution rate of 0.25 h^–1. In the second stage 49 mg vitamin B₁₂ 1^–1 was produced at a dilution rate of 0.03 h^–1.     

A heuristic approach to fed-batch optimisation of streptokinase fermentation

Previous studies have shown that the rate of formation of streptokinase, a secondary metabolite, in batch fermentation is proportional to the specific growth rate of the biomass, which in turn is inhibited by its substrate and the primary product (lactic acid). These kinetics suggest the suitability of fed-batch operation to increase the yield of streptokinase. A near-optimal feed policy has been calculated by the chemotaxis algorithm, and it shows a substrate feed rate decreasing nonlinearly and vanishing after 11 hours. This is followed by batch fermentation for a further 8 hours, at the end of which 12% more streptokinase is generated than by purely batch fermentation. Further improvements in productivity are possible.List of Symbols k _dh^–1 decay constant for active cells - k _ph^–1 decay constant for streptokinase - K _Igl^–1 inhibition constant for lactic acid - K_S gl^–1 inhibition constant for substrate - M gl^–1 lactic acid concentration - P gl^–1 streptokinase concentration - Q 1h^–1 substrate feed rate - S gl^–1 substrate concentration - S _ingl^–1 inlet concentration of substrate - t h time - t _bh end-point of batch fermentation - t _fh end-point of fed-batch fermentation - V l volume of broth in fermenter - V ₀ l initial value of V (at t=0) - V _ml maximum value of V - X gl^–1 total biomass concentration - X _agl^–1 concentration of active biomass - Y _MX yield coefficient for lactic acid from biomass - Y _PX yield coefficient for streptokinase from biomass - Y _XS yield coefficient for biomass from substrate Greek Letters h^–1 specific growth rate of biomass - _mh^–1 maximum specific growth rate     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

Production of thermostable xylanases in batch and continuous culture by Thermomonospora fusca KW 3

Summary The actinomycete Thermomonospora fusca KW 3 produced novel thermostable xylanases in batch and continuous cultures in media containing insoluble xylan. The production of xylanases could be induced with oat spelt or beech xylan. Very low activities were detected when the strain was grown on glucose or xylose. In continuous cultivations, mycelial wall growth could be prevented using a stirrer speed controller. Homogeneous mixing of the insoluble substrate was obtained by vibrating the flexible tubes. T. fusca KW 3 could be grown on insoluble xylan at growth rates as high as 0.23 h^–1, equivalent to a doubling time of 3 h. Xylanase activity decreased from maximum levels of 2.5 units (U) ml^–1 with increasing dilution rate and was nearly constant at a level of 0.5 U ml^–1 with dilution rates greater than 0.1 h^–1. Correspondence to: P. Röthlisberger     

Xylitol production from barley bran hydrolysates by continuous fermentation with Debaryomyces hansenii

The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l^–1 h^–1 were obtained for dilution rates of 0.008–0.088 h^–1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l^–1 h^–1) was reached at a dilution rate of 0.284 h^–1.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Growth kinetics of Thermus thermophilus

Summary The nonsporulating extreme thermophile Thermus thermophilus was grown in continuous culture at dilution rates up to 2.65 h^–1 at 75°C and pH 6.9 on complex medium. Concomitantly very low yield (Y=0.12 g cell dry weight g^–1 utilized organic carbon) and incomplete substrate utilization (always less than 45%) were found. In batch cultures T. thermophilus could be grown with _max =h^–1, in shake flasks only with _max =h^–1 with the same low yield and incomplete substrate utilization. Stable steady states at 84C and 45°C were realized at a dilution rate of 0.3 h^–1 whereas at 86°C and 40°C no growth could be detected. Artefacts arising from wall growth (in bioreactors) or improper materials must be ruled out. Inhibition of growth by organic substrates was demonstrated at low concentrations: a decrease in the yield obtained was found when more than 0.7 gl^–1 of meat extract were supplied in the medium. The maintenance requirement for oxygen is potentially very high and was determined to be 10 to 15 mmol g^–1 h^–1.     

Extractive fermentation of ethanol using membrane distillation

Summary During the anaerobic batch cultivation ofKluyveromyces fragilis on a complex medium containing glucose (100 gl^–1), ethanol was produced at levels resulting in the inhibition of growth and product (ethanol) formation. Continuous extraction of the ethanol using transmembrane distillation resulted in a 87% increase in ethanol productivity from 0.99 to 1.85 gl^–1h^–1.     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

Anaerobic degradation of p-cresol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of p-cresol under denitrifying conditions by a bacterial consortium was studied in batch and continuous cultures. Concentrations up to 3 mm were degraded within 5–6 days with 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde and 4-hydroxybenzoate as intermediates. Steady states could be maintained at only one dilution rate, D=0.04 h^–1. A further increase in the dilution rate to 0.0 8 h^–1 resulted in culture wash-out. An estimation of the Saturation constant was made (<1 mg/l), taking the maximum specific growth rate as 0.045 h^–1, thus yielding a value of 0.125 mg p-cresol/l. Correspondence to: N. Khoury     

n-3 PUFA productivity in chemostat cultures of microalgae

Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid productivities from chemostat cultures of an isolate of Isochrysis galbana have been studied. The productivities reached in the interval of dilution rates between 0.0295 h^–1 and 0.0355 h^–1 were 1.5mg·1^–1·h^–1 for lipids, 300 g·1^–1·h^–1 for EPA and 130g1·1^–1·h^–1 for DHA. Furthermore, light attenuation by mutual shading, and agitation speed influences on growth and fatty acid composition were analysed. A model relating steady-state dilution rates to internal average light intensity has been proposed, the parameter values of which obtained by non-linear regression were: maximum specific growth rate (_max)=0.0426 h^–1; the affinity of cells to light (I_k) = 10.92 W·m^–2; the exponent (n) = 5.13; regression coefficient (r ²)=0.9999. Correspondence to: E. Molina Grima     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.