Microcalorimetric investigations of the metabolism of yeasts. Growth in chemostat cultures on glucose

Summary As part of a project on the production of penicillin, the penicillin production of two strains of Penicillium chrysogenum which have a different penicillin productivity was investigated in bubble column bioreactors and for comparison in stirred fermenters. The main interest of this study were the complicated interrelations between the stirrer speed, the stirrer type, the shear stress, the morphology of the mycelium and broth viscosity as well as the effect of the oxygen transfer behavior on antibiotic productivity.Stirred tank reactors with different turbine stirrers as well as with a draught tube and propeller were employed.The main variable investigated was the stirrer speed. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply of pulpy mycelia is improved and more cell mass is formed. This result is the same for both strains in all three reactors.If the oxygen partial pressure is near the lower cirtical value, a high percentage of the carbon source is converted into penicillin but the penicillin productivity is low due to a low percentage of penicillin producing cells. At oxygen partial pressures just above 8% saturation, the absolute penicillin productivity is maximal. At higher stirrer speeds and dissolved oxygen concentrations the penicillin production phase is shorter, cell growth is higher and a higher percentage of the carbon source is converted into CO₂.In reactors with a draught tube and propeller, a lower productivity is attained than in those with turbine stirrers.The behavior of the two strains is fairly similar. The higher producing strain, however, has a more distinct separation between its periods of growth and production than does the low producing one. At high stirrer speeds the increase in the cell growth rate is less significant and the substrate yield coefficients are higher for the high producing strain than for the low producing one.Symbols C Dissolved oxygen concentration (mg l^–1) - C^* C at saturation (mg l^–1) - k_La Volumetric mass transfer coefficient (h^–1) - OTR Oxygen transfer rate (mg l^–1 h^–1) - OUR Oxygen uptake rate (mg l^–1 h^–1) - rpm Impeller speed (min^–1) - X (Dry) biomass concentration (g kg^–1) - V_g Volumetric gas flow rate (Nl min^–1) - CMC Carboxymethyl cellulose     

Decolourization of anaerobically digested and polyaluminium chloride treated distillery spentwash in a fungal stirred tank aerobic reactor

Decolourization of anaerobically digested and polyaluminium chloride treated distillery spentwash was studied in a fungal stirred tank aerobic reactor without dilution of wastewater. Aspergillus niger isolate IITB-V8 was used as the fungal inoculum. The main objectives of the study were to optimize the stirrer speed for achieving maximum decolourization and to determine the kinetic parameters. A mathematical model was developed to describe the batch culture kinetics. Volumetric oxygen transfer coefficient (k _L a) was obtained using dynamic method. The maximum specific growth rate and growth yield of fungus were determined using Logistic equation and using Luedeking–Piret equation. 150 rpm was found to be optimum stirrer speed for overall decolourization of 87%. At the optimum stirrer speed, volumetric oxygen transfer coefficient (k _L a) was 0.4957 min⁻¹ and the maximum specific growth rate of fungus was 0.224 h⁻¹. The values of yield coefficient (Y _x/s) and maintenance coefficient (m _s) were found to be 0.48 g cells (g substrate)⁻¹ and 0.015 g substrate (g cells)⁻¹ h⁻¹.     

The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum

The influence of mechanical forces resulting from the rotation of (multiple) turbine impellers on the morphology and penicillin production of Penicillium chrysogenum Panlabs P-1 was investigated in batch fermentations using semi-defined media. Experiments were carried out at three different scales of fermentation, 5 dm³,100 dm³ and 1000 dm³ working volume, with the impeller tip speed ranging from 2.5 to 6.3 m/s. Throughout all fermentations, the dissolved oxygen concentration never fell below the critical value for maximum penicillin production. Morphological measurements using image analysis showed that the mean main hyphal length and mean hyphal growth unit increased during the rapid growth period and then decreased to a relatively constant value dependent on the agitation intensity. The specific rate of penicillin production (q _pen)and the average main hyphal length during the linear penicillin production phase were lower at high agitation speed, which promoted more rapid mycelial fragmentation and a higher branching frequency. Comparison of the results from the three scales showed that impeller tip speed is a poor scale up parameter whereas a term based on mycelial circulation through the zone of high energy dissipation fitted the data well.List of Symbols C.E.R. mmol/(dm³h) Carbon dioxide evolution rate - D m Impeller diameter - D.O.T. % air saturation Dissolved oxygen tension - L _e m Mean effective length or main hyphal length - O.U.R. mmol/(dm³h) Oxygen uptake rate - P W Total power dissipation - q _pen units/(mg dry cell weight h) rate Specific penicillin production - R.Q. Respiratory quotient - 1/t _cs^–1 Circulation frequency     

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

Summary Limitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (q_pen). q_pen values were measured at different constant DOT levels. Below 30% air saturation q_pen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.     

Radioactive penicillin G production by immobilized fungal vesicles

Summary Radioactive penicillin G production from l-[1-¹⁴C]-valine (1.75 GBq · mmol^-1) by native and by calcium alginate gel immobilized mycelium of Penicillium chrysogenum PQ-96 in a medium for antibiotic production as well as by vesicles isolated from the protoplasts of the same strain in a well-defined reaction mixture was investigated. Specific radioactivity of the penicillin G produced by the native vesicles was 1.45 GBq · mmol^-1 and that of the antibiotic synthesized by the calcium alginate gel immobilized vesicles was 1.48 GBq · mmol^-1. By comparison, the specific radioactivity of penicillin G produced by native mycelium was 0.42 GBq · mmol^-1 and of that synthesized by the immobilized mycelium was 0.96 GBq · mmol^-1. Production of radioactive penicillin G by native and immobilized vesicles in repeated use was also investigated. At the beginning of the production phase, the radioactive penicillin G synthesized by the immobilized vesicles was 25 nmol · mg protein^-1 · h^-1 and decreased after 8 days to a level of 11 nmol · mg protein^-1 · h^-1. The half-life of the immobilized vesicles was 7 days. The native vesicles showed a rapid decrease in radioactive antibiotic production. In comparison, the penicillin G production in a repeated use of immobilized vesicles decreased during 40 days from 140 nmol · mg protein^-1 · h^-1 to 60 nmol · mg protein^-1 · h^-1. The half-life of the immobilized vesicles was 35 days. The native vesicles showed after 4 days a lack of activity of penicillin G production. The stability of immobilized mycelium or vesicles in the process of radioactive penicillin G production is discussed.     

Performance test of a 6-stage continuous reactor for palm methyl ester production

Effects of residence time (3-12 min), stirrer speed (0-800 rpm), and NaOH concentration (0.25-1.0 wt% of oil) on the production performance of the designed 6-stage continuous reactor (2.272 l) for transesterification of palm oil were investigated at molar ratio of methanol to oil of 6:1 and temperature of 60 degrees C. Higher stirrer speed increased the reaction rate up to an appropriate speed but excessive stirrer speed decreased the reaction rate. Inappropriate stirrer speed runs dramatically decreased the production capacity of the reactor. Higher NaOH concentration significantly increased reaction rate and production capacity of the reactor. The reactor had a residence time distribution equivalent to 5.98 ideal CSTRs in series and a production performance equivalent to a plug flow reactor. At NaOH of 1.0 wt% of oil, the reactor could produce saleable biodiesel within residence time of 6 min in which a production capacity was 17.3 l/h and a power consumption of stirrer was 0.6 kW/m(3).     

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     

Penicillin recovery from aqueous solutions by continuous foam flotation

Summary Penicillin G recovery is investigated in a continuous flotation column in the presence of different collectors which form a complex with penicillin. The performance of the penicillin recovery was investigated as a function of the mole ratio () of collector-to-penicillin and the aliphatic chain length of the collector. At =1 and low penicillin concentrations (e.g., 20 mg·1^-1), high foam liquid concentrations (680 mg·l^-1), low residue concentrations (12 mg·l^-1) and high penicillin separation (56) can be attained. At =4 the separation increases to 150, and 95% of the penicillin can be recovered.Symbols C_p penicillin concentration in feed (mg·l^-1) - C_R penicillin concentration in outlet liquid (mg·l^-1) - C_S penicillin concentration in foam liquid (mg·l^-1) - C_S/C_P penicillin enrichment (-) - C_S/C_R penicillin separation (-) - % Pen in S penicillin yield in foam liquid (%) - VV}_S foam liquid volume flow (ml·min^-1) - VV}_P feed (ml·min^-1) - VV_{N 2} nitrogen flow rate (ml·s^-1) - temperature     

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

In large-scale bioreactors, there is often insufficient mixing and as a consequence, cells experience uneven substrate and oxygen levels that influence product formation. In this study, the influence of dissolved oxygen (DO) gradients on the primary and secondary metabolism of a high producing industrial strain of Penicillium chrysogenum was investigated. Within a wide range of DO concentrations, obtained under chemostat conditions, we observed different responses from P. chrysogenum: (i) no influence on growth or penicillin production (>0.025 mmol L⁻¹); (ii) reduced penicillin production, but no growth limitation (0.013–0.025 mmol L⁻¹); and (iii) growth and penicillin production limitations (<0.013 mmol L⁻¹). In addition, scale down experiments were performed by oscillating the DO concentration in the bioreactor. We found that during DO oscillation, the penicillin production rate decreased below the value observed when a constant DO equal to the average oscillating DO value was used. To understand and predict the influence of oxygen levels on primary metabolism and penicillin production, we developed a black box model that was linked to a detailed kinetic model of the penicillin pathway. The model simulations represented the experimental data during the step experiments; however, during the oscillation experiments the predictions deviated, indicating the involvement of the central metabolism in penicillin production.     

Effect of shear on morphology and erythromycin production in Saccharopolyspora erythraea fermentations

A complex medium was used to investigate the effects of shear on the S. erythraea fermentation at 7-l scale. Maximum biomass was 11.1 - 0.5 g l^у at 1250 rpm (tip speed = 4.45 ms^у), whereas it was 12.7 - 0.2 g l^у at 350 rpm (tip speed = 1.07 ms^у). Specific erythromycin production was not stirrer speed dependent in the range of 350 to 1000 rpm and decreased by 10% at stirrer speed of 1250 rpm. Morphological measurements using image analysis showed that the major axis of the mycelia (both freely dispersed and clumps) decreased after the end of the rapid growth phase to a relatively constant value (equilibrium size) dependent on the stirrer speed. The mechanical properties of the cell wall were examined by disruption of fermentation broth in homogeniser and it was shown that mechanical strength of the cell wall increased in a large extent during deceleration phase.     

Effect of shear rate and culture pH on the production of xylanase by Thermomyces lanuginosus

Summary The pH-value and the stirrer speed during cultivation of the thermophilic fungus Thermomyces lanuginosus were found to have a pronounced influence on xylanase production using corn cobs as carbon source. The highest xylanase activity of 32500 nkat/ml was produced in labscale fermentation within 118 hours at a stirrer speed of 50 rpm and a controlled pH-value of 7.5.     

A model for penicillin production with and without temperature shift after the growth phase

Summary A strain of Penicillium chrysogenum producting about 8 g/l of penicillin V, was cultivated in a 10-1 bioreactor. Under carbon (C)-limitation during the production phase a glucose/ammonium sulphate mixture was fed using microprocessor control. When the temperature was shifted from 25° C to 30° C at the end of the active growth phase, the specific penicillin production rate was increased by 30%, while the yield remained constant. Maximal productivity without sporulation was obtained when the net growth rate of the active (respiring and producing) biomass, estimated by measuring the respiration rate under defined conditions, was equal to or higher than 0.004 h^–1. A model was developed for penicillin fermentation during C-limitation possessing the following properties: (1) the model is based on ordinary differential equations; (2) the influence of different nutrients is considered; (3) the model recognizes two cell types (active and inactive); (4) the model describes the influence of a temperature shift at the end of the vigorous growth phase. Offprint requests to: D. Siegmund     

Influence of pH and impeller tip speed on the cultivation of<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4 in a milk-based medium

Biomass production ofBifidobacterium pseudocatenulatum G4 in a milk-based medium was carried out in a 2- and 10-L stirred tank fermenters. The effects of impeller tip speed (0.28, 0.56, and 0.83 m/s) and pH control (6.0, 6.5, and 7.0) on the biomass production were investigated. The growth performance in the 2-L fermenter was significantly improved when the impeller tip speed was held constant at 0.56 m/s and the pH was controlled at 6.5. These conditions yielded a maximum biomass of 1.687×10⁹ cfu/mL, a maximum specific growth rate of 0.504 h⁻¹, a biomass productivity of 9.240×10⁷ cfu/mL·h, and a biomass yield of 9.791×10¹⁰ cfu/g lactose. The consumption of milk lactose resulted in the accumulation of 7.353 g/L acetic acid and 6.515 g/L lactic acid, with an acetic:lactic ratio of 1.129. Scale-up of the fermentation process to a 10-L fermenter based on a constant impeller tip speed of 0.56 m/s yielded reproducible results with respect to biomass production and cell viability.     

Identifying the conditions for development of beneficial mycelium morphology for chitosan-producing Absidia spp. in submersed cultures

Summary The first step when producing chitosan from fungi grown submersed, i.e. the cultivation technique of some chitosan-producing Abisidia spp/, was studied. Various strains have their own specific requirements for development of morphology beneficial for a high growth rate. Regarding the species studied (A. coerulea, CBS 100.38; A. fusca, CBS 102.35; A. glauca, Dep. Microbial Ecology, Lund; A. repens; CBS 102.32), these demands are primarily those concerning agitation. Increasing the number of spores (from 1.4·10⁷ to 5.8·10⁷ per liter) of A. coerulea increased the initial growth rate, which on the other hand (due to agglomeration of pellets) levelled off at a comparatively earlier stage. Too low stirrer speed generally caused the formation of a pulp with a strong decline in growth rate. Elevated stirrer speeds caused the formation of a more compact morphology, often in the form of small pellets. The use of an open paddle impeller with a large diameter (with satisfactory pumping and mixing characteristics) ran at a sufficiently high stirrer speed to avoid the formation of a viscous pulp allowed most of the species to grow satisfactorily. Continuous cultivations were hard to establish due to lack of new growth spots and accreted growth.     

Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

Scaling-up of a batch whey fermentation by Kluyveromyces fragilis

Summary A whey fermentation by Kluyveromyces fragilis was scaled-up to a 1000-dm³ stirred fermentor, by varying the stirrer speed, the air-flow rate and the initial concentration of lactose. Its evolution was simulated by applying the same unstructured model (consisting of a microbial specific growth rate of pseudo-first order with respect to the COD concentration and constant biomass yield per unit COD removed) set up in previous experiments using 8- to 80-dm³ fermentors. Despite the great scale-up ratios, very different operating conditions, and geometric dissimilarity, a series of empirical regressions previously developed allowed approximate, but acceptable prediction of the stoichiometric and kinetic coefficients of the above mathematical model, thus confirming the capability of this model to provide a reliable basis for further scale-up of this fermentation process to a production scale.     

Effective production of biologically active water-soluble β-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10?g?L^?1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0?g?L^?1 hr^?1 and 600?rpm, respectively, and at the w-glucan production stage, they were 0.5?g?L^?1 hr^?1 and 400?rpm, respectively. W-glucan production reached 17.31?g?L^?1, with a degree of polymerization of 19–25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.     

Isolation and characterization of a new strain of Achromobacter sp. with beta-lactam antibiotic acylase activity

A bacterial strain producing a -lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized. The isolate was identified as Achromobacter sp. using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence. The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l^–1. PAA at concentrations up to 12 g l^–1 had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate. Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme. The inducers were metabolized in all cases. Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G. A high stability of acylase activity was observed over a wide range of pH (5.0–8.5) and at temperatures above 55°C.     

Plant cell suspension culture in a bench-scale fermenter with a newly designed membrane stirrer for bubble-free aeration

In this paper, tests of an optimized membrane-stirrer geometry for bubble-free aeration of a plant cell suspension culture are described. Cell attachment and clogging of a previously described system [Piehl et al. (1988) Appl Microbiol Biotechnol 29:456–461] led to the development of a new stirrer. The volumetric oxygen transfer capacity has been measured in aqueous medium. The mass transfer coefficient, k _l a, was 3.75 h⁻¹ at 25 °C and at a stirrer speed of 34 rpm. The overall oxygen transfer capacity was investigated with a suspension culture of Aesculus hippocastanum. It was shown that the oxygen mass transfer was sufficient even at the maximum biomass of 10–12 g dry weight/l, which was obtained by using this system. Furthermore, special attention was given to medium components like C and N sources, to avoid growth limitation due to a shortage of nutrients. Received: 22 October 1996 / Revised version: 11 March 1997 / Accepted: 14 March 1997     

A modified unstructured mathemathical model for the penicillin G fed-batch fermentation

Summary In this paper, an updated unstructured mathematical model for the penicillin G fed-batch fermentation is proposed, in order to correct some physical and biochemical shortcomings in the model of Heijnen et al. (1979,Biotechnol. Bioeng.,21, 2175–2201) and the model of Bajpai and Reuß (1980,J. Chem. Tech. Biotechnol.,30, 332–344). Its main features are the consistency for all values of the variables, and the ability to adequately describe different metabolic conditions of the mould. The model presented here can be considered as the translation of the latest advances in the biochemical knowledge of the penicillin biosynthesis.Nomenclature t time (h) - S amount of substrate in broth (g) - X amount of cell mass in broth (g) - P amount of product in broth (g) - V fermentor volume (L) - F input substrate feed rate (L/hr) - C _s S/V substrate concentration in broth (g/L) - C _x X/V cell mass concentration in broth (g/L) - C _P P/V product concentration in broth (g/L) - s _F substrate concentration in feed stream (g/L) - E _m parameter related to the endogenous fraction of maintenance (g/L) - E _p parameter related to the endogenous fraction of production (g/L) - K _x Contois saturation constant for substrate limitation of biomass production (g/g DM) - K _s Monod saturation constant for substrate limitation of biomss production (g/L) - K _p saturation constant for substrate limitation of product formation (g/L) - K _i substrate inhibition constant for product formation (g/L) - m _s maintenance constant (g/g DM hr) - k _h penicillin hydrolysis or degradation constant (hr^–1) - Y _x/s cell mass on substrate yield (g DM/g) - Y _p/s product on substrate yield (g/g) - specific substrate consumption rate (g/g DM hr) - specific growth rate (hr^–1) - _substr specific substrate to biomass conversion rate (hr^–1) - _x maximum specific substrate to biomass conversion rate (hr^–1) - specific production rate (g/g DM hr) - _p specific production constant (g/g DM hr)