Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Design of a tubular loop bioreactor for scale-up and scale-down of fermentation processes

Microorganisms traveling through circulation loops in large-scale bioreactors experience variations in their environment such as dissolved oxygen concentration and pH gradients. The same changes are not experienced in small bioreactors, and it is suggested that herein lies one of the major reasons for the problems encountered when translating fermentation data from one scale to another. One approach to study this problem is to look at the circulation loop itself. The present work concerns an attempt to simulate the circulation loops inside stirred tank reactors, using a tubular loop reactor specially constructed for the purpose. The reactor carries a number of ports and probes along its length for the determination of concentration gradients within. The broth is circulated around the loop by the use of peristaltic pumps, and the circulation time (t(c), s) is used as a measure of simulated reactor size. The reactor system has been evaluated using the citric acid fermentation by Aspergillus niger as a test process. Acid production and fungal morphology, in terms of the mean convex perimeter of mycelial clumps quantified by image analysis, were used as the parameters of evaluation for the two systems in comparison. From comparative experiments carried out in 10 and 200 L stirred tank bioreactors, it appears that the loop reactor simulates the corresponding stirred tank representing a valuable tool in scaling up and scaling down of fermentation process.     

Optimization of agitation for production of swainsonine from Metarhizium anisopliae in stirred tank and airlift reactors

The optimal agitation rate for production of swainsonine from Metarhizium anisopliae grown in batch stirred tank reactors (2 to 20 l) was 400 rpm with a mixed hyphal and pelleted morphology where the specific swainsonine production rate was 9×10^–2 mg g^–1 cell dry wt h from 87 to 142 h. Culture of the fungus in a 6-l airlift reactor produced loose pellets and the production of swainsonine started at least 24 h earlier than in the stirred tank reactor. The final yield (5.9 mg swainsonine g^–1 cell dry wt) after 168 h in the airlift reactor was 18% less than those obtained in the stirred tank reactor with an agitation rate of 400 rpm.     

Optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger NGd-280

The present investigation is concerned with the optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger NGd-280 in a 15 l stirred tank bioreactor. Nutrients, especially nitrogen source have a marked influence on citrate productivity because it is an essential constituent of basal cell proteins. Citric acid has been known to be produced when the nitrogen source was the limiting factor. Ammonium nitrate was employed as a nitrogen source in the present study and batch culture experiments were carried out under various concentrations of ammonium nitrate. Specific growth rate was decreased and the biosynthesis of citric acid was delayed at higher concentrations of ammonium nitrate. Specific citric acid production rate was the highest when intracellular ammonium ion concentration was between 2.0 and 3.0 mmol g(-1) cells. Citrate production was however, stopped when intracellular ammonium ion concentration decreased below 1.0 mmol g(-1) cell.     

Pilot-scale citric acid production with Aspergillus niger under several conditions

Citric acid fermentations with Aspergillus niger in stirred tank reactors (500 l and 2.6 m³) were carried out using cane juice, cane molasses and a synthetic medium. Highest yields were obtained with the cane juice and synthetic medium. The rate of stirring had a pronounced influence on the morphology of A. niger.     

Scale‐up of citric acid fermentation by redox potential control

To obtain high citric acid productivity in Aspergillus niger fermentation on beet molasses substrate, a certain redox potential profile with two maxima (260 and 280 mV) and two minima (180 and 80 mV) must be maintained. The most effective regulation of redox potential is by regulation of aeration and agitation. It has been shown that control of redox potential by aeration and agitation is a most successful method for scale‐up from 10‐L laboratory scale to the 100‐ and 1000‐L pilot‐plant scale, even in geometrically dissimilar stirred‐tank reactors. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 552–557, 1999.     

Effect of pH on exocellular riboflavin production byEremothecium ashbyii

Summary The production of riboflavin byEremothecium ashbyii grown in a chemically defined medium in batch culture was affected by pH of the medium. Highest yields were obtained at constant pH of 4.5 and 5.5, while little or no riboflavin was detected at either pH 3.5 or 8.5. The medium pH also affected cell morphology. When the organism was grown in a stirred tank reactor and an airlift vessel at pH 4.5 very similar levels of riboflavin were obtained.     

Production of ethanol by a stirred catalytic basket reactor with immobilized yeast cells

A stirred catalytic basket reactor with immobilized yeast cells was used for the batchwise production of ethanol. Fractional conversions up to 0.99 in 10 h were attained, depending on the agitation rates, initial glucose, and cell densities. The volumetric productivity of the reactor was considerably better than that of conventional stirred tank reactors. Productivities were strongly dependent on the stirred speed.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.     

Dye removal from aqueous solution by adsorption on treated sawdust

Formaldehyde treated and sulphuric acid treated saw dusts were used to adsorb malachite green at varying dye concentration, adsorbent dose, pH and agitation time. Similar experiments were conducted with laboratory grade activated carbon to compare the results. The adsorption efficiency of sulphuric acid treated sawdust (SD) was higher than formaldehyde treated SD. The adsorption followed first order rate expression and Lagergren equation. An initial pH in the range of 6-9 was favorable for the dye removal by both the adsorbents. Dilute solutions were effectively decolorized by the adsorbents. It is proposed that in batch or stirred tank reactors, both adsorbents can be an attractive option for dye adsorption.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Hollow fiber bioreactors with internal aeration circuits

New hollow fiber bioreactors for aerobic culture were introduced and Aspergillus niger for citric acid production was cultivated as a model system. These reactors consisted of a bundle mixed of hydrophilic membranes for liquid nutrient transport and hydrophobic membranes for gaseous nutrient transport. The cells were successfully cultivated. However, the polymeric hollow fiber membranes were compressed and blocked by excessive fungal cell growth. Citric acid was produced with a high volumetric productivity compared with that of shake-flask fermentation, but the long-term operation was not successful due to a rapid decrease of the production rate.     

A novel multi-stage preculture strategy of Rhizopus oryzae ME-F12 for fumaric acid production in a stirred-tank reactor

This study has developed a novel multi-stage preculture strategy to control the morphology of filamentous Rhizopus oryzae in a 3.3-l stirred-tank reactor (STR), consequently enhancing the fumaric acid production. Using multi-stage precultures as inoculum, the morphology of R. oryzae was maintained as large clumps, small fluffy pellets, and entangled filaments. The highest fumaric acid production of 42.5 g/l with a yield of 55.6% was obtained in association with the small fluffy pellets which were formed in the third preculture at pH 3.0. The results indicated that the use of the multi-stage precultures as inoculum in the STR is a promising approach for commercial production of fumaric acid.     

Formation of gluconic acid at low pH-values by free and immobilized Aspergillus niger cells during citric acid fermentation

Summary A recently developed immobilization method, characterized by the adsorption of the mycelia onto a glass-carrier in a fixed-bed reactor, was applied for citric acid production by Aspergillus niger ATCC 9142, and compared with conventional culture techniques.In a fixed-bed reactor and in a stirred fermenter a rapid gluconic acid production started immediately after nitrate exhaustion, though the pH was below 2.5 During a second production phase a comparatively small amount of citric acid was formed.In surface and shaken-flask cultures nearly no gluconic acid could be found, whereas citric acid yields were significantly higher than in the fixed-bed reactor and in the stirred fermenter.Manganese (0.8×10^–7 Mol×dm^–3 after 6 days incubation) from the stainless steel parts of the vessel seemed to be responsible for both gluconic acid production and small citric acid yields in the stirred fermenter and in the fixed-bed reactor.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology

Background  

Although the citric acid fermentation by Aspergillus niger is one of the most important industrial microbial processes and various aspects of the fermentation appear in a very large number of publications since the 1950s, the effect of the spore inoculum level on fungal morphology is a rather neglected area. The aim of the presented investigations was to quantify the effects of changing spore inoculum level on the resulting mycelial morphology and to investigate the physiology that underlines the phenomena. Batch fermentations were carried out in a stirred tank bioreactor, which were inoculated directly with spores in concentrations ranging from 10⁴ to 10⁹ spores per ml. Morphological features, evaluated by digital image analysis, were classified using an artificial neural network (ANN), which considered four main object types: globular and elongated pellets, clumps and free mycelial trees. The significance of the particular morphological features and their combination was determined by cluster analysis.     

Fermentation pH in stirred tank and air-lift bioreactors affects phytase secretion by Aspergillus japonicus differently but not the particle size

Abstract

Phytases are mainly produced by filamentous fungi and have great potential for biotechnological use in animal feed treatment, because this enzyme hydrolyzes ester bonds of the phytic acid releasing inositol and inorganic phosphate. The aim of this work was to evaluate the effect of pH on the production of phytase by Aspergillus japonicus in two different bioreactors, known to have different mixing patterns—stirred tank and air-lift bioreactors. The maximum phytase production—53 U/mL—was obtained at 120 h in the stirred tank while in the air-lift the maximum value was 41 U/mL, observed at 144 h. In fermentations evaluated at controlled pH values (3.5, 6.0, and 7.5), the stirred tank was more efficient for production of phytase than the air-lift. Under these conditions, the highest value was measured at 24 h and pH 3.5. These results were not closely related to fungi particle size, because hyphae with a similar diameter (0.51–0.63 mm) and sphericity (0.78–0.87 mm) secreted different amounts of phytase under the conditions studied.     

Jet aeration as alternative to overcome mass transfer limitation of stirred bioreactors

In industrial biotechnology increasing reactor volumes have the potential to reduce production costs. Whenever the achievable space time yield is determined by the mass transfer performance of the reactor, energy efficiency plays an important role to meet the requirements regarding low investment and operating costs. Based on theoretical calculations, compared to bubble column, airlift reactor, and aerated stirred tank, the jet loop reactor shows the potential for an enhanced energetic efficiency at high mass transfer rates. Interestingly, its technical application in standard biotechnological production processes has not yet been realized. Compared to a stirred tank reactor powered by Rushton turbines, maximum oxygen transfer rates about 200% higher were achieved in a jet loop reactor at identical power input in a fed batch fermentation process. Moreover, a model‐based analysis of yield coefficients and growth kinetics showed that E. coli can be cultivated in jet loop reactors without significant differences in biomass growth. Based on an aerobic fermentation process, the assessment of energetic oxygen transfer efficiency [kgO₂ kW^?1 h^?1] for a jet loop reactor yielded an improvement of almost 100%. The jet loop reactor could be operated at mass transfer rates 67% higher compared to a stirred tank. Thus, an increase of 40% in maximum space time yield [kg m^?3 h^?1] could be observed.