Morphology and citric acid production of Aspergillus niger PM 1

Summary Aspergillus niger PM 1 was grown in a tubular loop and a stirred tank bioreactor. Batch fermentations were performed under various agitation conditions and pH. Citric acid, oxalic acid, extracellular polysaccharides and proteins were assayed. The following morphological parameters were measured: mean perimeter of clumps, mean perimeter of the central core of clumps, mean length of filaments and mean diameter of filaments. Citric acid production and morphology in both reactors were dependent on agitation intensity and pH. The length of the filaments was shown to be the only parameter that could be related to citric acid production in both reactors: the shorter the filaments the more citric acid was produced. However, for the same amount of citric acid produced the morphology of the organism grown in the stirred tank differed considerably from that grown in the loop reactor.     

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.     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

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 by immobilized Aspergillus niger in a fluidized bed reactor

Summary Citric acid production by immobilized of Aspergillus niger in a fluidized bed reactor was performed, evaluating the productivity and the stability of the process when pulsing device was used. The application of a pulsing flow to fluidized bed reactor and the feed nitrogen limited allow to control of bioparticles morphology avoiding bed compactation. When operated at optimum pulsation frequency (0.3 s^–1) the stability of the bioreactor was maintained for more than 30 days, increasing the citric acid production in more than 52.2%.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Effect of mode of operation of bioreactors on the biosynthesis of penicillin amidase in Escherichia coli

Different operational mode of bioreactors influence the biosynthesis of the enzyme and related products as well as the growth of industrial microorganisms. This communication deals with the effect of mode of operation of various bioreactors with different geometric configurations, viz., batch (includes commercially available batch stirred tank, and custom-designed cylindrical and tapered reactors), batch-fed, continuous flow stirred tank reactors on the biosynthesis of penicillin amidase in Escherichia coli. Experimental findings show that the biosynthesis of penicillin amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation. Further analysis show that the different reactor modes also influence periplasmic localization of the enzyme in the cell.     

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.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Reactor heterogeneity with saccharopolyspora erythraea airlift fermentations

Bioreactor heterogeneity has been studied in a multiconfigurable pilot-scale airlift reactor (0.25 m3) which created different degrees of heterogeneity. The impact of the two sparger configurations, i.e. in the draft tube or the annulus, in conjunction with a marine propeller fitted at the base of the downcomer, on the physiology of Saccharopolyspora erythraea was studied. Cellular growth, morphology, and productivity were compared between airlift and stirred tank reactors. Dissolved oxygen tension heterogeneity caused by differences in dissolved oxygen tension around the vessel did not affect growth, but the reduction of heterogeneity improved the specific erythromycin production rate and final specific production. Erythromycin production was shown to be proportional to the energy dissipation rate. The enhancement of bubble coalescence with increasing apparent viscosity led to the reduction of the sectional gas holdups and the improvement of liquid mixing. The extent of the changes with increasing apparent viscosity was dependent on the broth morphology, reactor configurations, and operating conditions. Copyright 1998 John Wiley & Sons, Inc.     

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.     

Performance of airlift bioreactors in the cultivation of some antibiotic producing microorganisms

The specific aspects of airlift reactors emphasizing their function relevance to particular application as bioreactors are presented. The two main groups of airlift reactors – external-loop and concentric-tube reactors – were investigated on a pilot-plant scale with regard to their performance during the cultivation of unicellular and filamentous microorganisms which produce Bacitracin, Cephalosporin C and Nystatin. Some results were compared to those obtained in conventional stirred tank bioreactors. The comparison was carried out based on physical properties (oxygen transfer rate (OTR), volumetric mass transfer coefficient (k_La) and efficiency of oxygen transfer (E)), cell mass, productivity and substrate consumption, secondary metabolite production, and efficiency of the product formation with regard to the specific power input. It was shown that B. licheniformis, C. acremonium and S. noursei fermentations occurred similarly to those performed in stirred vessels, proving that the capacity of the airlift bioreactors surpassed the problems which arise from the morphology and rheology of the broths. From the chemical engineering point of view, it was obvious that the primary tasks of a bioreactor (uniform distribution of microorganisms and nutrients over the entire fermenter volume, appropriate supply of biomass with nutrients and oxygen) were fulfilled by the airlift bioreactors tested. In addition, the efficiency of oxygen transfer (OTR referred to power input) in the airlift fermenters proved to be about 38% higher than in the stirred tank bioreactors (expressed as average values), while the sorption efficiency (OTR referred to antibiotic production) was found to be 22% greater in the airlift system than in an STR. Therefore, the biosyntheses were performed with about a 30–40% increase in energy efficiency and energy savings compared to the conventional system. Moreover, the lack of mechanical devices in the airlift system provides greater safety and a gentler environment for the cultivation of microorganisms.     

A High Performance Fermentation Process for Citric Acid Production by Aspergillus niger NGGCB-101, Using Vermiculite as an Additive in a Stirred Bioreactor

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG^GCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levels of vermiculite were added as an additive into the fermentation medium. The best results were observed when 0.20 g/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citric acid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelial pellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at different vermiculite levels was monitored for Q _p , Q _s and q _p , there was a significant enhancement (P 0.05) in these variables over the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basic parameters for high performance citric acid fermentation.     

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.     

Polysialic acid production using Escherichia coli K1 in a disposable bag reactor

Polysialic acid (polySia), consisting of α‐(2,8)‐linked N‐acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave‐induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h^?1. Investigation of stress‐related product release from the cell surface showed no significant differences between the disposable bag reactor with wave‐induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L^?1 and a polySia concentration of 245 mg L^?1 were reached. The total protein concentration in the supernatant was 132 mg L^?1. After efficient and time‐saving downstream processing characterization of the final product showed a protein content of below 0.04 mg_protein/g_polySia and a maximal chain length of ～90 degree of polymerization.     

Continuous production of α-amylase byBacillus amyloliquefaciens in a two-stage fermentor

Summary The production of a secondary metabolite (-amylase) by a highly aerobic bacterium (Bacillus amyloliquefaciens) was examined in batch, single-stage chemostat, two-stage stirred tank, and two-stage stirred tank/tubular reactor configurations. The relative performance of these reactor systems as measured by product concentration and volumetric productivity was compared, and the effect of aeration rate on the extent of plug flow in the tubular reactor was examined.     

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.     

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.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Production of optically pure L-alanine in batch and continuous stirred tank reactors using immobilized Pseudomonas sp.

A process to obtain optically pure l-alanine has been developed using batch and continuous stirred tank reactors with a new l-aminoacylase-producing bacterium Pseudomonas sp. BA2 immobilized in calcium alginate beads coated with glutaraldehyde. The maximum production of l-alanine in a continuous stirred tank reactor was 11.26 g after 2 days of operation which is higher than that previously reported.