Biochemical Engineering Journal 2 (1998) 197205

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor
Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow G1 1XW, UK Received 20 April 1998; accepted 11 September 1998

M. Papagianni1, M. Mattey, B. Kristiansen2,*

Abstract The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different congurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm3, respectively. Morphology was quantied 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 ( 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 congurations 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 identied a morphology parameter that was used successfully to characterize the process performance. # 1998 Elsevier Science S.A. All rights reserved.
Keywords: Aspergillus niger; Morphology; Citric acid; Loop bioreactor; Relative mixing time

1. Introduction In submerged fermentations, agitation is necessary for good mass and heat transfer rates. Mechanical forces resulting from the rotation of impellers can affect lamentous micro-organisms in various ways, causing rate variations of growth and product formation, development of different morphological forms and breakage of laments. Since lamentous micro-organisms are of great industrial importance, the relationship between mechanical parameters, morphology and productivity has attracted the attention of many investigators [13]. Particular morphological forms, often agitation induced, have been considered as more suitable for certain fermentations. Several authors have noted shorter
*Corresponding author. Tel.: +47-691-18972; fax: +47-691-18610; e-mail: bjorn_kristiansen@eunet.no 1 Department of Biosystems and Agricultural Engineering, College of Engineering, University of Kentucky, Lexington, KY 40546, USA. 2 Borregaard Industries, P.O. Box 162, N-1701 Sarpsborg, Norway.

and wider cells in fungal laments, with greater branching frequencies and reduced aggregate sizes under conditions of high agitation [47]. However, optimum stirrer speeds have been reported for many processes, while, side reactions and unwanted morphologies characterized stirrer speeds out of the ranges of interest [8,9]. Studying the behavior of three citric acid producing Aspergillus niger strains, Ujcova et al. [8] observed that higher speeds resulted in thicker, densely branched laments, while production was optimum within a narrow range of speeds and a drop in productivity resulted from higher stirrer speeds. In recent years, much effort has focused on the discovery of new metabolites and on improvement of the respective production methods. Information is, therefore, needed about the fundamental growth behavior with respect to morphology. The characterization of the morphology of lamentous micro-organisms started many years ago [6]. Early attempts to link mechanical parameters with morphology and production, were limiting by the lack of the means to quantify morphology. Nowadays, methods using automated image

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analysis permit the extraction of quantitative information on morphology parameters from lamentous fermentations, representing a useful tool for the design of modern model-based concepts of control engineering [10,11]. As a result, based on quantied morphology information, a number of interesting correlations have been published the last few years, especially for the penicillin fermentation. The impeller tip speed, the circulation frequency around the impeller, the power dissipation rate, are examples of parameters which been correlated with production rates and morphology parameters, as in the works of van Suijdam and Metz [5], Smith et al. [12] and Makagiansar et al. [2]. However, when the validity of such correlations was tested under different operation scales, it was either met with apparent success [2] or it was found difcult to prove the generality of the proposed relationships [12]. The behaviour of micro-organisms is difcult to predict under the constantly changing conditions in large-scale bioreactors and this, makes scale-up an exceedingly difcult task in fermentation technology. The complexity of the problem increases when lamentous micro-organisms are employed, with cells of different ages and subsequently different physiological stages. In order to simplify the highly complex ow patterns and general uid dynamics in a stirred tank bioreactor, loop reactors have been introduced as alternative fermenter designs with regard to scaleup of fermentation processes [13,14]. The loop reactor, is a well known reactor type in the chemical process technology. Its characteristics simple design, dened ow paths, large mass transfer rates for oxygen and substrates at low energy inputs attracted research for its suitability as a bioreactor [15,16] and reports have shown that loop congurations simulated successfully stirred tanks [13,14]. In these works, comparisons were based on growth kinetic parameters and production levels. When lamentous micro-organisms were used, morphology was either not given any consideration or was assessed only qualitatively. In the present work, the effect of agitation on citric acid production and A. niger morphology was investigated in a stirred tank (STR) and a tubular loop (TLR) bioreactor, over a range of circulation pump settings and stirrer speeds, respectively, in batch fermentations. Citric acid is one of the most important organic acids, in terms of bulk production, with wide use in the food and pharmaceutical industries [17]. Although, early observations suggested links between certain morphological types and citric acid production by A. niger [18], morphology was rarely quantied and also comparable data from different reactor systems is, to our knowledge, non-existing in the literature. Morphology in this study was quantied with image analysis. The mean convex perimeter of mycelial clumps served as the characteristic morphology parameter (P) to compare fermentations from the two systems. Since the two bioreactors had very different mixing regimes, circulation (tc) and mixing times (tm) were estimated and results compared on the basis of the dimensionless relative mixing time  m (tm/tc).

2. Materials and methods 2.1. Micro-organism An industrial strain of A. niger (PM1) was used in this study. For inoculum purposes, spores were produced on molasses agar plates (300 kg m3 cane molasses, 18 kg m3 agar, pH 6.8) after 7 days of incubation at 308C. Spores harvested from the plates in sterile water and the spore suspensions were used as inocula for shake asks at a level of 1 107 spore ml1. The composition of the medium for shake asks and bioreactor runs was as follows (kg m3): 150, D-Glucose; 0.252, CaCl26H2O; 2.5, NH4NO3; 0.25, MgSO47H2O; 4.5 103, ZnSO47H2O; 0.428, KCl; 0.1, KH2PO4; 0.75 103, FeSO47H2O. The fermentation inoculum was 300 ml of a 40 h-old shake ask culture at 308C and 200 rpm for the STR and 200 ml for the TLR. 3. Process The loop reactor (APV Chemical Machinery), especially built for scale-up purposes, was tted with a hydromechanical ow drive (pump) and a total operating volume of 6 dm3. The total length was 4 m; the internal diameter in the riser, downcomer and upper transverse sections was 5 cm and 2.5 cm for the lower transverse sections. The circulation pump was situated at the lower transverse part of the loop. Fermentations carried out at seven different pump settings 100, 150, 200, 300, 350, 400 and 500 rpm which covered the entire range of pump passing frequencies practical for this reactor. The corresponding approximate ow rates for the whole reactor were: 0.3, 0.4, 0.6, 0.7, 0.8, 1 and 1.5 dm3 s1. Fig. 1 shows a diagram of the loop reactor used in this study. The STR was a 10 l BIOSTAT, ED - ES10 (Braun Biotech International). The internal diameter of the culture vessel was 22 cm and the height to diameter ratio 3 : 1. The

Fig. 1. Schematic representation of the TLR.

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agitation system consisted of three disc turbine impellers (diameter 8 cm) with six at blades. The reactor was also equipped with four bafes. Six stirrer speeds were applied: 100, 200, 300, 400, 500 and 600 rpm and the operating volume was 8 dm3. In both reactors process temperature was 288C and the air ow rate 1 volume air per volume culture per minute. The pH was not controlled in TLR fermentations, while in the STR it was maintained at 2.1 by addition of 2 M NaOH solution. Dissolved oxygen tension (DOT) was monitored in both reactors (Ingold steam sterilizable probe, Life Science Labs, UK). Foam control was by addition of polypropylene glycol (MW 2000). Samples for assays were taken at inoculation time and thereafter two times per day to 168 h in total. Dry-weights were determined by ltering 20 ml of broth through preweighed glass microlters (grade GF/C, 4.25 cm diameter, Whatman, UK), washing with two volumes distilled water and drying in a microwave oven (600 W) on low power for 20 min and left in a dessicator for 24 h before reweighing. Citric acid was determined by the method of Marier and Boulet [19] and glucose was determined with an assay kit by Boehringer Mannheim. Viscosity measurements were made on broth samples at the end of the runs (168 h) using a Ferranti model VL concentric cylinder type viscometer. 4. The determination of mixing parameters tc and tm were determined using tracer response techniques. These are based on the fact that if a pulse of tracer is injected to the ow a decaying sinusoidal type of response is detected downstream of the injection point [20]. In this study, alkali was used as tracer and the procedure followed was based on the work of Verlaan et al. [21]. In the loop reactor the tracer (2 ml, 3 M NaOH) was injected to the top of the downcomer section with the pH probe placed immediately below the injection point. pH changes were recorded in both riser and downcomer sections, until the response of the pulse was completely damped and each experiment was carried out in triplicate. Response curves were drawn [22] and as tc, was regarded the time difference between two adjacent peaks, not taking the rst peak into account because of the incomplete radial distribution of the pulse during the rst circulation. As mixing time, was regarded the time required until the response oscillation was damped with all the subsequent measurements within 10% of the nal response. Tracer experiments carried out with the reactor lled with a solution of 50 mM potassium chloride in tap water (to create an adequate buffer for pH measurements) and also with the fermentation broth. There was no difference of the estimated mixing parameters between the two; however, to achieve a signicant change in response curves a larger number of titrations were required with the fermentation broth. In all cases, the rst two titrations were not considered since they did not give reproducible results.

A similar procedure was used for the estimation of mixing parameters in the STR. The alkali was injected from the top of the reactor and pH measurements were made at two different placements of the pH probe, while the response was monitored on line. Since in this reactor type the air bubble distribution (through the sparger) is more even compared to the loop reactor, where a single air inlet point exists, results for the mixing time were veried to nd no difference by using a dye as a tracer and measuring the absorbance from samples taken at regular intervals. 4.1. Quantification of morphology Morphology was quantied using an image analysis system consisting of a phase contrast microscope (Olympus CH), a CCD camera (Sony XC77CE), a PC with a frame grabber and the image analysis software (Aquitas, Dynamic Data Links, Cambridge). The CCD camera captured images of 512 512 pixels, with a grayness level from 0 to 255. In all fermentations the bulk of the mycelium was in the form of clumps from the rst 24 h following inoculation. Samples for morphological characterization were taken from 24 h, two times per day for a total of 168 h. Samples were immediately xed with an equal volume of xative, as described by Righelato et al. [23] (13 ml 40% formaldehyde and 5 ml glacial acetic acid added to 200 ml of 50% w/v ethanol) and used for image analysis measurements by Tucker et al. [24]. The xed sample was further diluted with xative prior to measurements and the dilution was adjusted to separate the mycelial clumps on the microscope slide. The length of the perimeters was measured for at least 50 clumps per sample by joining the tips of the peripheral laments in each clump. This was the `convex perimeter' of clumps according to the denition given by Paul and Thomas [11]. A magnication of 100 was used for this work and mean values were estimated for each sample. Examination of fresh and xed samples showed that the xation method did not affect the morphology. Fixed samples remained unchanged for prolonged periods. The ne structure of the mycelium seemed also perfectly preserved, as repetitive measurements on the characteristics of vacuoles (e.g., diameters and volumes) showed very good reproducibility for over a year (unpublished results). 5. Results and discussion In each bioreactor, citric acid production and morphology were clearly dependent on culture conditions. Fig. 2 and Fig. 3 show the time courses for citric acid, as functions of the circulation pump setting (rpm) and the stirrer speed (rpm) in the TLR and the STR, respectively. Increasing the agitation intensity citric acid production increased, although in the STR the same nal citric acid concentrations obtained from fermentations carried out at 400, 500 and 600 rpm. In both bioreactors, no direct effect of agitation on biomass

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Fig. 2. Time courses for citric acid production in the TLR at different circulation pump speeds.

Fig. 3. Time courses for citric acid production in the STR at different stirrer speeds.

levels was observed (not shown). Biomass concentrations were rather low. The highest biomass concentration obtained was 7.2 kg m3, in the STR run at 300 rpm, while the lowest was 5.2 kg m3, in the TLR run with a pump speed of 200 rpm. The pH in the TLR was not controlled by titrants, since from pH 3 at the inoculation time it dropped to around 2.32.1 within 72 h and remained unchanged thereafter. In the STR fermentations the pH maintained at 2.1 during fermentations since, when left uncontrolled it dropped to 1.8. In all fermentations in this study the bulk of the mycelium was in the form of clumps, as particles of intertwining

laments around a small core, not having the compact structure of what is usually referred to as pellets [25] and not macroscopically visible. A. niger morphology responded to agitation changes and a range of different clump sizes obtained in both systems, each corresponding to a certain degree of agitation. Fig. 4 shows the characteristic morphology of the fungus at the end (168 h) of the STR run at 500 rpm. The characteristic morphology parameter P decreased as circulation frequency through the pump increased in the TLR and as stirrer speed increased in the STR. This effect is shown in Fig. 5Fig. 6. DOT were lower but never fell below 40% at low agitation levels in both bioreactors. For the particular fermentation aeration is an important parameter for a good yield [26], however, morphology does not seem to be directly inuenced. Research has shown that variations in DOT were not accompanied by signicant changes in morphology [5,27,28]. Gomez et al. [28] in their work on citric acid production by A. niger showed that no difference in morphology for pellets and laments could be ascribed to dissolved oxygen levels although production was enhanced by an increase in dissolved oxygen. Since in all fermentations in this study the DOT never fell below 40%, the lower yields that was obtained at low rpm could be regarded as the result of the certain morphology developed under such agitation conditions and not as the result of any mass transfer limitations. Fig. 7 shows the plots of the nal citric acid concentrations versus the corresponding convex perimeters of clumps from fermentations carried out in both bioreactors. It is obvious that the size of clumps should not exceed a given value in order to have maximum productivity of citric acid, so, for this fermentation morphology and productivity were closely linked. Early studies by Clark [18], showed that increased citric acid productivities were associated with a certain morphological type: small pellets and short, thick densely branched laments and it is a general belief that pelleted morphology is required for a successful citric acid fermentation [26]. Obviously this is not the case for the strain used in this study. High yields were obtained without pellets, however, the characteristic morphology of the citric acid producing lament was apparent. Many investigators have discussed effects of agitation on morphology and biosynthesis. There are cases where growth rates and productivities were linked to morphology, as discussed by Marckl et al [29], while others, where no link was observed, as in the work of BelmarBeiny and Thomas [1]. Often, fragmentation resulted from increased shear stress applied to the mycelium was characteristic at high stirrer speeds, as in the work of Ayazi Shamlou et al. [30]. The authors showed that hyphal breakage was the dominant mechanism that determined the hyphal length of Penicillium chrysogenum under conditions of intensive agitation in 7 and 150 dm3 mechanically agitated bioreactors. The impeller damage to mycelia was also investigated by Justen et al. [31] and it was shown to be dependent on the impeller type and agitation intensity. Moreover, fragmentation data,

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Fig. 4. Photograph of a mycelial clump: typical morphology of A. niger in the STR at 500 rpm and 168 h of fermentation.

Fig. 5. The effect of agitation on morphology in the TLR; mean convex perimeter of clumps (P) at different circulation pump speeds (168 h fermentation).

Fig. 6. The effect of agitation on morphology in the STR; mean convex perimeter of clumps (P) at different stirrer speeds (168 h fermentation).

derived from mean projected areas and total hyphal lengths, was successfully correlated with the energy dissipation/ circulation function given by the authors, at different scales. Lower productivities have been reported at high agitation rates and were attributed to cell damage, as in the work of Smith et al. [12]. In the present study, fragmentation is likely to have occurred at increased agitation levels in both bioreactors. The broader standard deviations of mean values of P at high rpm can be an indication of fragmentation (Figs. 4 and 5). Also, unpublished results on the timecourses of morphology parameters, for example, mean

lament length and vacuole diameters, suggest that fragmentation controlled morphology at stirrer speeds higher than 400 rpm. However, the damage to laments should have been small, compared to other studies [12] and no direct effect was noticed upon the specic rate of citric acid production and the nal product yield. For the range of pump passing frequencies practical for the TLR, microscopic observations and plots of time courses of clump perimeters (not shown here) did not support any particular harmful pump effect on morphology [22].

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Fig. 7. The relationship between mean convex perimeter of clumps and citric acid production in the TLR and the STR.

As Figs. 46 show, the curves obtained from the two bioreactors resemble each other and there are no fundamental differences in the results from two systems with different congurations. It would be interesting, therefore, to nd a parameter with which the performance of both reactor systems could be compared. Mixing intensity could be such a parameter. To characterize mixing, tc and mixing times tm were estimated for both bioreactors, as described in the previous section. Circulation times for the TLR (for 500100 rpm) were: 4, 6, 7, 8, 10, 14 and 18 s and for the STR (for 600 to 100 rpm): 1.1, 1.8, 2.2, 3, 3.8 and 6 s. The corresponding mixing times for the TLR were: 32, 36, 40, 45, 50, 61 and 72 s, while for the STR: 8, 12, 14, 15, 16 and 22 s. Both tc and tm values were smaller in the STR. As also mentioned in the previous section, there was no difference in the estimated mixing parameters when the tracer experiments were carried out with tap water and fermentation broth. A comparison of the rheologies of fermentation broths, using the concentric cylinder viscometer in samples taken at 168 h, showed in all cases Newtonian behaviour and this can be attributed to the low biomass concentrations. In loop reactors two fundamentally different mixing effects are superimposed on each other: longitudinal mixing and bachmixing due to recycling of the circulation ow. For the characterization of ow in tubular loop reactors the diffusion model has often been used [16]. In this model, ow through tubes is described as plug ow with diffusion like disturbance caused by molecular diffusion, small edies, dead spaces and radial velocity gradients. The model is characterized by the Bodenstein number (Bo VL/Dacca), which expresses the degree of axial mixing. According to Blenke [16], Bo 200 corresponds to a medium longitudinal mixing while a Bo 50 to a high one. In the present study, Bodenstein numbers were calculated from the tracer response experimental data for the downcomer section of the loop bioreactor [22]. For the tc of 4, 6, 10 and 18 s the

corresponding Bo were 100, 80, 60 and 40, which were indicative of high longitudinal mixing intensities. In an attempt to nd a parameter that brings together the results from the two systems in a form more suitable for direct comparison, the relative mixing time was used as the parameter that characterizes the mixing intensity.  m, equal to the tm divided by its corresponding tc, was calculated and found to be between four and eight in both bioreactors. This dimensionless parameter has been used by Seipenbush and Blenke [15] on studies of the effect of ow pattern on productivity in yeast cultivations in a loop bioreactor. Verlaan et al. [21], studying the axial dispersion in an airlift loop reactor with a working volume of 0.165 m3 and height 3.23 m, correlated the relative mixing time with the Bo to obtain the empirical correlation:  m 0.093 Bo. With the Bo obtained for the TLR in the present study, the relative mixing times calculated with the equation of Verlaan et al. were very close to those obtained from the experimental data. The equation derived numbers were 9.3, 7.4, 5.5 and 3.7 while the corresponding experimentally estimated were 8, 6, 5 and 4 and this suggests that the relative mixing time is a parameter that can be used with condence in characterization of mixing in the TLR. For the two bioreactors with dissimilar congurations and ow regimes used in this study, relative mixing times in the same range strongly indicate that an equilibrium between the effects of micro and macro-mixing was the case. Mixing in bioreactors determines the process performance. For example, Fields and Slater [32], investigating the inuence of liquid mixing on the respiration of micro-organisms in an airlift bioreactor found that the respiratory quotients were affected by the local mixing behaviour. In a system where changes in agitation intensity reect to the morphology of the fungus, it can be expected that the mixing properties will inuence the morphology and given that morphology and production are closely linked production. Correlating the dimensionless  m with the nal citric acid concentrations, the curves obtained from the two bioreactors were very similar (Fig. 8(a)). As Fig. 8(a) shows that citric acid production increases with the relative mixing time, accordingly, the clump perimeter should decrease as the relative mixing time increases and Fig. 8(b) shows this indeed to be the case. Fig. 8((a) and (b)) show that on the basis of  m results from the two systems agree even quantitatively. It appears that the amount of citric acid produced is a function of the relative mixing time and the size of clumps, irrespective of the reactor type. It also becomes clear that agitation controls production through morphology. Comparing the performance of the two bioreactors, the results suggest that the loop reactor simulates the STR behavior for the given conditions and scales of operation. Tubular loop reactors have been introduced in some cases as alternative fermenter designs that may have advantages with regard to scale-up of fermentation processes. Unfortunately, only a limited number of detailed investigations on

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For this purpose a set of empirical correlations has been formulated and plotted for the TLR. The relationship between  m and lnp (p is the product of citric acid concentration kg m3) for that reactor is shown in Fig. 9. This gure suggests that lnp can be regarded as a linear function of tm described by the equation: p e3:40:21m r 0:88 (1) The relationship between the morphology parameter P and lnp for the TLR may also be regarded as a linear function, as Fig. 10 shows, with the equation describing it being: p e5:340:001P ; r 0:92 (2) To obtain one expression for p, depending on the  m and P, the two equations were multiplied: p2 e3:40:21m e5:340:001P or 0:5 p e8:740:21m 0:001P a simpler form of which is: p e4:370:105m 0:005P (5) The above (Eq. (5)) was formulated from the experimental data of TLR fermentations. In Fig. 11 and Fig. 12 experimental values of lnp from both bioreactors, along with values calculated with the Eq. (5) for known tm and P from STR fermentations, were plotted against the relative mixing time and the mean convex perimeter of clumps. Both gures show a good t between correlation and experimental derived points, suggesting that the correlation could describe the data points for experiments performed under different culture conditions and that the relative mixing time can be used successfully in comparison of two systems with (3)

(4)

Fig. 8. (a) Citric acid concentrations and (b) mean perimeters of clumps as functions of the relative mixing time in the TLR and the STR (168 h).

loop bioreactors are available in the literature. Such studies have shown that small loops can behave like stirred tanks. In both reactors cells experience similar changes in their macro and micro environment producing a response which could be independent of the reactor type. Russel et al. [13] designed a tubular loop reactor, tested its performance with Saccharomyces cereviciae and outlined a design procedure for a large scale tubular reactor which had considerable advantages over the more complex scale-up of a STR. The same growth kinetics results obtained in the loop and a conventional STR. Growth and product formation of three metabolite producing mycelial organisms were studied and compared in TLR and STR by Ziegler et al. [33]. Results from the two reactor types were comparable, however, morphology was poorly assessed and any observations made were not quantied. Another mycelial microorganism, Aurobasidium pullulans, was the test micro-organism in studies of the performance of TLR and STR, in the work of Kristiansen and McNeil [14]. From comparative experiments in 10 and 200 dm3 STR fermentations, it appeared that the loop behaved as the corresponding STR representing in this way a valuable tool in scale-up and down of fermentation processes. So far, morphology has not been used in such comparisons and results in the present study suggest that for the particular fermentation it represents a valuable parameter in the comparison of the two reactors.

Fig. 9. Loop bioreactor: correlation of lnp with the relative mixing time.

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Fig. 12. Relationship between lnp and  m in the TLR and the STR; experimental data and correlation derived points

Fig. 10. Loop bioreactor: correlation of lnp with the morphology parameter P (mean convex perimeter of clumps).

dependent and closely linked. Since, the results from two systems with different congurations were of the same trend, even quantitatively, the bioreactors performance compared on the basis of the dimensionless mixing parameter relative mixing time. It appeared that the loop reactor simulates the stirred tank reactor's behavior. Both, the morphology parameter P (mean size of clumps) and  m, were used successfully as parameters for comparison and empirical correlations derived from the loop reactor fermentations showed a good t when plotted along with the experimental data from stirred tank fermentations. The need of quantied morphology information in studies of process development and scale-up, has been stressed by many authors; the present study demonstrates how, in a fermentation in which morphology and productivity are linked, morphology can be used to characterize process performance. 7. List of symbols Bo D D Dax L N p P STR TLR tc tm m V Bodenstein number (-) Impeller diameter (m) Pipe diameter (m) Axial dispersion coefficient (m2 s1) length (m) Stirrer speed (rpm) Product concentration (kg m3) Convex perimeter of clumps (mm) Stirred tank reactor Tubular loop reactor Circulation time (s) Mixing time (s) Relative mixing time (-) Velocity (m s1)

Fig. 11. Relationship between lnp and the morphology parameter P in the TLR and the STR; experimental data and correlation derived points.

different congurations. The above expressions are of value only for the particular strain and the considered scale of operation and for this case they support our conclusion that the loop simulates the stirred tank. The loop reactor in this study was developed as a scale-up tool [34] and the present results show it does simulate the STR behavior. 6. Conclusions In each bioreactor used in the present study, morphology and citric acid production of A. niger PM1 were agitation

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References
[1] M.T. Belmar-Beiny, C.R. Thomas, Biotechnol. Bioeng. 37 (1991) 456462. [2] H.Y. Makagiansar, P. Ayazi Shamlou, C.R. Thomas, M.D. Lilly, Bioprocess Eng. 9 (1993) 8390. [3] M. Papagianni, M. Mattey, B. Kristiansen, Biotechnol. Lett. 16(9) (1994) 929934. [4] W.M. Dion, A. Carilli, G. Sermonti, E.B. Chain, Rend. Ist. Super.de Sanita 17 (1954) 187205. [5] J.C. Van Suijdam, B. Metz, Biotechnol. Bioeng. 23 (1981) 111148. [6] B. Metz, E.W. de Bruijn, J.C. van Suijdam, Biotechnol. Bioeng. 23 (1981), 23 and 149162. [7] A. Mitard, A. Riba, Biotechnol. Bioeng. 32 (1988) 835840. [8] E. Ujcova, Z. Fencl, M. Musilcova, L. Seichert, Biotechnol. Bioeng. 22 (1980) 237241. [9] F. Vardar, M.D. Lilly, Eur. J. Appl. Microbiol. Biotechnol. 14 (1982) 203211. [10] C.R. Thomas, TIBTECH 10 (1992) 343348. [11] G.C. Paul, C.R. Thomas, Adv. Biochem. Eng. Biotechnol. 60 (1998) 159. [12] J.J. Smith, M.D. Lilly, R.I. Fox, Biotechnol. Bioeng. 35 (1990) 10111023. [13] T.W.F. Russel, I.J. Dunn, H.W. Blanch, Biotechnol. Bioeng. 16 (1974) 12611272. [14] B. Kristiansen, B. McNeil, in: G.W. Moody, P.B. Baker (Eds.), Proc. Int. Conf. Bioreactors and Biotransformations, 1987, p. 321. [15] R. Seipenbuch, H. Blenke, Adv. Biochem. Eng. 15 (1980) 140. [16] H. Blenke, Adv. Biochem. Eng. 13 (1979) 121214. [17] M. Mattey, Crit. Rev. Biotechnol. 98 (1992) 133138.

[18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]

D.S. Clark, Can. J. Microbiol. 8 (1962) 133136. J.R. Marier, M.J. Boulet, Dairy Sci. 41 (1956) 16831692. J. Bryant, Adv. Biochem. Eng. 5 (1977) 101123. P. Verlaan, A.M.M. van Eijs, J. Tramper, K. van't Riet, K.Ch.A.M. Luyben, Chem. Eng. Sci. 44(5) (1989) 11391146. M. Papagianni, Ph.D. Thesis, 1995, University of Strathclyde, Glasgow, UK. R.C. Righelato, A.P.J. Trinci, S.J. Pirt, A.J. Peat, Gen. Microbiol. 50 (1968) 399412. G.K. Tucker, T. Kelly, P. Delgrazia, C.R. Thomas, Biotechnol. Prog. 8 (1992) 353359. R. Steel, S.M. Martin, C.P. Lentz, Can. J. Microbiol. 1 (1954) 150 157. hr, Citric acid fermentation, CRC Crit. Rev. C.P. Kubicek, M. Ro Biotechnol. 3(4) (1989) 331373. B.L.A. Carter, A.T. Bull, Biotechnol. Bioeng. 11 (1969) 779785. R. Gomez, I. Schnabel, J. Garrido, Enzyme Microb. Technol. 10 (1988) 188191. H. Markl, R. Bronnenmeier, in: H. Brauer (Ed), Fundamentals of Biochemical Engineering, 2 (1985), VCH Verlagsgesellschaft, Germany, p. 370392. P. Ayazi Shamlou, H.Y. Makagiansar, A.P. Ison, M.D. Lilly, Chem. Eng. Sci. 49 (1994) 26212631. P. Justen, G.C. Paul, A.W. Nienow, C.R. Thomas, Biotechnol. Bioeng. 52 (1996) 672684. P.R. Fields, N.K.H. Slater, Chem Eng. Sci. 38 (1983) 647653. H. Ziegler, I.J. Dunn, J.R., Bourne, Proc. Eur. Congr. Biotechnol. I (1978), Part 1, Interlaken, pp. 7477. B. Kristiansen, Proc. Int. Symp. Bioreactor performance, Helsingor, Denmark, 1993, pp. 209220.