growth morphology and 2 hydrodynamics of filamentous fungi ... · growth of aspergillus oryzae on a...

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Advances in Agricultural and Food Biotechnology, 2006: 17-34 ISBN: 81-7736-269-0 Editors: Ramón Gerardo Guevara-González and Irineo Torres-Pacheco

2 Growth morphology and hydrodynamics of filamentous fungi in submerged cultures

García-Soto Mariano J.1, Botello-Álvarez Enrique1, Jiménez-Islas Hugo1 Navarrete-Bolaños José1, Barajas-Conde Eloy1 and Rico-Martínez Ramiro2 1Instituto Tecnológico de Celaya, Departamento de Ingeniería Bioquímica 2Departamento de Ingeniería Química, Ave, Tecnológico y A. García-Cubas S/N, Col. FOVISSSTE, Celaya, Gto, C.P. 38010, México

Abstract A submerged culture of microorganisms involves their propagation in a liquid medium inside a vessel commonly called bioreactor: the bioreactor main objective is to provide suitable physical conditions for microorganism growth. These conditions include optimal pH, dissolved oxygen levels, agitation, temperature, and substrate availability, for both microbial growth and development leading to high yields of specific metabolites.

Correspondence/Reprint request: Dr. Navarrete-Bolaños José, Instituto Tecnológico de Celaya, Departamento de Ingeniería Bioquímica, Ave. Tecnológico y A. García-Cubas, S/N, Col. FOVISSSTE, Celaya, Gto, C.P. 38010 México. E-mail: [email protected]

García-Soto Mariano J. et al. 18

Submerged cultures in bioreactors make possible to achieve high microbial biomass production allowing metabolite production for commercial applications. In this sense, submerged cultures in bioreactors are one of the leading tools for practical implementation of recent biotechnology advances leading to novel bioprocesses. However, in the bioreactors, the microorganisms are generally subjected to conditions that can differ significantly from those present on their natural habitats. These changes may lead to microbial stress, and low yields of specific desired products. As a consequence, the design and operation of bioreactors, along with culture media selection, must be continuously fed by technological advances and original research. Introduction Among the concerns during submerged cultures in bioreactors are the mechanical interactions fluid-microorganism, considering the latter as a solid-disperse phase, as well as the interactions between such disperse phase with the walls and mobile parts of the bioreactor. The microorganism movement inside the bioreactor is the result of fluid shear forces over the microorganisms’ surface, leading to their incorporation into the convective streams. When a microorganism is exposed to a spatial velocity field, a velocity gradient is generated over its surface, inducing microorganism’s surface erosion as consequence of the shear stresses. Sahoo et al., (2003) cultured Bacillus subtilis in a Couette flow bioreactor. The microorganisms were exposed to shear rates in the range 0.028-1482 s-1 which affected their morphology and size, growth velocity, and enzyme biosynthesis. The shear rate inherent in continuous stirred tanks can also affect enzyme activity. Gunjikar et al., (2001) studied the effect of agitation speed of a mixed tank (16.67, 33.33, 50 rpm) on cellulase activity (exo-1, 4-β-D-glucan-4-cellobiohydrolase, endo-1, 4-β-D-glucanhydrolase, 1, 4-β-glucosidase). After several assays, a considerably reduction of cellulase activity and hydrolysis potential was found. Michaud et al., (2003) found that the superficial gas velocity through a fluidized reactor, with biogas recirculation and biofilm covered particles (used for waste water treatment), had a significant effect on growth and activity of the biofilms due to the shear rate on their superficial area. These are some examples on the effect of the mechanical interaction fluid-microorganism. However, the most dramatic cases are those manifested during filamentous fungi cultures. The filamentous fungi culture in bioreactors, at commercial levels, started with penicillin production during the Second World War. Today there is a large variety of products obtained by fungi submerged cultures, such as enzymes (glycosidases, amylases, proteases, tannases), antibiotics (cephalosporins,

Agricultural bioengineering 19

cyclosporins) and several organics acids (citric, fumaric, gluconic), among others (Papagianni, 2004). One common feature of filamentous fungi is their polarized growth pattern in the form of filaments, called hyphae. The basic growth structure involves tubular filaments (hyphae) generated from reproductive spores. These hyphae grow in the form of branched networks, called mycelia. The usual morphologies presented in submerged cultures can be classified in two extremes: dispersed mycelium, in turn classified as free mycelium or clumps, and dense agglomerates denominated pellets. The pellets may be spherical or ellipsoidal agglomerates, consisting of branched and intertwined networks of hyphae. Nevertheless, their structure is also variable depending on intertwined hyphae compactness, which can be slight or strong, generating smooth pellets (compact agglomerates with smooth surface) or hairy pellets (loose agglomerates with hairy surface). (Li et al., 2000, Papagianni, 2004). The morphology developed by filamentous fungi during submerged culturing has a great impact on both culture productivity and bioreactor handling. The two principal morphologies described before exhibit significant differences during cultivation (Cui et al., 1998):

• When fungi grow as free mycelia, a significant increase of apparent viscosity is observed. This increase may change the rheological behavior; transforming the culture media to a Non Newtonian fluid. This fact induces an important reduction on mixing and air dispersion, which causes limitations on solid-fluid mass transference and oxygen availability within the liquid medium.

• When fungi grow in pellet form, limitations on intraparticle mass transference may be present. Such limitations may produce regions (microenvironments) with different grow patterns and substrate availability. Large pellets are an example of these events: their external zone is metabolically active, while low viability zones exist within the pellets (Figure 1).

On the other hand, the fungi morphology developed during fermentation, may lead to the synthesis of particular metabolites; reflecting both phenotypic characteristics and metabolism “inducibility” of filamentous fungi, as consequence of fluid-microorganism interaction inside the reactor. Papagianni (2004) observed that when Aspergillus niger grows as disperse mycelium, peptic enzymes production is favored, while a great citric acid production is achieved when the fungus grows in pellet form. He concluded that three main factors determine the morphologic development of filamentous fungi: 1) genetic material from specific fungal species, and its interaction with, 2) chemical factors (constituents, micro and macro, of the culture media), and 3) physical factors (temperature, pH, mechanical forces, etc.).


a b

Figure 1. Rhizopus nigricans, pellet form. a) Spherical aggregated. Scale: bar = 10 mm. b) Longitudinal cut under stereoscopy view; the central cavity is characteristic of large pellets. Scale: bar = 5 mm.

This chapter will be focused on several issues related to filamentous fungi morphological development during submerged culture processes. These include the use of digital image analysis to study the growth of fungi, hydrodynamics effects on fungal morphology in bubbling column bioreactors, and mathematical modeling of intraparticle phenomena in filamentous fungal pellets. Digital image analysis for filamentous fungi growth characterization in submerged cultures Sometimes, during the course of experiments, is important to make detailed descriptions of visual aspects such as color, appearance, shape and size, as well as object number, density, position, direction, etc. Formerly, both photography and cinematography were used only as documentation tools. Today, digital photographic systems allow not only the gathering of graphic evidence, but also the numerical assessment of the previously described characteristics. Several studies about morphological development of filamentous fungi have taken advantage of digital image analysis. Spohr et al. (1998) studied the growth of Aspergillus oryzae on a plate placed directly on a microscope deck. They followed the gradual changes with a digital video using 10 minutes as sampling interval. After image processing, parameters like spore sphericity and volume, specific growth rate, and primary hyphae growth velocity, including branching and bifurcations, were obtained.


Li et al. (2000) employed image analysis for studying A. oryzae morphology inside a 80 m3 mixed tank reactor, finding both dispersed mycelia and clumps as principal growth morphologies. Additional parameters like projected area (indirect measuring of biomass), and average hyphae size and thickness were analyzed. The number of bifurcation tips was used to estimate the average hyphae branch length. In the case of clumps, sphericity measurements were performed. These measurements were used to study mycelia growth and fragmentation for different operation conditions. Cui et al. (1998) studied the growth morphology of A. awamori (CBS 115.52) in a 3L mixed tank, under energetic dissipation and dissolved oxygen tension changes. They observed the growth of fungal pellets with superficial mycelium, hairy pellets. Parameters like compact core diameter of the pellet, and hypha average length, from the hairy zone, were obtained using digital image analysis. In all of the aforementioned references, the measurements were performed by taking samples outside the bioreactor for image analysis. Lucatero et al. (2003) implemented an image acquisition technique mounted directly on the bioreactor. Good quality images were obtained, observing the presence of pellets, castor’s oil drops (used as substrate) and air bubbles interacting. This kind of techniques allows object parameter characterization, following its progression directly; without requiring sample removal from the bioreactor. In this section, the growth morphology of Rhizopus nigricans cultivated in a bubbling column bioreactor is used as study case. The microorganism was isolated during pigment extraction studies from Marigold flowers (Tagetes erecta). It produces an enzymatic complex with cellulase activity, employed in marigold flower pretreatments for colorant extraction of commercial importance (Navarrete-Bolaños et al., 2004). Rhizopus nigricans has been maintained on solid medium, using potato dextrose agar (PDA, Becton Dickinson) incubated at 28oC. Inoculum propagation was made in 250 mL conical flasks containing 100mL potato dextrose broth (PDB), taking superficial aerial mycelium from the PDA slants with 36 h growth. Culture flasks were incubated in an orbital shaker (4520, Forma Scientific Co.) at 100 rpm and 28o C for 24 h, obtaining compact mycelial pellets. Submerged cultures, starting from flasks cultivations, were performed in the bubbling column schematically described in Figure 2. The bioreactor was sterilized, using overheated steam, prior to each assay. The bioreactor was operated at 28 oC with PDB medium. Cellulase productivity was measured indirectly by viscosity reduction of carboxymethyl-cellulose (CMC, Fluka BioChemika GmbH) solutions. For such measurement 2mL of supernatant from the bioreactor were added to 100mL of the CMC solution, and allowed to react for 24 h at 28° C and 180rpm (Navarrete-Bolaños et al., 2004).

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Figure 2. Schematic of the column bioreactor; external accessories, for measuring DO2 concentration and images acquisition, are also depicted. Digital image analysis The global steps involved during image analysis processing commonly are: image acquisition, gray scale processing, object detection, binary image processing, image editing, measurements and calculations, and data analysis (15). The first step, for image analysis, involves capturing specific objects. A digital video camera recorder (VCR, Sony Handycam DVC-VTR730, Sony Corp), with shutter speed control and adjustable zoom, was used. The digital VCR was located in front of the reactor at 0.5 m height from the bioreactor’s base, and at 0.02 m from the cubic glass jacket. The glass jacket was installed in order to avoid curvature effects from the cylindrical column wall. Image acquisition was performed at an average depth (dp) of 0.01 m with respect to the bioreactor’s internal wall (Figure 3). Behind the reactor, at the same level than the VCR, two light sources, with appropriate inclination, were located. A thin paper filter was placed, between the reactor and the light sources, in order to absorb the excess light from the sources, providing a better contrast during image acquisition.

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Figure 3. Equipment distribution for image acquisition. The VCR was user-programmed, adjusting the build-in special effects included by the manufacturer. For fungi analysis, very sharp resolution is required in order to capture well defined outlines. From the VCR menu, the fastest shutter speed was selected: Program AE (Auto Exposure) Sports lesson (1/250 – 1/1000 sec.) for image acquisition. Additionally successive fixed image series at constants intervals was attained with Flash Motion Digital Effect, and Black/White high contrast images with B&W Picture Effect. In each sampling interval, throughout the experimental assays, 15 to 20 pictures were processed. The images obtained were transferred to the analysis module system. This module system consist in a Pentium III (Intel Corp.) personal computer with a FireWire IEEE 1394 DV 32-bit PCI device, for image and video download; supplied with DVTools 1.6 (Pinnacle Systems Inc.) as transferring software. This software allowed saving, changing and modifying video sequences to successive series of pictures in bitmaps (*.bmp). During image analysis, an image edition step was necessary in order to get a good contrast between target objects and picture noise. Invert filter from Corel PhotoPaint 9.0 (Corel Corporation) software, was used as main editing tool. This filter inverts image colors automatically, producing a photographic negative with high quality contrast. Duotone filters, like dcolor2.cpd and tgray4.cpd both from Corel PhotoPaint 9.0, were also used for best contrast improvement, obtaining even more detailed objects. Figure 5 shows an edited image. In the edited image, the fungus’ compact center can be easily distinguished from its external mycelial zone; a reference scale is used for reporting measurements.

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Figure 4. Rhizopus nigricans photography taken directly from the reactor.

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Figure 5. Edited photography of Rhizopus nigricans. A well defined compact center and mycelial zone are observed. Once the editing step is concluded, the images are ready for measurement. The measurements were performed using the software SigmaScan (Pro 5.0 © SPSS Inc.) which has programmed routines for object detection, pre-edition and measurement. Firstly, a reference scale is obtained by marking two points into a picture belonging to a specific object of known dimensions. Secondly, an object recognition subroutine based on intensity thresholds allows the identification of the objects of interest. Finally, based on the chosen geometric characteristics a measurement subroutine is run. The results are reported in a file with *.xls format for subsequent analyses. Growth kinetics of Rhizopus nigricans In what follows, the results from the growth kinetic study of Rhizopus nigricans in submerged culture in the bubbling column previously described


are presented. The bubble column was operated with a superficial air velocity of 2.80 x10-3 m/s. In addition to the image acquisition and analysis, several other variables including reducing sugar consumption, cellulase production, pH evolution, and dissolved oxygen uptake were also monitored. Figure 6 illustrates the growth morphology throughout the fermentation assay. From the image sequence alone very few conclusions can be drawn. However, the image analysis techniques allow us to gain further insight through the results of the type exemplified in Figure 7. This figure describes the growth and evolution of R. nigricans, starting from small pellets, as function of quantifiable geometric characteristics. The average growth of both core pellet and hairy mycelia is observed. In the first hours, up to the first 16 h, both core and hairy mycelia grow exponentially. Then a grow rate decrease is observed, more drastic for the hairy zone. The filamentous mycelia conforming the hairy zone tends to surround the core mycelia, compacting itself over the core. Figure 8 presents the substrate uptake and cellulase production kinetics, while Figure 9 shows the pH and dissolved oxygen profile changes. The information obtained from the digital image analysis can be thus linked to the biomass production, as well as other important variables in the culture. For example, it is evident that the principal period of cellulase production coincides with the exponential growth period (projected area). Such period is also characterized by a steady pH decrease, and high oxygen consumption. In Figure 9, one can observe sharp declines in the dissolved oxygen measured;

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a) 00.0 h, 100.0 % res. susbt. 100.0 % enz. actv. b) 08.0 h, 158.5 % res. susbt. 168.9 % enz. actv. c) 24.0 h, 130.3 % res. susbt. 195.5 % enz. actv. d) 36.0 h, 116.0 % res. susbt. 195.9 % enz. actv. e) 48.0 h, 104.6 % res. susbt. 196.2 % enz. actv.

Figure 6. Growth sequence of Rhizopus nigricans during submerged culture. Remaining substrate percentage in culture medium, and enzymatic activity measurements have been included for reference. Scale: bar = 3 mm.


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Figure 7. Growth morphology of Rhizopus nigricans as function of projected area: (□) core center with mycelia, (○) core center without mycelia and (▲) filamentous or mycelial zone.

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Figure 8. Kinetic of substrate consumption and cellulose production: (○) sugar concentration and (■) enzymatic activity, expressed in percentage. these peaks were generated by stopping the oxygen supply for 5 minutes in order to monitor the oxygen demand from the culture. As it can be observed in the Figure, after 12 h, the peaks become progressively shallower, indicating less oxygen demand as the fermentation progresses.

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Figure 9. Dissolved oxygen (DO2) and pH changes in culture medium as a function of the fermentation time. Effect of hydrodynamic conditions on the morphology of fungal cultures in a bubbling column Several studies on filamentous fungi cultures in mixed tank bioreactors have been reported in the literature. These studies focused in explaining how hydrodynamic conditions influence the observed macroscopic mycelial morphology diversity. Li et al., (2002) described two mechanisms for disperse hyphae fragmentation: one dominated by inertial forces and the second by viscous forces. For both mechanisms, the hydrodynamic effect can be explained through interactions of the hyphae and variable length scale eddies, locally within the fluid. If the hyphae average length is larger than the local eddies, normal forces are exerted over the branched hyphae producing their fragmentation (inertial effect). On the other hand, if the hyphae are smaller than the average eddy´s length, the hyphae is dragged by the eddy, undergoing shear rates that erode their surface and lead to their fragmentation (viscous effect). The hyphae exhibit a characteristic resistance to the fragmentation; it must be surpassed by the momentum transferred by either mechanism in order to lead to the hyphae fragmentation. Bubbles flowing through a liquid medium always take the form that minimizes the amount of energy that need to be transferred in order to continue rising (Bozzano and Dente, 2001). For filamentous fungi, however, the momentum transfer does not come from the usual primary sources: bubbles rising or impellors moving. The momentum transfer is confined to the liquid-solid interface and is related to viscous and convective forces. Furthermore, since the density differences between the liquid medium and the fungal

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conglomerates are usually negligible, one can also discard any gravitational effect. Thus, if a fungal conglomerate travels on a unidirectional flow, it should grow as if suspended in a stagnant fluid medium. Finally, since hyphae growth is driven by substrate gradients, one would expect uniform radial mycelial growth on a homogeneous fluid, and thus spherical and spongy fungal conglomerates (Figure 10). A bubbling column is a bioreactor consisting mainly of a cylinder with air diffusers at the bottom. The bubbles are formed at the diffuser, and as they are dispersed and rise through the column the oxygen contained in them is transferred to the liquid medium. When the bubbles pass through the diffusers, they possess a large amount of dynamic pressure. Such energy allows them to drag the liquid as they rise, and it is partially dissipated through transfer to the surrounding liquid. The bubbles rise preferentially around the column´s center, where the viscous resistance is minimal, and away from the walls resulting in an inhomogeneous radial distribution of bubbles. Close to the walls, the liquid displaced by the bubbles undergo recirculation (Ekambara and Joshi, 2005). Such recirculation is present for low superficial air velocities. Increasing the air flow beyond a characteristic threshold leads to turbulence in the liquid phase, and induces high interaction zones with locally ascending and descending liquid. In what follows, a closer look of the growth morphology of Rhizopus nigricans in bubbling columns as a function of variable superficial air velocity (0.0965x103-11.377x103 ms-1), inoculums percentage (2.5-15 % (v/v)), and initial morphology is presented. The range of air velocities selected covers both turbulent and laminar flow regimes. A simple way to characterize the hydrodynamic regime, observed in a bubbling column, is through the friction factor. The friction factor is a measure

Figure 10. Spherical spongy pellets of Rhizopus nigricans.


of the kinetic energy transferred to a solid moving parallel to the flow due to viscous effects. It can be estimated by a momentum balance, as a function of the pressure drop and the fluid kinetic energy, as long as the energy transferred due to collisions is negligible. The friction factor is generally presented as a function of the Reynolds number, characterizing two main flow regimes, laminar and turbulent, with a transitional regime between them (Bird et al., 2002). Experimentally, the friction factor can be estimated from Eq. (1), under the assumptions previously noted. The term on the left hand side of Eq. (1) corresponds to the hydrostatic pressure drop, while the term on the right is the kinetic energy that introduces the friction factor.

airair

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)(2

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In this equation, (h – h0) is the water level difference in a U-tube, ρH2O and ρair are the water and air densities, and <vair> is the superficial air velocity. Figure 11, illustrates the calculated friction factor against the superficial gas velocity. Note that the reported values are significantly higher than those reported in the literature (Bird et al., 2002). This apparent discrepancy is due to the assumptions under which the friction factor is calculated; besides the viscous character of the bubble-fluid interaction, one also accounts in this calculation for the normal stresses responsible of the bubbles rising and local mixing (Nieuwenhuys, 2003). However, the calculation is still useful for classifying the flow regime. By analogy with the classic plots (Bird et al., 2002)) one can conclude that for superficial air velocities below 3 x 10-3 ms-1 one encounters a laminar flow regime (constant slope), whereas for velocities

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Figure 11. Friction factor in bubbling column as descriptor of the flow regime.


beyond 8 x 10-3 ms-1 one observes full turbulent flow (vanishing slope), the transitional flow regime is located in between. Based on this tentative classification two superficial air velocity values for each flow regime were chosen: 0.965 X 10-3 and 3.18 X 10-3 ms-1 for the laminar regime, 5.41 X 10-3 and 7.63 X 10-3 ms-1 for the transitional regime, and 9.58 X 10 and 37 X 10-3 ms-1 for the turbulent regime. Inoculum preparation was described previously. For starter inoculum in the form of pellets the orbital agitation was operated at 100 rpms. Each flask containing100 mL PDB exhibits an average mycelia dry weight of 0.1435 +/- 0.0129 g/L. The experiments were performed sequentially gradually increasing superficial air velocity and inoculum percentage. Some experiments breaking this sequence were also performed for control. Figure 12 summarizes in graphical form the different morphological development observed. Images (a) and (b) correspond to the laminar regime. In (a) the mycelia grew in the form of spongy loose pellets; while in (b) although the pellet form persists, the pellets have lost most of their hairy appearance and they are more compact, in addition one observes the development of amorphous clumps. In the remaining flow regimes, the mycelia grows in the form of small, very compact nuclei, highly crosslinked. For these flow regimes one also observes the presence of small, compact amorphous mycelia clumps. From these results, one can conclude that the superficial air velocity, and thus the hydrodynamic regime, has a significant effect on the morphological

Figure 12. Observed morphology of cultures at different operational conditions (Superficial air velocity (m/s) and inoculum percent (v/v)). a) 9.65 x 10-4 m/s, 2.5%; b) 3.19 x 10-3, 5%; c) 5.41 x 10-3, 7.5%; d) 7.64 x 10-3, 10%; e) 9.58 x 10-3, 12.5%, f) 1.14 x 10-2, 15%.


development of the fungi. For the laminar regime the initial mycelial morphology was not significantly affected, the pellet form was conserved. As it was previously indicated, for this regime one observes segregation between ascending and descending liquid streams, due to the bubbles being localized in the central part of the column, and mostly homogenous flow inside the column (Ranade, 1997). Under these conditions, the pellets travel along the fluid bulk streams with negligible slipping velocities with respect to the surrounding liquid. The mycelia exhibit radial growth, without suffering significant erosion which can lead to fragmentation or undergoing a compactness response for diminishing their stress. For the remaining regimes, one observes the destruction of the initial pelleted forms giving rise to the presence of small compact nuclei, highly crosslinked by mycelial hair and more compact. The very few pellets that remained intact acquired a compact and ovoid form. For the turbulent and transitional regimes, one observes a globally persisting profile of recirculation flows, with intense mixing zones. In these zones, the mycelia are exposed to large velocity gradients and viscous forces. Under these conditions the mycelia are stretched and rolled into themselves, in a manner similar to a thread rubbed between the hands. The analysis over the hydrodynamic regime effect on the cultures is not complete unless we link the morphological changes to the productivity indexes. Figure 13 illustrates the cellulase production. Cellulase production decreases when the superficial air velocity and inoculum percentage are increased. In order to test the effect of morphology over the cellulase productivity, experimental essays with disperse mycelia as starter inoculum were made. The inoculum required 180 rpm on the orbital shaker to exhibit this morphology. Figure 13 also shows the cellulase production with the disperse mycelia inoculum. As it can be observed from the figure, the cellulase productivity exhibits a significant decrease with respect to the values reached with the pelleted inoculum. These results are observed also for changes in the relationship between superficial air velocity and inoculum percentage that has been used for the construction of Figure 13. In general, low superficial air velocities lead to spongy pellet morphologies with high cellulase productivity, while superficial air velocities in the turbulent regime lead to disperse mycelia with low cellulase productivity, more or less independently of the inoculum percentage used. One can conclude, then, that the superficial air velocity, and thus the hydrodynamic regime, has a dominant effect, over inoculum morphology, if one wishes to increase the cellulase productivity. Similar conclusions were reached in terms of the biomass duplication measurement. This measurement is obtained dividing the generated biomass by the initially inoculated biomass. Figure 14 reveals that biomass growth is once again controlled by the superficial surface velocity and inoculum percentage.


The effect is more significant for disperse mycelia as inoculum morphology. One can observe from Figure 14 that the biomass duplication reaches a constant value for turbulent flows and disperse mycelia as initial inoculum morphology.

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Figure 13. Effect of superficial air velocity and inoculum percentage on cellulase productivity. Two different initial morphologies were used: pellets and disperse mycelia.

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Figure 14. Effect of superficial air velocity and inoculum percentage on biomass duplication. Two different initial morphologies were used: pellets and disperse mycelia.


In summary, the hydrodynamic regime has a significant effect on filamentous fungi morphology and productivity variables. However, although mechanical interactions on cultures appear to play a secondary role, they are not fully understood and may yet prove to be important in improving the culture productivity.

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