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NOTES Pellet Formation and Fragmentation in Submerged Cultures of Penicillium chrysogenum and Its Relation to Penicillin Production Jens NieIsen,*Claus L. Johansen, Michael Jacobsen, Preben Krabben, and John Villadsen Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark
The spores of Penicillium chrysogenum are of the noncoagulating type, and afier spore germination a culture of disperse mycelia is obtained. In this study, it is shown that when the hyphal elements increase in size, they may agglomerate, and depending on the operating conditions, these agglomerates may develop into pellets with a dense core. The influence of initial spore concentration and agitation rate on agglomeration, leading to pellet formation, was studied. For a low concentration of spores in the inoculum, only a few hyphal elements agglomerate and pellets with a small diameter are obtained. At higher spore concentrations, many hyphal elements agglomerate and develop into large diameter pellets. Finally, at a very high spore concentration in the inoculum, the final hyphal element size is small and agglomerates therefore are not formed. With a high agitation rate, the agglomeration of hyphal elements is reduced. In a repeated fed-batch cultivation, where there was a shift from pellet morphology to disperse mycelia, it was found that there is no relation between macroscopic morphology and penicillin production by P. chrysogenum. The morphology was quantified throughout the repeated fed-batch cultivation, and both the pellet diameter and the concentration of pellets were affected by the agitation rate.
Introduction During the submerged growth, many filamentous fungi may grow either as free mycelia or as pellets, and the growth form is determined by a number of factors, e.g., the pH of the medium, the power input, the spore concentration in the inocolum, and the presence of surfactants (Pirt and Callow, 1960; Metz and Kossen, 1977; Braun and Vecht-Lifshitz, 1991). The exact mechanism behind pellet formation is not known, and the mechanism probably varies from species to speciesperhaps even from strain to strain. However, two types of pellet-forming microorganisms are traditionally recognized (Takahashi and Yamada, 1959): (1)the coagulating type and (2) the noncoagulating type. For the coagulating type, spores coagulate in the early stage of the cultivation, germinate, and gradually develop into pellets. For the noncoagulating type, a single spore is able to develop into a pellet, as recently illustrated by King and co-workers for a Streptomyces species (Yang et al., 1992). Penicillium chrysogenum is normally considered to be of the noncoagulating type (Takahashi and Yamada, 1959; Metz, 19761, but the development of pellets from hyphal elements has been observed (Pirtand Callow, 1960; van Suijdam et al., 1980). Recently, Thomas and co-workers showed that during submerged cultivations with P. chrysogenum, loose clumps of mycelia are formed by the agglomeration of hyphal elements (Packer and Thomas, 1990; Tucker et al., 1992). Pellets of P. chrysogenum therefore are likely to be formed as a result of the agglomeration of several hyphal elements to.form loose clumps, which may gradually develop into a pellet with a dense core due to growth of the hyphae 8756-7938/95/3011-0093$09.00/0
within the clumps. Agglomeration of hyphal elements is determined both by the properties of the individual hyphal elements, e.g., the total hyphal length, the branching pattern, and the cell wall surface, and by the environmental conditions, e.g., the medium composition and the shear forces acting on the clumps. In studies of pellet formation, it is therefore necessary to include an analysis of the development of freely dispersed mycelia from spores. This paper communicates the results from studies of the formation of pellets in submerged cultures of P. chrysogenum. Image analysis was used to describe the development of mycelia, the agglomeration of hyphal elements to clumps, and finally the formation of pellets from the loose clumps. The mechanisms for pellet formation in submerged cultures of P. chrysogenum are discussed on the basis of experimental results. Furthermore, the morphology of P. chrysogenum was quantified during a repeated fed-batch cultivation lasting more than 450 h, and the relation between macroscopic morphology and penicillin production is discussed.
Materials and Methods Strain. The strain of Penicillium chrysogenum used
was donated by Novo Nordisk NS (Bagsvaerd, Denmark). It is a high-yielding (former production) strain giving about 20-25 glL penicillin V after 200 h of fed-batch cultivation (Johansen, 1993). Freeze-dried spores were used to inoculate rice cultures, and 15-20 days after inoculation, the rice grains were covered with a thick layer of spores. The inoculum for the submerged cultivations was taken from these cultures by washing the grains with sterilized water, whereafter the spore con-
0 1995 American Chemical Society and American Institute of Chemical Engineers
Biotecbnol. Prog., 1995, Vol. 11, No. 1
94 Table 1. Conditions in the Five Batch Cultivations" inoculum agitation rate cultivation (spored.,) medium (rpm) CULT4 3.5 107 complex 500 CULT5 3.7 x 107 complex 500 CULT9 8.6 x lo7 defined controlled BO27 6.0x 107 complex controlled BO28 2.4 107 complex controlled a
From Johansen (1993).
centration in the suspension was quantified in a Thoma counting chamber. Media. One batch cultivation (CULTS) was carried out in a defined medium, and four batch cultivations (CULT4, CULT5, B027, and B028) were carried out in a complex medium. The defined medium contained 25 g/L eucrose, 1.6 g/L KH2P04, 7.0 g/L (NH&S04, 0.04 g/L FeSOc7Hz0,O.l g/L MgS04-7Hz0,0.5 g L KC1,0.05 I , trace metal CaClz*2Hz0,0.2mLJL pleuronic, and 5 " solution. The trace metal solution contained 1.0 g/L CuSO&Hz0,4.0 g/L &'&04*7HzO,and 4.0 g/L hhso&o. The complex medium contained 50 g/L corn steep liquor, 30 g/L sucrose, 2.0 g/L KH2P04,lO.O g/L (NH.&S04,0.06 g/L CaC12-2Hz0, and 0.2 m L n pleuronic. One repeated fed-batch cultivation (FB027)was carried out in a complex medium containing 100 g/L corn steep liquor, 3.0 g/L sucrose, 5.7 g/L phenoxyacetic acid (as a precursor for penicillin V production), 1.0 g/L KHzPO~, 12.0 g/L (N&)2SO4, 0.06 g/L CaClz.2Hz0, and 0.2 " I , pleuronic. The feed contained 450 g/L glucose, 33.3 (N&)zS04, and 33.3 g/L phenoxyacetic acid. Cultivation Conditions. All cultivation experiments were carried out in a standard 41 L Chemap bioreactor equipped with three Rushton turbines and having an aspect ratio of 3. The working volume was 25 L. The temperature was kept at 25 "C, the head space pressure was 1.5 bar, and the aeration rate was 1wm. An overview of the cultivation conditions for the five batch experiments is shown in Table 1. In two of the batch cultivations (CULT4 and CULT5) the agitation rate was kept constant at 500 rpm, whereas in all other experiments the dissolved oxygen tension was kept constant at 45% of saturation by set point control of the agitation rate. However, the agitation rate was never below 175 rpm. The batch cultivations were inoculated with spores of approximately constant age to the concentrations shown in Table 1. The repeated fed-batch cultivation was inoculated with 4 kg of medium from the batch cultivation B027. The mode of operation was switched from batch to fed-batch when the glucose and sucrose were exhausted (48 h after inoculation of the batch). After 144 and 286 h of cultivation, respectively, 80% of the spent medium was withdrawn and replaced with fresh medium (same composition as the initial medium). The feed profile during the repeated fed-batch cultivation was as follows: 0-6 h, no feed; 6-48 h, 70 gh;48-144 h, 90 gh;144-168 h, 70 g/h; 168-286 h, 90 g/h; 286-310 h, 70 g/h; 310-406 h, 90 g/h. During the cultivations, cell-free samples were collected by means of an in situ membrane module from ABC (Munich, Germany) (Christensen et al., 1991). The cell-free samples were collected in a fraction collector placed in a refrigerator and analyzed off-line. Analysis. The biomass concentration was measured by filtering the sample on a sterile, dry, preweighed filter, followed by drying in an oven for 24 h at 105 "C and measurement of the weight gain. The exhaust gas was analyzed for oxygen and carbon dioxide by paramagnetic (Magnos 6G) and infrared (Uras 3G) analysis (both from Hartmann & Braun, Germany), respectively. Penicillin
V was measured by HPLC as described in Christensen et al. (19941, using analytical grade K-penicillin V as a standard. Quantification of the Morphology. The morphology was quantified using a manual image analysis system consisting of a microscope (Nikon Optiphot-2), a CCD camera, a PC with a frame-grabber, and image analysis software (Image H o w , Copenhagen, Denmark). The CCD camera captured images of 512 x 512 pixels, each with a grayness (brightness) level from 0 (representing black) to 255 (representing white). Normally a magnification of 2000 was used, and with this magnification the standard deviations on measurements of the same hyphal element were found to be less than 1%for the total hyphal length and less than 5% for the hyphal diameter. Average values were obtained from the measurement of 50 individual hyphal elements. This gave a relative standard deviation below 5% for the average total hyphal length. For each sample, the morphology of 100 pellets was characterized by measuring the area and the perimeter of the pellet core and the maximum diameter of the pellet. The pellet core was analyzed using a semiautomated procedure. The pellet core was bracketed within a certain grayness interval, which was subsequently amplified by means of the image analysis software. The equivalent core diameter was calculated from the core area, and the core circularity was calculated from (Cox and Thomas, 1992) core circularity =
(perimeter of the core12 4 4 a r e a of the core)
The pellet concentration (the number of pellets per volume medium) was determined using a counting chamber.
Experimental Results Batch Cultivations. The morphology was studied in each of the five batch cultivations. Typical profiles for the biomass concentration and the carbon dioxide evolution rate are shown in Figure 1A for CULT4 and CULT5. These two cultivations were carried out at the same operating conditions (both with a constant agitation rate of 500 rpm), and the initial spore levels were approximately the same. Apart from some scatter in the biomass concentration measurements (these measurements of biomass in a viscous medium containing clumps of solid feed material are very diflicult to handle), the reproducibility is very satisfactory; thus, even the details of the carbon dioxide evolution profile are mimicked in the two independent experiments. CULT5 was further analyzed in Nielsen et al. (1994) with respect to the cellular metabolism. Results of the morphological characterization of the two batch cultivations CULT4 and CULT5 are shown in Figure 1B. ARer spore germination, the average total hyphal length increased exponentially with a specific growth rate of 0.20 h-l, which corresponds to the maximum specific growth rate for the applied strain (Nielsen and Krabben, 1994). ARer approximately 32 h of cultivation, significant clump formation occurred as a result of agglomeration of the large hyphal elements in the culture. This resulted in a rapid decrease in the average total hyphal length of the remaining population of freely dispersed mycelia. Thomas and co-workers (Packer and Thomas, 1990; Tucker et al., 1992) also observed a decrease in the average total hyphal length when the
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Time [h] Figure 1. Batch cultivations CULT4 and CULT5 (A) Measurements of the biomass concentration (g of DWL) and the carbon dioxide evolution rate (CER, mmol/l/h): (0) biomass concentration during CULT4;).( biomass concentration during CULT5. (B) Measurements of the average total hyphal length and the pellet concentration: (0)average total hyphal length during CULT4;).( average total hyphal length during CULT5; (*) pellet concentration during CULT4; (A)pellet concentration during CULT5. The lines represent the exponential growth of the average total hyphal length and a linear increase in the pellet concentration, respectively.
fraction of clumps increased in a submerged culture of P. chrysogenum. Shortly after the formation of clumps, pellets appeared in the culture. The first pellets were no more than dense clumps, but gradually pellets with a spherical, dense core emerged. After the appearance of the first pellets, the pellet concentration continued to increase linearly for another 15 h, as shown in Figure 1B. The final pellet concentration was about 1.5 x lo7 pelletsL If it is assumed that all of the spores germinate, which may be reasonable in a complex medium (Nielsen and Krabben, 19941,there are approximately two hyphal elements in each pellet. If some fraction of the spores fails to develop into hyphae, the final pellet concentration is even closer to the spore concentration in the inoculum (see Table 11, which may explain why early studies of P. chrysogenum characterized the organism to be of the noncoagulating type. Figure 2A shows the profiles of the CER and the agitation rate during the two fixed oxygen tension batch cultivations BO27 and B028. The higher spore concentration used for the inoculation of BO27 compared with BO28 gives faster progress of the cultivation. However, spore germination occurred a t approximately the same
100 0
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Time [h] Figure 2. Batch cultivations BO27 and B028. (A) Carbon dioxide evolution rate (CER, mm0lA.h) and agitation rate. (B) Measurements of the average total hyphal length and the pellet concentration: (0)average total hyphal length during B027;).( average total hyphal length during B028; (A)pellet concentration during B027. The lines represent the exponential growth of the average total hyphal length in BO28 and a linear increase of the pellet concentration in BO27 after 33 h, respectively.
time in the two experiments. Except for the time difference, the profiles for the CER and the agitation rate are very similar for the two experiments. The results of the morphological characterization of these two batch cultivations are shown in Figure 2B. During B028,the morphology developed very similar to that observed in the fxed agitation rate cultivations CULT4 and CULT5 (pellet formation occurred after 35 h; data not shown). Hence, from the lower limit 175 rpm to 500 rpm, the agitation rate has no significant influence on the morphology during the rapid growth phase. In BO27 some incipient agglomeration of hyphal elements was observed already after 29 h of cultivation, and the measurements of the free mycelia therefore were not representative for the population. This explains the very different profile of the average total hyphal length during BO27 compared with the other batch cultivations. The fluffy clumps could not be dispersed even after dilution and vigorous mixing of the sample, but true pellet formation with a dense pellet core did not develop until 35 h after the start of the cultivation, indicating that the time required for full pellet morphology to appear is approximately constant for the strain. Since the specific growth rate of the hyphal elements is likely to be the same when they are agglomerated and when they are dispersed freely in the medium, the average total hyphal length a t the time of pellet formation was probably
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Time [h] Figure 3. Equivalent pellet core diameter during three batch cultivations. CULT4 and BO27 were carried out on the complex medium and CULT9 was carried out on a defined medium.
approximately the same in BO27 and B028. Thus, the higher spore concentration used in BO27 increases the probability of agglomeration of hyphal elements, leading to an early formation of clumps, but dense core pellets are formed only when the average total hyphal length of the hyphal elements (whether they are present as freely dispersed mycelia or in fluffy clumps) has reached a certain size. Figure 3 shows the equivalent pellet core diameter during the three batch cultivations CULT4, CULTS,and B027. The equivalent pellet core diameter increased linearly with approximately the same rate in all three cultivations, but the initial core diameter was different. In CULT4, where the pellets were formed almost directly from hyphal elements, the pellet core was initially very small, whereas in B027, where the pellets were formed from "older" clumps, which may have been more dense, the pellet core was initially larger. Furthermore, on an average basis, the pellets in BO27 originated from agglomerations of approximately four hyphal elements, whereas the pellets in CULT4 originated from the agglomeration of at most two hyphal elements. In CULTS, the spore concentration was even higher than that in B027, but on the defined medium, a smaller fraction of the spores germinated (Nielsen and Krabben, 19941, and the maximum concentration of disperse hyphal elements is very likely to be smaller than that in B027. Throughout all of the batch experiments, the ratio between the outer pellet diameter and the equivalent pellet core diameter was about 2, but with a tendency to decrease at the end of the cultivations where the pellets became larger and less flu@. Fed-Batch Culkivation. The macroscopic morphology was studied during a repeated fed-batch cultivation (FB027). The inoculum was biomass from the batch cultivation B027, which was present mainly as pellets. After 144 and 286 h of cultivation, respectively, 80% of the spent medium was withdrawn and fresh medium was added, whereafter the fed-batch process was continued. Figure 4A shows the time course of the biomass and penicillin concentrations. In each of the three growth periods, there was a rapid growth phase of approximately 20 h, where amino acids originally present in the complex medium were consumed, and thereafter the biomass concentration increased approximately linearly to about 40 g/L at about 80 h of cultivation. After 80 h, the biomass concentration stayed at a constant level. Initially the penicillin V concentration increased very slowly, but after 20 h of cultivation it increased approximately linearly throughout the first growth period to about 18
= I
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Figure 4. Repeated fed-batch cultivation FB027. (A) Measurement of the biomass and the penicillin V concentration: (0) biomass concentration (g of DWL); (A)penicillin V concentration (g/L). (B) Pellet concentration and equivalent core diameter: (m) pelIet concentration (mL-l); (0)equivalent core diameter (mm). g/L. In the second period the volumetric rate of production was slightly lower, but the specific penicillin production rate was approximately the same as in the first period. In the third and final period the penicillin production was very low, and at the end the concentration of penicillin V decreased due to degradation to penicilloic acid. The low productivity must be in some way connected with a degeneration of the cells, since the environmental conditions were approximately the same in the three periods (Johansen, 1993). Pellet concentration and the equivalent pellet core diameter are shown in Figure 4B for the whole repeated fed-batch experiment. The inoculum was a culture of pellets giving a pellet concentration of about 4000 mL-l at t = 0. In the initial phase with rapid growth, the pellet concentration increased to a level of about 5000 pellets/ mL, and in the same period the equivalent pellet diameter increased to about 0.4 mm. In the linear growth phase the pellet concentration decreased slowly to about 4000 pellets/mL, and thereafter the pellet concentration decreased rapidly to about 500 pellets/mL a t the end of the first period. T h i s decrease is explained by the breakup of the pellets due to substrate limitation in the center of the pellet, resulting in cell lysis and hereby loss of structural stability. In the second period there was an initial rapid increase in the pellet concentration, but only to a lev4 of about 1000 pelletdml. Thereafter, the pellet concentration decreased slowly down to about 700 pellets/mL a t the end of the second period. In the third
Biotechnol. Pfog., 1995, Vol. 11, No. 1
Equivalent pellet dlameter [mm]
Figure 5. Histograms representing the distribution of the equivalent core diameter during the first period of repeated fedbatch cultivation FB027. Open bars represent the distribution after 5 h of cultivation, and filled bars represent the distribution after 110 h of cultivation.
period, no new pellets were formed and the pellet concentration steadily decreased to below 100 pelletdml. After the initial increase, the equivalent pellet diameter stayed approximately constant at 0.4 mm throughout the rest of the experiment, except for small increases a t the beginning of the second and third periods. In the submerged culture, the equivalent pellet diameter was determined by growth and by fragmentation of hyphae growing out from the pellet. In the beginning of each period there was rapid growth, which explains the increase in the equivalent pellet diameter. Later on in each period the specific growth rate was low, and the equivalent pellet diameter therefore was determined mainly by fragmentation. Pellet fragmentation has been modeled by van Suijdam and Metz (19811, and they introduced the concept of an equilibrium pellet diameter, which is the minimum pellet diameter that fragments due to the action of shear forces. Thus, pellets with a size below this equilibrium value will not fragment. This equilibrium pellet diameter is determined by the agitation rate, which was low (about 300 rpm) at the beginning of each period and higher (500-600 rpm) at the end of each period. Thus, at the beginning of each period, the equilibrium pellet diameter increased and at the same time pellet growth was faster than pellet fragmentation. This resulted in an overall increase in the pellet diameter. With the applied bioreactor and an agitation rate of about 500 rpm, the equilibrium pellet diameter seems to be 0.4 mm. Fragmentation of hyphae growing out from the pellet resulted in almost spherical pellets, as seen by a significant decrease in the core circularity from an initial value of about 3.5 to 1.5 a t the end of the first period. In the second and third periods, the core circularity increased slightly in the beginning, indicating the formation of more fluffy pellets during the rapid growth phase (as discussed earlier, fragmentation was also reduced a t the beginning of these two stages). However, when the growth slowed down, the core circularity again decreased to about 1.5. The fragmentation of pellets resulted in a skewed pellet size distribution, as seen from the histograms for 5 and 110 h of cultivation (see Figure 5). The distributions can be described quite well with a log-normal distribution (Reichl et al., 1992). Initially the pellet distribution was quite narrow, wheareas later in the cultivation the spread was larger. With a large variance, more pellets have to
97
be measured in order to obtain a good estimate (i.e., an estimate with a low standard deviation) of the average pellet diameter. However, measurements of about 100 pellets generally are sufficient to obtain a good estimate of the average pellet diameter. At the beginning of the first period, most (more than 95%) of the biomass was present as pellets. In the rapid growth phase the pellet diameter stayed more or less constant and the number of pellets per volume medium decreased. Hence, the fraction of biomass present as pellets decreased drastically. This decrease continued through the second and third periods, and at the end of the experiment a very low fraction of the biomass (less than 5%) was present as pellets. Thus, during the repeated fed batch, an increasing fraction of the biomass was present as disperse mycelia, with a morphology that was approximately constant throughout the experiment. The average total hyphal length was about 200 pm, and the average number of tips was about four per hypha. The relatively small size of the disperse mycelia was due to fragmentation of the hyphal elements, and there was an equilibrium size for the hyphal elements as for the pellets. For the repeated fed-batch experiment, the equilibrium hyphal length was around 200 pm. Fragmentation resulted in the formation of inactive tips, but for many of the hyphal elements outgrowth from septa was observed.
Discussion For P.chrysogenum, pellet formation is found to occur by the agglomeration of disperse mycelia, and the number of hyphal elements in a pellet is on the order of 2-4, depending on the spore concentration. This corresponds with observations for Aspergillus niger, even though pellet formation of this organism is by the agglomeration of a large number of spores (Takahashi et al., 1958). The initial pellet diameter is determined by the number of hyphal elements that agglomerate. Thus, the initial pellet diameter is found to increase with spore concentration. This is different from what is observed for microorganisms where pellets are formed by the agglomeration of spores (Takahashi et al., 1958; Vecht-Lifshitz et al., 1990). As reported by Tucker and Thomas (19921, it is found that no (or very few) pellets are formed at high spore concentrations (above lo8 spores/L), but some agglomeration is observed at low agitation rates. Thus, on the basis of our present experimental data, it is concluded that agglomeration leading to pellet formation is not simply determined by the probability of physical contact between hyphal elements. When a fed-batch culture was inoculated with a culture of pellets, an initial increase in the pellet concentration was observed. However, when the specific growth rate of the biomass decreased, the pellet concentration decreased rapidly due to breakup of the pellets. This breakup was most likely caused by cell lysis within the pellets, whereby the stability of the pellet was lost. Besides pellet breakup, hyphal elements were torn off at the pellet surface. This process determined the pellet size, and for the applied strain relatively small pellets resulted (with a diameter of about 0.4 mm). Due to the pellet breakup and the loss of hyphal elements from the pellet surface, a drastic change in the macroscopic morphology was observed during the first and second periods of a repeated fed-batch cultivation, i.e., there was a shift from a pellet culture to a culture of disperse mycelia. However, the specific penicillin productivity was found to be approximately the same in these two growth periods, and it is therefore concluded that the macro-
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scopic morphology, i.e., whether the culture is in the form of pellets or disperse mycelia, has no direct influence on the penicillin productivity. In large-scale production processes there may, however, be an indirect influence since, at the same biomass concentration, a culture of pellets is much less viscous than a culture of disperse mycelia, and problems associated with insufficient gasliquid mass transfer are therefore reduced.
Literature Cited Braun, $.; Vecht-Lifshitz, S. E. Mycelial morphology and metabolite production. Trends Biotechnol. 1991, 9,63-68. Christensen, L.H.; Nielsen, J.; Villadsen, J. Delay and dispersion in an in-situ membrane probe for bioreactors. Chem. Eng. Sci. 1991,46,3304-3307. Christensen, L. H.; Mandrup, G.; Nielsen, J.; Villadsen, J. A robust LC method for studying the penicillin fermentation. Anal. Chin. Acta 1QS4,in press. Cox, P. W.; Thomas, C. R. Classification and measurement of fungal pellets by automated image analysis. Biotechnol. Bweng. 1992,39,945-952. Johansen, C. L. Monitoring and modelling of the penicillin fermentation. Ph.D. Thesis, Technical University of Denmark, Lyngby, Denmark, 1993. Metz, B. From pulp t o pellets. Ph.D. Thesis, Technical University of Delft, Delft, The Netherlands, 1976. Metz, B.; Kossen, N. W. F. The growth of molds in the form of pellets-A literature review. Biotechnol. Bioeng. 1977,19, 781-799. Nielsen, J. A simple morphologically structured model describing the growth of filamentous microorganisms. Bwtechnol. Bioeng. 1993,41,715-727. Nielsen, J.; Krabben, P. Hyphal growth and fragmentation of P. chrysogenum in submerged cultures. Bwtechnol. Bweng. 1944,submitted for publication. Nielsen, J.;Johansen, C. L.; Villadsen, J. Culture fluorescence measurements during batch and fed-batch cultivations with Penicillium chrysogenum. J.Biotechnol. 1994,submitted for publication.
Packer, H. L.; Thomas, C. R. Morphological measurements on filamentous microorganisms by fully automatic image analysis. Biotechnol. Bioeng. 1990,35,870-881. Pirt, S. J.; Callow, D. S. Continuous-flow culture of the filamentous mould Penicillium chrysogenum and the control of its morphology. Nature 1969,184,307-310. Reichl, U.;King, R.; Gilles, E. D. Characterization of pellet morphology during submerged growth of Streptomyces ten& by image analysis. Biotechnol. Bweng. 1892,39,164-170. Takahashi, J.;Yamada, K Studies on the effect of some physical conditions on the submerged mold culture. Part 11. On the two types of pellet formation in the shaking culture. J.Agric. Chem. SOC.1969,33,707-709. Takahashi, J.; Yamada, K; Aasai, T. Studies on the effect of some physical conditions on the submerged mold culture. Part I. The process of pellet formation of Asp. niger under the shaking culture, and the effect of the inoculum size on the shape of pellet. J.Agric. Chem. Soc. 1968,32,501-506. Tucker, K. G.; Thomas, C. R. Mycelial morphology: The effect of spore inoculum level. Biotechnol. Lett. 1992,14, 10711074. Tucker, K. G.; Kelly, T.; Delgrazia, P.; Thomas, C. R. Fullyautomatic measurement of mycelial morphology by image analysis. Bwtechnol. Prog. 1992,8, 353-359. van Suijdam, J. C.; Kossen, N. W. F.; Paul, P. G. An inoculum technique for the production of fungal pellets. Eur. J.Appl. Microbiol. 1980,10,211-221. van Suijdam, J. C.; Metz, B. Fungal pellet breakup as a function of shear in a fermentor. J.Ferment. Technol. 1981,59,329333. Vecht-Lifshitz, S.E.;Magdassi, S.; Braun, S. Pellet formation and cellular aggregation in Streptomyces tendae. Biotechnol. Bioeng. 1990,35,890-896. Yang, H.;Reichl, U.; King, R.; Gilles, E. D. Measurement and simulation of the morphological development of filamentous microorganisms. Biotechnol. Bioeng. 1992,39,44-48. Accepted July 5, 1994.@ Abstract published in Advance ACS Abstracts, September 1, 1994. @
