Rheological properties of culture broth of Acremonium … · 2010. 4. 8. · Abstract The effects of glass ... best fluid models have been sought through the compar- ... Korea-Australia

Korea-Australia Rheology Journal March 2010 Vol. 22, No. 1 51

Korea-Australia Rheology JournalVol. 22, No. 1, March 2010 pp. 51-58

Rheological properties of culture broth of Acremonium chrysogenum M35 in

baffled flasks with glass beads

Hwan Hyo Lee, Yoon Seok Song, Jeong Yong Lee, Hyun Wook Jung and Seung Wook Kim*

Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Korea

(Received October 1, 2009; final version received December 25, 2009)

Abstract

The effects of glass beads on the rheological properties of culture broths of Acremonium chrysogenum M35grown in baffled flasks was investigated in this study. The addition of glass beads to cultures of A. chrysoge-num M35 led to considerably higher levels of shear rheological properties such as shear stress versus shearrate, which are essential for controlling the performance of culture broths. The shear properties of culturebroths of A. chrysogenum M35 evaluated in this study were also found to be better predicted by the power-law and the Herschel-Bulkley fluid models than by the Bingham plastic and the Casson fluid models. Theconsistency index (K) and the flow behavior index (n) data from the power-law fluid model for various cul-ture broths clearly substantiated the usefulness of glass beads for effectively promoting the cell growth andcell differentiation, because they were directly affected by the morphological changes of A. chrysogenumM35 induced by the glass beads.

Keywords : Acremonium chrysogenum, rheological models, glass beads, shear stress, shear viscosity, mor-

phology

1. Introduction

Acremonium chrysogenum is an important fungi for the

production of cephalosporin C (CPC), which is a β-lactam

antibiotic with some biological activity (Demain and

Elander, 1999; Kumar et al., 2008; Lejon et al., 2008;

Junker et al., 2009). The morphological differentiation of

A. chrysogenum in culture broth is intimately related to

CPC production. In addition, CPC is usually activated

against both gram-positive and gram-negative bacteria

(Suárez et al., 2008; Sundsfjord et al., 2008).

The morphology of cells is critically influenced by the

limiting nutrient, viscosity and dilution rate, which all play

a key role in determining the differentiation of filamentous

fungi (Lee et al., 2001; Schmitt et al., 2004; Zahar et al.,

2009). The morphology of A. chrysogenum is character-

ized by three features; hyphae, swollen hyphal fragments,

and arthrospores. Interestingly, the differentiation of

hyphae into highly swollen hyphal fragments obviously

occurs prior to the production of CPC, and highly swollen

hyphal fragments are gradually differentiated into

arthrospores during CPC production (Matsumura et al.,

1980; Grimm et al., 2005).

The systematic investigation on the morphological

change of A. chrysogenum is academically and industrially

important because it affects the rheological properties of

the fermentation broth and consequently its mass and heat

transfer characteristics (Olsvik and Kristiansen, 1992). For

instance, free filamentous form can lead to viscous fer-

mentation broths with pseudoplastic or shear thinning

nature, whereas the pelletized form ensures Newtonian

behavior with low viscosity (Karaffa et al., 1977; Bhargava

et al., 2005).

The rheological behavior of culture broth, which is influ-

enced by the morphological change and biomass concen-

tration, is indispensable to enhance the yield of the desired

product because the morphological differentiation of A.

chrysogenum in culture broth is closely related to its rheo-

logical properties (Riley et al., 2000; Cho et al., 2002).

While the basic understanding about morphological and

rheological properties in cultures of A. chrysogenum has

been greatly advanced (Oncu et al., 2007; Petersen et al.,

2008), there still remains the need to eloquently establish

the relationship between both properties for high produc-

tivity of CPC.

It has also been reported that changes in the composition

and structure of cell walls may be closely linked with the

resistance to shear (Olsvik and Kristiansen, 1994). To

enhance CPC production, morphological characteristics

and stimulation factors in the fermentation process should

be simultaneously considered to control the flow behavior

of culture broths.

In this study, experiments were conducted to elucidate*Corresponding author: kimsw@ korea.ac.kr© 2010 by The Korean Society of Rheology

Hwan Hyo Lee, Yoon Seok Song, Jeong Yong Lee, Hyun Wook Jung and Seung Wook Kim

52 Korea-Australia Rheology Journal

the influence of glass beads on morphological changes and

rheological properties of A. chrysogenum M35 cultures in

baffled shake-flasks. Furthermore, rheological models to

accurately delineate the cultivation of A. chrysogenum

M35 were compared.

2. Materials and methods

2.1. StrainA. chrysogenum M35 mutated from A. chrysogenum

ATCC 20339 was used for this study (Kim et al., 2006).

2.2. Media and culture conditionsThe basal seed medium was composed of 2.5% sucrose,

1.0% glucose, 2.5% corn steep liquor, 0.4% (NH4)2SO4,

3.0% soy bean meal, 1.0% cotton seed flour and 0.5%

CaCO3 (Lee et al., 2001). The main medium consisted of

1.95% glucose, 5% corn steep liquor, 0.8% (NH4)2SO4,

0.3% KH2PO4, 0.5% K2HPO4, 0.5% DL-methionine and

0.4% trace element solution (Lee et al., 2001a; Cuadra et

al., 2008).

Sugars, corn steep liquor (CSL), (NH4)2SO4 and DL-

methionine were sterilized separately from the other com-

ponents. CaCO3 was added after setting the pH to 7.0 prior

to sterilization. Finally, 4% (v/v) linoleic acid was added to

the main medium, which was already proven to remark-

ably elevate the CPC production (Kim et al., 2006; Kim et

al., 2007).

A. chrysogenum M35 was cultured in advance in a 2 L

Erlenmeyer flask containing 200 mL of the basal seed

medium at 27oC while shaking at 280 rpm for 72 hours.

Next, A. chrysogenum M35 was cultured in 250 mL baf-

fled shake-flasks containing 10 mL of the main medium on

a rotary shaking incubator (Rhee et al., 2003). Flask cul-

tures were agitated with different numbers of glass beads

(glass bead 3, Glastechnique Mfg., Germany) at 130 rpm

and 27oC (Rhee et al., 2003; Tollnick et al., 2004).

2.3. Measurement of rheological propertiesRheological properties such as shear viscosity and shear

stress were measured using an AR 2000 controlled stress

rheometer (TA instruments). The effect of glass beads on

the rheological behavior of culture broths has been quan-

titatively scrutinized from the shear stress data plotted

against shear rate and fluid constitutive models. Among

many possible fluid models, the Bingham (Eq. (1) in shear

flow), Casson (Eq. (2)), power-law (Eq. (3)) and Herschel-

Bulkley (Eq. (4)) models have primarily been employed to

scrutinized rheological properties in submerged cultures of

microorganisms (Kim et al., 2005; Kim et al., 2006). The

best fluid models have been sought through the compar-

ison of aforementioned models for predicting the rheo-

logical behavior of the culture broths with A. chrysogenum

M35 (Chu and Constantinides, 1988; Lim et al., 2002; Pol-

lard et al., 2002; Kim et al., 2005)

Bingham: (1)

Casson: (2)

Power law: (3)

Herschel-Bulkley: (4)

where τ denotes the shear stress, the shear rate, the

Bingham yield stress, the plastic viscosity, the Cas-

son yield stress, the Casson viscosity, K the consis-

tency index, n the flow behavior (power-law) index,

the Herschel-Bulkley yield stress, the Herschel-Bulk-

ley consistency index and the Herschel-Bulkley flow

behavior index (Pollard et al., 2002).

3. Results and discussion

In general, the flow behavior of the culture broths hinges

on the type of microbes, media and cultivation conditions.

Indeed, the cell growth and metabolite production exhibit

interestingly different patterns among basal seed medium

according to the composition of culture media and culture

conditions (Lee et al., 2001; Rhee et al., 2003).

The typical morphology of A. chrysogenum M35 in a

250 mL baffled shake-flask with glass beads was photo-

graphed on the fourth day, when the growth of culture

broths is actively maximized, using a camera mounted on

an optical microscope (Fig. 1). The morphological char-

acteristics of cells attested a wide variety of differentiation

patterns in baffled flasks with glass beads. That is, the cul-

τ τy B,– µPγ·

=

τ0.5

τy C,( )0.5

µP C, γ·0.5

+=

τ Kγ·n

=

τ τy HB,KHBγ

·HB

n+=

γ·

τy B,

µP τy C,

µP C,

τy HB,

KHB

nHB

Fig. 1. Typical morphology of A. chrysogenum M35 after four

days of cultivation in a 250 mL baffled shake-flask with

(a) no glass beads, (b) 2 glass beads, (c) 4 glass beads and

(d) 6 glass beads.

Rheological properties of culture broth of Acremonium chrysogenum M35 in baffled flasks with glass beads


ture broth of A. chrysogenum M35 grown in baffled flasks

with glass beads contained many swollen hyphal fragments

and arthropores. Furthermore, the dispersion of A.

chrysogenum M35 in the center of the cell pellets was

greatly improved by the addition of glass beads.

Fig. 2 displays plots of the shear stress versus the shear

rate of culture broths grown in the main media containing

different numbers of glass beads. The results revealed that

the culture broth had a non-Newtonian (shear thinning)

nature and also its shear stress is raised as increasing glass

beads. In the main medium without glass beads, as cell

growth and differentiation occurred, the shear stress of the

culture broth increased until the sixth day, at which point

the swollen hyphal fragments differentiated into

arthrospores (Fig. 2(a)). When 6 glass beads were added to

the culture broth, the maximum value of the shear stress

was significantly increased up to 1.61 Pa (Fig. 2(d)). Espe-

cially, the values of the shear stress in cultures that include

4 or 6 glass beads increased abruptly on the third day (Figs.

2(c) and (d)).

These rheological data were also implemented to seek

the fluid model for satisfactorily evaluating the flow

behavior of culture broths, among the four different non-

Newtonian viscous models (Figs. 3~6). The Bingham plas-

tic model fitted the experimental data well until the fourth

day; however, it could not predict the data after the fifth

day (Fig. 3). Fig. 4 represents the shear stress-shear rate

plots of the culture broths as determined by the Casson

model, showing that the Casson model is a little better than

the Bingham plastic for prediction of the cultivation of A.

chrysogenum M35 in baffled flasks containing glass beads.

The power-law model, which is one of the most useful

models in transport systems, supports the great rheological

estimation of the cultivation of A. chrysogenum M35 with

and without glass beads (Fig. 5). Finally, the Herschel-

Bulkley equation also well reflected the experimental data

throughout the entire experimental period for samples with

and without glass beads (Fig. 6).

Fig. 2. Rheological properties (shear stress vs. shear rate) of the main culture broths in baffled flasks with (a) no glass beads, (b) 2 glass

beads, (c) 4 glass beads and (d) 6 glass beads at different culture times.



Fig. 3. Prediction of rheological properties of the main culture broths in baffled flasks by the Bingham model with (a) no glass beads,

(b) 2 glass beads, (c) 4 glass beads and (d) 6 glass beads at different culture times.

Fig. 4. Prediction of rheological properties of the main culture broths in baffled flasks by the Casson model with (a) no glass beads,




Fig. 5. Prediction of rheological properties of the main culture broths in baffled flasks by the power-law model with (a) no glass beads,


Fig. 6. Prediction of rheological properties of the main culture broths in baffled flasks by the Herschel-Bulkley model with (a) no glass

beads, (b) 2 glass beads, (c) 4 glass beads and (d) 6 glass beads at different culture times.



The rheological study convinced that the power-law and

the Herschel-Bulkley models remarkably portrayed the

rheological profiles of the culture broths of A. chrysoge-

num M35 in baffled shake-flasks with glass beads (Figs. 5

and 6) in contrast to the Bingham plastic and the Casson

models. For instance, the consistency index (K) and the

flow behavior index (n) of the power-law fluid model, as

useful indicators for culture growth and differentiation,

were compared according to the number of glass beads,

exhibiting that the culture broth becomes more shear-thin-

ning as glass beads increases (Fig. 7). Specifically, in the

culture broths of A. chrysogenum M35 without glass bead,

the consistency index (K) increased from 0.03 Pa ·sn on the

first day to 0.21 Pa ·sn on the fifth day, after which it

decreased to 0.09 Pa ·sn on the sixth day. However, the cul-

ture broths that contained 4 and 6 glass beads displayed

rapid changes in K and n along with the evolution of time,

comparing with no glass bead case. When 6 glass beads

were added to the main culture broth, the consistency index

(K) was drastically increased from 0.04 Pa ·sn on the first

day to 0.27 Pa ·sn on the fifth day, but it decreased to

0.21 Pa ·sn on the sixth day. And also, flow behavior

(power-law) index (n) is decreased as the glass bead

increases, representing that the main culture is getting more

shear thinning.

The progress of cell growth is clearly reflected in rheo-

logical data. Variation of rheological properties such as the

consistency index and the flow behavior index, as indi-

cated in Fig. 7, might be due to the influence of the phys-

ical or chemical stimulation to cell wall or membrane

structure in main culture by glass beads (Kim et al., 2007),

leading to the secretion of compounds that change the cell

properties for enhancement of oxygen or mass transfer

(Bai et al., 2003). Specifically noting, during the expo-

nential growth phase (from the third to the fourth day) of

A. chrysogenum M35, swollen hyphal fragments were

sharply increased, resulting in the upturn of the consistency

index (K) in main culture with 4 or 6 glass beads. It is

believed that the shear stress would be greatly raised by the

enhanced dispersability of mycelia owing to the addition of

glass beads. In the stationary period (about fifth day)

between the exponential growth phase and the death phase,

the morphology of A. chrysogenum M35 was considerably

altered in submerged culture with glass beads, because

swollen filamentous hyphae in A. chrysogenum M35 were

transformed to arthrospores due to the active secretion of

the antibiotic.

Taken together, rheological information of the culture

broths with glass beads, predicted by reasonable models,

e.g., power-law model, accurately accounts for the growth

or differentiation of cells as well as morphological changes

of A. chrysogenum M35, a filamentous fungus, in sub-

merged culture with glass beads.

4. Conclusion

In this study, rheological properties of culture broths of

Acremonium chrysogenum M35 have been correlated with

the progress of the cell growth and differentiation, focusing

the effect of glass beads in baffled flasks. Glass beads

effectively increase the stress level and non-Newtonian

characteristics in cultures under shear flow condition, con-

firming that cells are vigorously differentiated by the stim-

ulation of the cell wall or membrane and also the active

secretion of compounds for enhancing transport perfor-

mance. Also, material parameters in the power-law model

(or the Herschel-Bulkley fluid model), which is superior to

predict rheological behavior of the culture broths in this

Fig. 7. Changes in the consistency index and the flow behavior

index of the main culture broth of A. chrysogenum M35

in a 250 mL baffled flask with glass beads.



study, well describe the cell growth and differentiation

along with the evolution of time.

Acknowledgments

This study was supported by research grants from the

Korea Science and Engineering Foundation (KOSEF)

through the Applied Rheology Center (ARC), an official

KOSEF created engineering research center (ERC) at

Korea University, Seoul, Korea.

References

Bai, Z., L. M. Harvey, and B. McNeil., 2003, Oxidative stress in

submerged cultures of fungi. Crit. Rev. Biotechnol. 23, 267.

Bhargava, S., K. S. Wenger, K. Rane, V. Rising, and M. R. Mar-

ten, 2005, Effect of cycle time on fungal morphology, broth

rheology, and recombinant enzyme productivity during pulsed

addition of limiting carbon source. Biotechnol. Bioeng. 89,

524.

Cho, Y. J., H. J. Hwang, S. W. Kim, C. H. Song, and J. W. Yun,

2002, Effect of carbon source and aeration rate on broth rhe-

ology and fungal morphology during red pigment production

by Paecilomyces sinclairii in a batch bioreactor. J. Biotechnol.

25, 13.

Chu, W. B. Z. and A. Constantinides, 1988, Moedling, opti-

mization, and computer control of the cephalosporin C fer-

mentation process. Biotechnol. Bioeng. 32, 27.

T. Cuadra, F. J. Fernández, A. Tomasini, and J. Barrios-González,

2008, Influence of pH regulation and nutrient content on ceph-

alosporin C production in solid-state fermentation by Acre-

monium chrysogenum C10. Lett. Appl. Microbiol. 46, 216.

Demain A. L. and R. P. Elander, 1999, The beta-lactam antibiotics:

past, present, and future. Antonie Van Leeuwenhoek 75, 5.

Grimm, L. H., S. Kelly, R. Krull, and D. C. Hempel, 2005, Mor-

phology and productivity of filamentous fungi. Appl. Micro-

biol. Biotechnol. 69, 375.

Junker, B., A. Walker, M. Hesse, M. Lester, D. Vesey, Chris-

tensen, J., Burgess, B. and Connors, N, 2009, Pilot-scale pro-

cess development and scale up for antifungal production.

Bioprocess Biosyst. Eng. 32, 443.

Karaffa, L., E. Sandor, Kozma, and A. Szentirmai, 1977,

Methionine enhances sugar consumption, fragmentation, vac-

uolation and cephalosporin C production in Acremonium

chrysogenum. Process Biochem. 32, 495.

Kim, N. R., J. S. Lim, S. I. Hong, and S. W. Kim, 2005, Opti-

mization of feed conditions in a 2.5-l fed-batch culture using

rice oil to improve cephalosporin C production by Cepha-

losporium acremonium M25. World J. Microbiol. Biotechnol.

21, 787.

Kim, J. C., S. W. Kang, J. S. Lim, Y. S. Song, and S. W. Kim,

2006, Stimulation of cephalosporin C production by Acre-

monium chrysogenum M35 using fatty acids. J. Microbiol. Bio-

technol. 16, 1120.

J. C. Kim, J. S. Lim, J. M. Kim, C. Y. Kim, and S. W. Kim, 2005,

Relationship bween morphology and viscosity of the main cul-

ture broth of Cephalosporium acremonium M25. Korea-Aus-

tralia Rheol. J. 17, 15.

Kim, J. C., Y. S. Song, D. H. Lee, S. W. Kang, and S. W. Kim,

2007, Fatty acids reduce the tensile strength of fungal hyphae

during cephalosporin C production in Acremonium chrysoge-

num. Biotechnol. Lett. 29, 51.

Kumar, S., S. Kundu, K. Pakshirajan, and V. V. Dasu, 2008,

Cephalosporins determination with a novel microbial biosensor

based on permeabilized Pseudomonas aeruginosa whole cells.

Appl. Biochem. Biotechnol. 151, 653.

Lee, M. S., J. S. Lim, C. H. Kim, K. K. Oh, D. R. Yang, and S.

W. Kim, 2001a, Enhancement of cephalosporin C production

by cultivation of Cephalosporium acremonium M25 using a

mixture of inocula. Lett. Appl. Microbiol. 32, 402.

Lee, M. S., J. S. Lim, C. H. Kim, K. K. Oh, S. I. Hong, and S.

W. Kim, 2001, Effects of nutrients and culture conditions on

morphology in the seed culture of Cephalosporium acremo-

nium ATCC 20339. Biotechnol. Biopro. Eng. 6, 156.

Lejon, S., J. Ellis, and K. Valegård, 2008, The last step in ceph-

alosporin C formation revealed: crystal structures of deace-

tylcephalosporin C acetyltransferase from Acremonium chry-

sogenum in complexes with reaction intermediates. J. Mol.

Biol. 377, 935.

Lim, J. S., J. H. Kim, C. Y. Kim, and S. W. Kim, 2002, Mor-

phological and rheological properties of culture broth of Ceph-

alosporium acremonium M25. Korea-Australia Rheol. J. 14,

11.

Matsumura, M., T. Imanaka, T. Yoshida, and H. Taguchi, 1980,

Morphological differentiation in relation to cephalosporin C

synthesis by Cephalosporium acremonium. J. Ferment. Tech-

nol. 58, 197.

Olsvik, E. and B. Kristiansen, 1994, Rheology of filamentous fer-

mentations. Biotechnol. Adv. 1, 1.

Olsvik, E. S. and B. Kristiansen, 1992, Influence of oxygen ten-

sion, biomass concentration, and specific growth rate on the

rheological properties of a filamentous fermentation broth. Bio-

technol. Bioeng. 20, 1293.

Oncu, S., C. Tari, and S. Unluturk, 2007, Effect of various pro-

cess parameters on morphology, rheology, and polygalactu-

ronase production by Aspergillus sojae in a batch bioreactor.

Biotechnol. Prog. 23, 836.

Petersen, N., S. Stocks, and K. V. Gernaey, 2008, Multivariate

models for prediction of rheological characteristics of fila-

mentous fermentation broth from the size distribution. Bio-

technol. Bioeng. 1, 61.

Pollard, D. J., G. Hunt, T. K. Kirschner, and P. M. Salmon, 2002,

Rheological characterization of a fungal fermentation for the

production of pneumocandins. Bioprocess Biosyst. Eng. 24,

373.

Rhee, J. I., G. Seidel, and K. Schugerl, 2003, Modeling of the

biosynthesis of cephalosporin C by Acremonium chrysogenum.

Eng. Life Sci. 3, 392.

Riley, G. L., K. G. Tucker, G. C. Paul, and C. R. Thomas, 2000,

Effect of biomass concentration and mycelial morphology on

fermentation broth rheology. Biotechnol. Bioeng. 20, 160.

Schmitt, E. K., B. Hoff, and U. Kück, 2004, Regulation of ceph-

alosporin biosynthesis. Adv. Biochem. Eng. Biotechnol. 88, 1.



C. Suárez, and F. Gudiol, 2008, Beta-lactam antibiotics. Enferm.

Infecc. Microbiol. Clin. 27, 116.

Sundsfjord, A., G. S. Simonsen, B. Haldorsen, E. W. Lundblad,

and O. Samuelsen, 2008, Broad-spectrum beta-lactamases in

Gram-negative bacteria. Tidsskr Nor Laegeforen. 128, 2741.

Tollnick, C., G. Seidel, M. Beyer, and K. Schügerl, 2004, Inves-

tigations of the production of cephalosporin C by Acremonium

chrysogenum. Adv. Biochem. Eng. Biotechnol. 86, 1.

Zahar, J. R., O. Lortholary, C. Martin, G. Potel, P. Plesiat, and P.

Nordmann, 2009, Addressing the challenge of extended-spec-

trum beta-lactamases. Curr. Opin. Investig. Drugs. 10, 172.

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Rheological properties of culture broth of Acremonium … · 2010. 4. 8. · Abstract The effects of glass ... best fluid models have been sought through the compar- ... Korea-Australia