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Process Biochemistry 37 (2002) 1257–1262 www.elsevier.com / locate / procbio Effect of aeration rate on

Process Biochemistry 37 (2002) 1257–1262

Process Biochemistry 37 (2002) 1257–1262 www.elsevier.com / locate / procbio Effect of aeration rate on the

www.elsevier.com/locate/procbio

Effect of aeration rate on the mycelial morphology and exo-biopolymer production in Cordyceps militaris

Jong Pil Park a , Young Mi Kim a , Sang Woo Kim a , Hye Jin Hwang a , Youn Jeung Cho a , Yong Se Lee b , Chi Hyun Song a , Jong Won Yun a, *

a Department of Biotechnology, Taegu Uni ersity, Kyungsan, Kyungbuk 712-714, South Korea b Department of Natural Resources, Taegu Uni ersity, Kyungsan, Kyungbuk 712-714, South Korea

Received 23 October 2001; accepted 4 December 2001

Abstract

The influence of aeration rate on Cordyceps militaris morphology and exo-biopolymer production was investigated in a 5-l jar fermentor. The mycelial morphology of C. militaris was characterized by image analysis, which included mean diameter, circularity, roughness, and compactness of the pellets. Cells were observed to form mainly pellets during the entire culture period irrespective of aeration conditions. There existed a notable variation in morphological parameters between the pellets grown on different aeration conditions, by which exo-biopolymer production yields were correspondingly altered. The mean diameter and compactness of the pellets indicated higher values at 2 vvm (volume of air per volume of culture per minute), which was closely related to exo-biopolymer biosynthesis. The more compact pelleted form was favourable for exo-biopolymer production. Under extremely low and high aeration conditions (e.g. 0.5 and 4 vvm), severe deformations of pellets (autolysis of core and shaving off the outer hairy region) were observed at the later stages of fermentation associated with a decrease in morphological parameters. © 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Aeration rate; Cordyceps militaris; exo-biopolymers; Morphology; Submerged culture

1. Introduction

Mushrooms have been regarded as popular folk or effective medicines used to treat human diseases such as hepatis, hypertension, hypercholesterolemia, and gastric cancer [1–5]. Both crude exo-biopolymers produced by submerged culture of Cordyceps species and mycelial biomass have recently been regarded as desired prod- ucts with perceived health benefits [6]. Cordyceps mili- taris, belonging to the class ascomycetes, has received special attention for medicinal purpose due to its vari- ous physiological activities [5,6]. Fungal morphology is an important parameter that affects the rheological properties of the fermentation broth, and control of the morphology is highly desired in industrial fungal fermentation [7–11]. In general, two

* Corresponding author. Tel.: +82-53-850-6556; fax: + 82-53-850-

6559.

E-mail address: jwyun@taegu.ac.kr (J.W. Yun).

growth forms, the filamentous and the pelleted form, can be observed in most fungal fermentations and the pelleted form is usually less viscous than the filamen- tous form [12,13]. Pellets are characterized by the myce- lia developing into stable, spherical aggregates consisting of a more or less dense, branched and par- tially intertwined network of hyphae [14]. A number of reports have been documented concerning factors influ- encing fungal morphology, rheology and production of microbial exo-biopolymers (e.g. polysaccharides), ma- jor products from many fungi [15–19]. Amongst many aspects of morphology, the effect of aeration has been studied by a number of investigators [17,19–22]. By varying the aeration rate, the shape of morphology changed from filamentous to pellet, and vice versa [23]. The aim of this study was to characterize the mor- phology of C. militaris and thus to determine the favourable mycelial form for exo-biopolymer produc- tion by varying the aeration rates in a 5-l batch bioreactor.

0032-9592/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.

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2. Materials and methods

2.1. Microorganism and media

C. militaris was kindly provided by Dr JM Sung, Kangwon National University, Korea. The microor- ganism was maintained on potato dextrose agar (PDA) plates and subcultured every month. Slants were incu- bated at 20 °C for 7 days and then stored in a refriger- ator. The seed culture was grown in a 250 ml ask containing 50 ml of PMP medium at 20 °C in a shake incubator at 150 rpm for 5 days.

2.2. Inoculum preparation

C. militaris was initially grown on PDA medium in a petridish, and transferred into the seed culture medium by punching out from the agar plate culture with a cutter [24]. Flask culture experiments were performed in a 250 ml ask containing 50 ml of the media after inoculating with 4% (v/v) of the seed culture.

2.3. Fermentation conditions

The medium composition for the fermentation was as follows: 40 g sucrose, 10 g corn steep powder, 0.5 g K 2 HPO 4 , 0.5 g KH 2 PO 4 , 0.5 g MgSO 4 ·7H 2 O, 0.1 g FeSO 4 ·7H 2 O in 1-l distilled water [24]. The fermenta- tion was carried out in a 5-l batch bioreactor (KOBIO- TECH Co., Seoul, Korea) with a working volume of 3-l. The production medium was inoculated with 4% (v/v) ( 0.4 g/l dry equivalent of cells) of the seed culture and then cultivated for 10 days at 20 °C. The pH and agitation rates were controlled at 6.0 and 150 rpm, respectively. All experiments were performed in triplicate to ensure the trends observed were reproducible.

2.4. Analytical methods

The dry weight of mycelium was measured after repeated washing (with distilled water) of the mycelial pellet, obtained after the rst centrifugation (10 000× g for 10 min) and then dried at 90 °C for 12 h, to constant weight. This weight was compared to the total weight obtained from the ltrates. To measure the exo-biopolymer concentration, the samples collected at various intervals from the bioreactor were centrifuged at 10 000× g for 15 min, and the supernatant was ltered through a membrane lter (0.45 m). The re- sulting culture ltrate was mixed with four times vol- ume of absolute ethanol, stirred vigorously and left overnight at 4 °C. The precipitated exo-biopolymers were centrifuged at 10 000× g for 10 min and the supernatant was discarded. The residue was re-precipi- tated with four times volume of ethanol, the precipitate

of crude exo-biopolymers was freezedried in a lyophilizer and the weight of the polymer was esti- mated. For a quantitative measurement of sucrose, the ltrate from membrane ltration (0.45 m, Millipore) was analyzed by high performance liquid chromatogra- phy, using an Aminex HPX-42C column (0.78×30 cm; Bio-rad, USA) equipped with a refractive index detec- tor (Shimadzu Co., Kyoto, Japan).

2.5. Characterization of the morphology

The morphological properties of the samples col- lected were evaluated using an image analyzer (Matrox Electronic System, Canada) with software linked to a light microscope (Olympus, USA) through a CCD cam- era. Samples were xed with an equal volume of xa- tive (13 ml of 40% formaldehyde, 5 ml glacial acetic acid, 200 ml of 50% ethanol). An aliquot (0.1 ml) of each xed sample was transferred to a slide, air dried, and then stained with methylene blue (0.3 g of methyl- ene blue, 30 ml of 95% ethanol in 100 ml water) [25]. For each sample, the morphology of 50 pellets was characterized by measuring the area and perimeter of the pellet core and the maximum diameter of the pellet. Normally, a magnication of 100 was used. The mor- phology of the pellets was characterized by their mean diameter, circularity, roughness and compactness. The circularity or shape factor was estimated as the ratio of the Fierets minimum diameter to the Fierets maxi- mum diameter of the pellets or aggregates. The com- pactness was estimated as the ratio of the projected area of the hyphae in a clump to the projected convex area of that clump, the latter being the area after lling internal voids and concavities in the clumps external perimeter. In addition, the roughness (R) was measured using the following equation: R = (pellet/aggregate perimeter) 2 /(4 × pellet area) [26].

3. Results and discussion

3.1. Morphological obser ation under different aeration

rates

Pellet morphology has been cited as one of the key factors determining fermentation productivity [8]. In this study, the pellet morphology was characterized with respect to pellet diameter, circularity, roughness, and compactness of a pellet. Pellets were differentiated from fungal fragments and clumps by their grayness level. The amount of grayness, which was used as a critical threshold level or cut-off, was previously ob- tained by analyzing many images. The pellets were stained as described earlier, which were easy for deter- mination of the core pellets and the outer hairy regions accurately. Fig. 1 shows the typical morphological

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changes in C. militaris cell during the entire fermenta- tion period under various aeration conditions. The cells were observed to form mainly pellets during the entire culture period irrespective of aeration conditions. How- ever, as the fermentation proceeded, the outer hairy region of the pellets were shaved off and the core area was reduced, resulting in a corresponding decrease in pellet circularity and roughness, although the cell con- centration remained the same in the stationary phase. At the later stages of fermentation, typically after day 8 in this work, the pellets not only broke up but also the mycelia lost rigidity and appeared somewhat withered (Fig. 1). Furthermore, pellet autolysis was accelerated at 0.5 vvm due to deletion of dissolved oxygen (starva- tion), consequently to form a looser mycelial clump (Fig. 1A). Moreover, higher aeration (4 vvm) also caused pellet autolysis because complete consumption of substrate (sucrose) resulted from rapid mycelial growth. The population of the free mycelia in the fermentation medium was also least during the growth phase, which indicated that pellets, rather than free mycelia, were the more productive morphological forms of C. militaris for optimal cell growth and exo-biopoly- mer production as described later. Cui et al. [27] re- ported that high DO tension produced denser pellets of Aspergillus awamori, whereas weak and uffy pellets were formed under very low DO tension.

3.2. Characterization of mycelial morphology

Morphological properties of a lamentous fungus play an important role in their metabolism during fermentation processes [710,28]. Fig. 2 shows the mean diameter, circularity, roughness, and compactness

of the pellets during submerged culture of C. militaris under different aeration conditions. There was a signi- cant variance in the morphological parameters between the pellets grown under different aeration conditions. The pellet diameter increased rapidly from the rst day to the end of fermentation at an aeration rate of 2 vvm. However, pellet sizes decreased at a low and a high aeration conditions after day 6, which indicated that pellet lyses occurred due to either higher oxygen starva- tion (0.5 vvm) or rapid depletion of substrate associated with facilitated oxygen supply into core of the pellets (4 vvm). The maximum value of the pellet mean diameter grown at 0.5, 2 and 4 vvm were about 0.53, 0.71 and 0.58 mm, respectively. The circularity and roughness observed at aeration rates of 0.5 and 4 vvm increased from the early stage of fermentation (till day 6) and thereafter slowly decreased as in previous morphologi- cal observations (Fig. 2B and C). This result was prob- ably caused by loosening of the pellet cores, after which the pellets rapidly broke up into hyphal fragments and ocks (Fig. 1A and C). In other words, the outer hairy region of the pellets were shaved off, which resulted in corresponding decrease in pellet roughness and pellet diameter although the cell concentration remained the same in the stationary phase. Since shaving off hyphae is a function of hydrodynamic forces, minimum rough- ness was observed under high aeration condition (4 vvm). The decrease in pellet roughness can be explained by severe hyphal fragmentation as a result of the reduc- tion of the hyphal tensile strength due to a successive vacuole formation [29]. Taking into account that circu- larity mean a shape factor describing the deviation of the pellet image from a true circle, and roughness mean the irregularity of the perimeter of pellet, compact

mean the irregularity of the perimeter of pellet, compact Fig. 1. Morphological changes in C .

Fig. 1. Morphological changes in C. militaris in a 5-l batch reactor. 2 10 d mean fermentation period in day.

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et al . / Process Biochemistry 37 (2002) 1257 – 1262 Fig. 2. (A) Mean diameter,

Fig. 2. (A) Mean diameter, (B) circularity, (C) roughness, and (D) compactness of C. militaris pellets growing in a 5-l batch reactor at the aeration rates of 0.5 vvm ( ), 2 vvm ( ), and 4 vvm ( ).

pellet formation with delaying pellet deformation was favourable at an aeration rate of 2 vvm. This agreed with the result that compactness (fullness) of the pellets indicated a maximum and remained nearly constant at 2 vvm (Fig. 2D). Riley et al. [26] have pointed out that compactness and roughness of Penicillium chrysogenum pellets were kept nearly constant during the entire period of fermentation. In a review article by Paul and Thomas [30], compactness and circularity of Aspergillus niger pellets decreased slowly while the core area sharply increased during the exponential growth of mycelia. Olsvik et al. [31] have reported similar result to the present ndings; i.e. A. niger pellets showed de- creased roughness as the DO level increased, whereas maximum compactness was observed at an intermedi- ate DO level examined (12%). Low roughness of the pellets observed at a high oxygen level in the vessel might imply some effect on hyphalhyphal interaction within the mycelial clump.

3.3. Effect of aeration rate on mycelial growth and exo-biopolymer production

In a previous paper [24], it was reported that sucrose was the most suitable carbon source for both mycelial growth and exo-biopolymer production in C. militaris. Fig. 3A shows the time prole of sucrose consumption at different aeration rates. The concentration of resid-

ual sucrose sharply decreased during the fermentation with corresponding increase in biomass and exo-bio- polymer production. Almost complete sugar depletion was observed for the culture grown at an aeration rates of 4 vvm, whereas 17 and 10 g/l sucrose remained in the fermentation broth in cultures grown at aeration rates of 0.5 and 2 vvm for the same fermentation period. High aeration rate of 4 vvm clearly resulted in sub- stantially increased mycelial growth (Fig. 3B). In cul- tures under the examined aeration rates (0.5, 2 and 4 vvm), the maximum mycelial biomass yield increased up to 5, 8, and 8 days, respectively, and then remained constant during the later stages of fermentation. Maxi- mum exo-biopolymer production (14.5 g/l) was achieved at 2 vvm, whereas a further increase in aera- tion rate at values over 2 vvm or below, resulted in a decrease in exo-biopolymer production (Fig. 3C). It is noteworthy that the combination of dissolved oxygen concentration with favourable morphology played an important role in higher exo-biopolymer production. The DO level at an aeration rate of 2 vvm was reduced from 100% saturation at the beginning of fermentation to around 15% saturation at days 23, thereafter high DO level (76%) was maintained throughout the fermentation, allowing the pellets to show a more compact form (Fig. 3). These results are also in good agreement with those of other investiga- tors [32]. Gibbs and Seviour [16] and Wecker and

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Onken [19] have reported that the inhibitory effect of high dissolved oxygen concentration was observed on polysaccharide production in Aureobasidium pullulans. Therefore, the decrease in exo-biopolymer production at aeration rates higher than 2 vvm or below was due either to the change in morphology of the microorgan- ism during fermentation or substantial cell damage or change in a mycelial regulatory mechanism [33] (Fig. 3A and C).

4. Conclusion

phological parameters may depend on the fungal spe- cies, the growth medium or the physical environment within the culture vessel. Extensive studies on the exo- biopolymers, including molecular characterization and structure identication, are ongoing in this laboratory.

Acknowledgements

This work was supported by the RRC program of MOST and KOSEA.

From the above results, a critical conclusion was derived that the aeration rate was an important factor for exo-biopolymer production controlling the growth of C. militaris in a more compact pelleted form. Mor-

of C . militaris in a more compact pelleted form. Mor- Fig. 3. Time pro fi

Fig. 3. Time proles of (A) sucrose consumption, (B) mycelial growth, and (C) exo-biopolymer production in C. militaris using a 5-l batch reactor at various aeration rates of 0.5 vvm ( ), 2 vvm ( ), and 4 vvm ( ).

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