Indian Journal of Biotechnology Vol I. October 2002, pp 357-362

Morphological Changes in Submerged Cultivation of Aspergillus awamori for Glucoamylase Production

David K Daniel', Nabanita Biswas2 and Debabrata Das3*

'Oepartment of Chemical Engineering, Vellore Institute of Technology, Vellore 632 014. India 20epartment of Microbiology, Calcutta University. Kolkata 700 019, India

' Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India

Received 26 February 2002; accepted 15 May 2002

Industrial processes involving submerged fermentation of fungal cultures require greater attention due to complex morphologies. Growth of Aspergillus awamori was investigated in submerged culture using different parameters such as pH and agitator speed with a goal to assess the morphology and fragmentation behaviour during the fermentation. Microscopic examination of the mycelia revealed that the length of the branches decreased with increasing pH. Pellet formation was significant at pH 5.5 and the diameter of the pellet was found to vary with fermentation time. A maximum pellet diameter of 1.69 mm was observed at a pH of 7.5, which decreased to 0.38 mm for cultivations at pH of 3.5. During mechanical agitation, fragmentation was found to dominate fungal growth and branching. Agitator speeds higher than 600 rpm resulted in decreased cell growth and glucoamylase production.

Keywords: Aspergillus awamori, fungal morphology, glucoamylase, fragmentation, pellets, agitation

Introduction Filamentous micro-organisms in submerged

aerobic cultivations lead to suspension characteristics quite different from bacterial and yeast cultures. The morphological feature of filamentous micro­organisms, which has a significant effect on the rheological properties of the cultivation medium are reflected in the production and excretion of different microbial metabolites (Braun & Vetch-Lifshitz, 1991). In submerged cultivation involving filamentous organisms, the morphology can vary from discrete compact pellets of hyphae to homogenous suspension of dispersed mycelia (Metz & Kossen, 1977). The filamentous form of mycelia easily causes entanglement, and the cultivation broth becomes very viscous. Increasing the agitation rate improves the overall homogeneity, but this also raises the power consumption and damages the cell due to high shearing (Van Suijdam & Metz, 1981; Tanaka et aI., 1974). On the other hand, pellet type of growth has the advantages of decreased viscosity of the cultivation broth and renders the rheological properties to become Newtonian. Moreover, the pellet formation facilitates the separation of fluid in downstream processes (Nielson et ai, 1995).

*Author for correspondence: Tel : +91-3222-778053, 82248; Fax: +91-3222-778707, 755303 E-mail : ddas@hijli .iitkgp.ernet.in

Control of the morphology of fungi is an important pre-requisite for their industrial application because it strongly influences the physical properties of a fungal cultivation broth, which in turn causes numerous problems in an industrial fermentation process . Fungal morphology is therefore considered as one of the key parameters in industrial production and hence it becomes necessary to study the influence of process conditions on the morphology of fungi in a fermentation process. The aim of the present work was to assess morphological changes and fragmentation behaviour in a laboratory scale batch fungal fermentation process. To accomplish this , A. awamori NRRL 3112, a potential glucoamy lase producer was grown in a fermenter under different process conditions and the effects of these process parameters on the morphology and enzyme production are reported. Such studies on morphology are essential for successful modeling during fermentations.

Materials and Methods Organism

Aspergillus awamori NRRL 3112, a strain that produces glucoamylase (Soni et ai, 1995) was used in this study. The strain has been procured from the U S Department of Agriculture, Illinois, USA as a gift. The organism was maintained on MYGP agar slants and subcultured regularly every month.

3S8 INDIAN J BIOTECHNOL. OCTOBER 2002

Media alld Inoculum Development The strain was maintained as spores at -20°e. The

fungus was grown in a medium containing the following ingredients per litre: Maltose, 20g; NaN03,

Sg; KH2P04 , Ig; MgS04.7H20, O.Sg; KCI, O.Sg; Peptone, 8g; FeS04.7H20, O.Olg. The pH of the medium was adjusted to S.S , prior to autoclaving (120°C for 20 min). Spores from slants were scrapped off and suspended in 10ml of sterile water. The spore suspension was shaken thoroughly to break up any aggregates. The spore count was determined microscopically using I ml of the above suspension after making the serial dilutions. S% v/v spore suspension containing 2xl07 spores per ml was used as the inoculum in 100 ml medium using SOO ml Erlenmeyer flasks on a shaker (Grant Instruments Cambridge Ltd, England) at ISO rpm. The cultures were incubated at 30°C.

Submerged Cultivation in Controlled Bioreactor A stirred tank bioreactor (Bioengineering AG,

Switzerland) with a working volume of 1.2 I was used. Agitation was performed using two 6-bladed disc turbine impellers. Airflow rate was maintained at O.S vvm using a dust and oil free compressor. The pH of the culture broth in the fermenter was controlled by the addition of I (N) NaOH or 1 (N) HCI. Foam was automatically controlled by the addition of anti foam reagent (Silicone oil, Merck, Germany). Sampling was done at regular intervals followed by centrifugation at SOOO rpm (Sigma, Germany) for 15 min. The supernatants were used for the determination of the enzyme activity .

Microscopic Studies Samples from fermenter were diluted to give a

biomass concentration of less than O.S g/l. A sample taken from the bioreactor after dilution was directly treated with fixative solution (9% w/v acidic formaldehyde in SO% ethyl alcohol) to stop the metabolic activities and followed by adsorption on a microscopic slide. The adsorbed samples were stained using lacto phenol cotton blue and the dye solution was removed by washing with distilled water and samples allowed to dry again at room temperature. For pellets, the sample was suspended in a petri plate to preserve the three-dimensional structure of the pellets. The mycelial morphology was observed using an inverted research microscope (Olympus Optical Co Ltd, UK). Magnifications were selected so that optimal vi sualization and accuracy of the samples was

ensured. About ten sets in each sample were observed to get the total hyphal length and number of tips. The process was repeated at least three times using different positions on each microscopic slide. The number of pellets per ml of medium was determined manually by counting.

Analytical Methods Biomass. For the determination of biomass

concentration, S ml broth sample was diluted with 40 ml distilled water and centrifuged at 5000 rpm for IS min. The biomass sample was then placed on a pre weighed · Whatman No I filter paper (Whatman International , England). The biomass was washed with distilled water several times to remove the soluble materials. The mycelia were then dried in an oven at lOsoC to a constant weight.

Glucoalllylase activity. The enzyme activity was determined using the method described by Mase et al (1996) with slight modifications. O.S ml of 4% (w/v) soluble starch solution in 0.1 M citrate buffer (PH 4.0) was mixed with the crude enzyme (sui tably diluted) in a test tube. The reaction mixture was incubated at S.soC for 30 min and subsequently the reaction was stopped by heating the mixture in a boiling water bath for S min. The reducing sugars released were estimated by the method reported by Miller (1959). One unit of glucoamylase activity was defined as that, which catalyzed the generation of one micromole of glucose/min/ml enzyme solution under the above conditions.

Results and Discussion The aim of this work was to understand the factors

affecting morphology and analyzing their effects on bioreactor performance during a submerged glucoamylase fermentation process. Factors affecting the above can generally be categorized as strain dependent, nutntIOn dependent, and cultivation condition dependent factors (Chiqueuo et ai, 1992: Barton et al, 1972; Smith et al, 1990). The interaction between these factors has to be considered because it is difficult to determine or discuss the effect of these factors separately. Hence in the present work, only the pH of the medium and speed of the agitator in the biorcactor have been considered as the major factors causing morphological changes during glucoamylasc fermentation process. The ranges of these selected process parameters were chosen based on earlier work in the author's laboratory.

DANIEL et at: GLUCOAMYLASE FROM ASPERGILLUS A WAMORI 359

Studies Oil the Effect of pH Cell morphology. The influence of pH on the

morphology of A. awal1lOri NRRL 3112 was investigated in a lab scale fermenter with three different pH of the medium. In all the experiments, the aeration and agitation was maintained at 0.5 vvm and 600 rpm, respectively. The spores started to germinate 10 hrs after inoculation and microscopic examination of the samples withdrawn at few hours after inoculation showed that the morphology of the culture depends on pH of the medium. The observations of the number of tips revealed that significant branching of hyphal elements starts after 15 hrs. Fig. I depicts the microscopic appearance of the mycelium grown at pH values 3.5, 5.5 and 7.5. The length of the branches decreased from 185 flm to 50 flm with increase in pH of the medium. The thickness of the hyphae at pH 7.5 (Fig. 2a) covered a wider range than that at pH 5.5 (Fig. 2b). At low pH (3.5) high vacuolation of the mycelium was prominent, resulting in very poor growth (Fig. 2c). Pellet formation was marked at pH 5.5 and above but it did occur to a lower extent at pH value 3.5. Furthermore, it was observed that the diameter of the pellet increased with cultivation time. The maximum pellet diameter observed was 1.69 mm at a pH of 7.5 and this decreased to 0.38 mm for cultivations at a pH of 3.5. Galbraith & Smith (1969) have also reported similar observations for A. niger in shake flask cultures. After inoculation of spores, the pellet concentration was constant until fragmentation sets in, which is confirmed by the presence of fewer branches and shorter lengths of hyphae. The fragmentation of pellets probably depends on a combination of the pellet size, the age of the pellets and the stirring rate in the bioreactor (Justen et ai, 1996; Shamlou et al, 1994).

Glucoamylase and biomass production. The effect of pH of the medium on glucoamylase activity is shown in Fig. 3. The glucoamylase activity reached a maximum of 1.95 Vim I at pH of 5.5 after 48 hrs of growth. The biomass profiles at different pH are shown in Fig. 4. The biomass concentration was found to be maximum at 16.89 g/l after 120 hrs of fermentation at the pH 5.5 .

Studies on the Effect of Agitator Speed In an effort to determine the morphological and

fragmentation behaviour of A. awamori NRRL 3112, studies were conducted at three agitator speeds, viz. 300, 600 and 900 rpm. The relations between cell

growth, morphology and enzyme production as a function of different agitator speeds (300, 600 and 900 rpm) were investigated. In all the experiments. the aeration rate was maintained constant at 0.5 vvm. The temperature and pH during the course of fermentation were maintained at 30°C and 5.5. respectively. After about 20 hrs of cultivation, agglomeration between the hyphal elements was very high and the growth form was characterized as pellets. In the case of 300 rpm culture, distinct pellet morphology was observed with a small fraction of mycelium, which decreased with time (Fig. Sa). The number of pellets increased with the increase of

(at

(b)

(cl

Fig. I-Mycelial form of Aspergillus aW(ll1lori NRRL 3112 (x 40 magnification) in cultures grown at (a) 3.5 pH. (b) 5.5 pH & (c) 7.5pH.

360 INDIAN J BIOTECHNOL, OCTOBER 2002

Fig. 2-Hyphal forms produced during the growth of A. awamori NRRL 3112 (x 100 magnification) at (a) 7.5 pH, (b) 5.5 pH & (c) 3.5 pH.

agitation speed. During the period of 20 to 40 hrs the average pellet diameter was found to be 0.51 mm and increased at constant rate for the 300 rpm agitated culture. In the case of 900 rpm agitated culture the growth was mainly in the micro pellet form (Fig. 5b). However, in the 600 rpm agitated culture, the growth was in the form of small pellets with mycelial network (Fig. 5c).

Cell growth. The variation of cell growth with time in all the cultures has been observed (Fig. 6). The cell growth increased exponentiaIly in all the cases for the first 70 hrs of cultivation. In the cultures with an agitation speed of 300 rpm, the cell dry weight increased with time until it reached 17.53 g/I after 120

hrs and decreased for the rest of the cultivation. On the other hand, in cultures with an agitation speed of 600 rpm, no further increase in biomass was observed after 100 hrs and the maximum cell concentration (13.55 g/l) was found to decrease for the rest of the cultivation. To investigate further, the volumetric oxygen transfer coefficient at 300 rpm was 274.3 h-' at 600 rpm. The highest value of volumetric oxygen transfer coefficient of 648.3 h-1 was found to be at an agitator speed 900 rpm. This indicates the presence of increased mass transfer at higher agitator speeds that should eventually lead to higher cell growth. However, in the present study, decreased cell growth was observed at 900 rpm, which may probably be due to the fragmentation and spowlation of the mycelia at this speed.

GLucoamyLase production. The glucoamylase production under different agitation speeds is shown

E100

2-.?;-"> 80 U ro <D

~ 80 >. E ro 8 40 :::l rn ~ 20 ~ Oi 0:::

o 2 3 5

Time (days)

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6 7

Fig. }--Effect of pH on glucoamylase activi ty during culture of A. awamori NRRL 3112.

25~--------------------~======~

~2O

~ c o 'iii 15

E ~ c 810

ll! ro E ~ 5

2 345

Time (days)

6 8

Fig. 4--Effect of pH on biomass production of A awamori NRRL 3112.

DANIEL et al: GLUCOAMYLASE FROM ASPERGILLUS A WAMORI 361

1:3) .-, I

~ .. , . .

'J' , ~. : ~ \

.4 I ~."t

, .. -~

"' . 'to " r

• .. ~ w

(b)

... I --,. /

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Fig. 5-Effect of agitation on pellet morphology of A awamori NRRL 3 112 (x 40 magnification) at (a) 300 rpm, (b) 900 rpm & (c) 600 rpm.

in Fig. 7. During the first 20 hrs of fermentation, the accumulation of the extra cellular enzyme increased with the time. Increase in agitator speed inhibits the aggregation of both spores and hyphal cells resulting in a micro pellet growth in hyphal network. The differences in enzyme excretion rate were due to morphological differences during this phase. The growth under high agitation was characterized by a higher hyphal extension and branching (Fig. 8). Therefore, it showed a higher number of apical tips, however, the glucoamylase production at higher agitation was decreased. A decrease in citric acid

~.--------------------------------.

O~~-r~~~--r-~.-~-r~~~--~

o ~ ~ 100 1~ 1~

Time (h)

Fig. &-Effect of agitation on biomass production during culture of A. awamori NRRL 3112 at (a) 300 rpm, (b) 900 rpm & (c) 600 rpm.

~ 100

~ 'S 15 80 ro Ql

'" ~~ E ro 8 :::l 40 0> Ql

,~ ro 20 Qj a::

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.-300 rpm

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80 100

Fig. 7-Effect of agitation on glucoamylase activity during culture of A. awamori NRRL 3112 at (a) 300 rpm, (b) 600 rpm & (c) 900 rpm.

production by A. niger was observed by Ujcova et at (1980) at high agitator speeds. Similar observations for penicillin fermentation by Penicillin chrysogenum have also been made (Konig et ai, 1981).

Cell morphology. An inoculum concentration of 2x107 spores per ml was used in all cultures under study. In the case of 300 and 600 rpm cultures distinct pellet morphology was observed with a small fraction of free mycelium that decreased with time. The number of pellets was 30±5 and 150±50 per ml at 300 and 600 rpm agitation, respectively. During 20 to 40 hrs of fermentation, the average pellet diameter increased at 300 and 600 rpm agitation. The growth of cells at 900 rpm agitation was mainly as mycelial network with a higher number of cell aggregates of

362 INDIAN J BIOTECHNOL, OCTOBER 2002

Fig. 8--Hyphal extension & branching patterns during culture of A. awall/ori NRRL 3112 (x 100 magnification) at 900 rpm .

diameter. After about 40 hrs, the fraction of free mycelial cells decreased through the fragmentation of cells in this culture. Thus, the increase in agitator speed inhibited the aggregation between the germinated particles, which resulted in a significant decrease in the pellet diameter (Taguchi et ai, 1968) . Therefore, the differences observed in the glucoamylase production could be attributed to the shear force in the bioreactor and the resulting morphological changes during the cultivation.

Conclusion Laboratory scale fermentations with A. awamori

NRRL 3112 at different process conditions were performed to study the changes in fungal morphology, fragmentation behaviour and glucoamylase production. This study has shown that pellet formation was significant at pH 5.5 whereas poor growth was observed at higher pH. The glucoamylase activity reached a maximum of 1.95 Vlml at pH 5.5 after 20-40 hrs of fermentation, while the maximum growth was observed after 120 hrs of fermentation. The cell growth and glucoamylase production were reduced at higher agitator speed while extensive sporulation of the organism was observed at an agitator speed of 900 rpm. On the other hand, it was found that increased agitation led to higher biomass concentration. Thus, this work has revealed a preliminary insight into the relation between morphology and glucoamylase production.

Acknowledgement

The authors thank Or S K Ghosh and Mr Sanjay Pal , Department of Biotechnology, IlT Kharagpur for their experimental support and assistance during the microscopy. References Barton L L et ai, 1972. Effect of maltose on glucoamylase

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