ORIGINAL PAPER

Changes in morphology of Rhizopus chinensis in submergedfermentation and their effect on production of mycelium-boundlipase

Yun Teng Æ Yan Xu Æ Dong Wang

Received: 23 May 2008 / Accepted: 19 August 2008 / Published online: 9 September 2008

� Springer-Verlag 2008

Abstract In order to control suitable mycelium mor-

phology to obtain high lipase productivity by Rhizopus

chinensis in submerged fermentation, the effects of fungal

morphology on the lipase production by this strain both in

shake flask and fermentor were investigated. Different

inoculum level and shear stress were used to develop dis-

tinctive morphologies. Analyses and investigations both on

micromorphology and macromorphology were performed.

Study of micromorphology reveals that micromorphologies

for dispersed mycelia and aggregated mycelia are different

in cell shape, biosynthetic activity. Macromorphology and

broth rheology study in fermentor indicate that pellet for-

mation results in low broth viscosity. Under this condition,

the oil can disperse sufficiently in broth which is very

important for lipase production. These results indicate that

morphology changes affected the lipase production sig-

nificantly for R. chinensis and the aggregated mycelia were

suggested to achieve high lipase production.

Keywords Lipase � Morphology � Rheology �Rhizopus chinensis � Mycelia aggregation

Introduction

Fungal morphology is an important parameter for fila-

mentous fungus fermentations, is often considered as one

of the key parameters of physiological and engineering

studies of filamentous fermentations used in the design and

operation of such systems [1]. In submerged culture,

morphology of filamentous microorganisms usually varies

between ‘‘pellet’’, ‘‘clump’’ and ‘‘filamentous’’ forms,

depending on the respective culture conditions and geno-

type of the strain [2]. Often, morphology is correlated with

production of enzymes or secondary metabolites [3].

Filamentous microorganisms are good source for many

primary metabolites and secondary metabolites such as

organic acids and antibiotics. They are also good producer

for many enzymes or microbial proteins production. The

change of mycelial morphology was found to have

remarkably effect on these primary or secondary metabo-

lites [4–7] production, and also on enzymes production.

Therefore, many researchers have focused on this research

field, and some valuable results were obtained. Papagianni

[8] and her coworkers reported that growth of Aspergillus

niger in the form of large pellets was associated with lower

specific protease activities and increased specific gluco-

amylase activities compared with filamentous

morphologies. Jin [9] and his coworkers found that the

formation of small compact pellets under designed culti-

vation conditions favored higher fungal protein yields. El-

Enshasy [10] reported that culture conditions resulting in

the formation of smaller fungal pellets with an increased

mycelial density result in higher yields of extracellular

Y. Teng � Y. Xu � D. Wang

Laboratory of Brewing Microbiology and Applied Enzymology,

School of Biotechnology, Jiangnan University, 1800 Lihu Rd,

214122 Wuxi, Jiangsu, People’s Republic of China

Y. Teng � Y. Xu � D. Wang

Key Laboratory of Industrial Biotechnology,

Ministry of Education, Jiangnan University, 1800 Lihu Rd,

214122 Wuxi, Jiangsu, People’s Republic of China

e-mail: tengyunflight@yahoo.com.cn

Y. Xu (&)

State Key Laboratory of Food Science and Technology,

Jiangnan University, 1800 Lihu Rd, 214122 Wuxi,

Jiangsu, People’s Republic of China

e-mail: yxu@jiangnan.edu.cn

123

Bioprocess Biosyst Eng (2009) 32:397–405

DOI 10.1007/s00449-008-0259-8

glucose oxidase for recombinant A. niger. Other enzymes

production were also affected by the change of mycelial

morphology, they were amylase [11], lipase [12, 13], pol-

ygalacturonase [14], neo-fructosyltransferase [15], and

phytase [16]. The relation between fungal morphology and

process productivities has attracted interest from both

academic and industry and attempts have been made to

manipulate morphology to achieve maximal performance.

Most of researchers focused on two aspects of mor-

phology changes: one is the change of microscopic

morphology which can influence the enzyme biosynthesis

and secretion capacity directly; another one is the change

of macroscopic morphology which can influence broth the

rheology and the mass transfer properties [17]. The

micromorphology deals with the morphology of the

individual hyphal elements, e.g., the diameter, the length

of the hyphal elements and the number of tips on an

individual hyphal. Peberdy suggested that protein secre-

tion in filamentous fungi is intimately associated with the

process of growth at the hyphal tip [18]. Spohr and his

coworkers [11] reported that the enzyme secretion

capacity for A. oryzae have a correlation with the number

of tips. The change of micromorphology for recombinant

strain A. oryzae was also proved to have remarkable

influence on the lipase production [12]. Compare with

micromorphology, the macromorphology could be easily

distinguished by eyes or simple observation methods,

these morphological growth forms can have a significant

effect on the rheology of the fermentation broth and thus

the bioreactor performance. For example, pellet mor-

phologies result in Newtonian broths and better mass

transfer rates compared to viscous and often pseudoplastic

filamentous broths [19]. The different of mass transfer

rate will lead to a different metabolic pathway and dif-

ferent enzyme production.

Rhizopus chinensis CCTCC M201021 is a typical fila-

mentous fungus, and the mycelium-bound lipase produced

by this strain was proved to be an efficient biocatalyst in

biosynthesis of short-chain fatty esters in n-heptane [20]. In

our previous studies, we found that the change of mor-

phology could effect the mycelium-bound lipase

production for the mutant strain R. chinensis Y92-M [21]

and the lipase with synthetic activity was membrane-bound

[22]. In order to find out the relationship between the

change of morphology and lipase production by R. chin-

ensis, both effects of micromorphology and

macromorphology on mycelium-bound lipase production

were investigated. The study of micromorphology was

performed using mycelia obtained from shake-flask. And

the rheology study under different macromorphology was

performed in stirred tank reactor. Different morphologies

were manipulated by different inoculum level and stirring

speed.

Material and methods

Microorganism and media

Rhizopus chinensis CCTCC (China Center for Type Cul-

ture Collection) M201021 was isolated from Da Qu (a kind

of traditional leaven for production of Chinese liquor) by

our laboratory. The composition of medium contained

olive oil (23.7 g/L), MgSO4.7H2O (0.5 g/L), K2HPO4 (3 g/

L), maltose (1 g/L), peptone (40.6 g/L), pH was adjusted to

5.5. The medium was inoculated with spores with different

spore concentrations after sterilization. The spores were

washed from a fresh potato dextrose agar slant (72 h) by

deionized water, and the spore concentration was deter-

mined hemacytometer count.

Culture conditions

Shake flask cultures

Erlenmeyer flasks of 250 mL, containing different volume

of medium, were sterilized at 121 �C for 20 min. After

inoculated by different inoculum levels, the flasks were

incubated on shaker incubator for different rotation speed

at 30 �C for 72 h. The cultivated mycelium was separated

from the culture by filtration, washed twice with tap water

and once with 25 mM phosphate buffer (pH 7.0). The

mycelia were then lyophilized for 24 h by a freeze drying

system (Labconco, USA). Biomass was determined from

100 mL fermentation broth.

Stirred tank reactor (STR) cultures

Fermentation was carried out in a stirred-tank fermentor

(7L, Bio Flo110, New Brunswick scientific, USA) at 30 �C

under different stiring speed and different inoculum levels,

the working volume was 4.5 L, and the aeration rate was

1 vvm.

Analytical methods

Synthetic activity determination

Synthetic activity was measured by the ester-synthesis

method in heptane according to the procedure described

previously [22]. Octanoic acid (1.2 M) and ethanol (1.2 M)

in heptane each of 0.5 mL were mixed. The reaction was

started by adding 20 mg dry cell and incubated for 30 min at

40 �C with a shaking speed of 200 rpm. The reaction mix-

ture was then filtered using 0.15 lm membrane to remove

the cells. Samples (400lL) were then drawn and mixed with

2-hexanol of 100lL, as internal-standard, then analyzed by

subsequent gas chromatograph (Agilent 6820, Agilent

398 Bioprocess Biosyst Eng (2009) 32:397–405

123

Technologies Company Ltd, Shanghai, China) which

equipped with a polyethylene glycol capillary column

(AC20, 30 m 9 0.22 mm 9 0.25 lm, SGE International

Pty. Ltd, Australia). Nitrogen was used as a carrier gas, and

the injector and detector temperatures were set at 250 �C.

Oven temperature by programmed temperature was started

at 90 �C for 1 min before being elevated to 200 �C for 5 min

at 10�C/min. One unit of lipase synthetic activity was

defined as the amount of enzyme which catalyzed to produce

1 lmol of ester per minute.

Glucose and maltose determined

Glucose and maltose concentration was determined by

HPLC (Waters 650E, Waters Corp., USA), which equipped

with Sugarpak1 (6.5 mm 9 300 mm) columen (Waters

Corp., USA) and refractive index detector (Waters 2410,

Waters Corp., USA). Pure water was used as mobile phase,

flow rate was 0.5 mL/min and columen temperature was

85 �C. Before injection, the sample was prepared by cen-

trification and filtration by 0.25 lm microporous

membrane, and diluted to a certain concentration.

Image analysis

Sample preparation was carried using method described by

Haack [12]. Two-milliliter samples were taken during the

cultivations for image analysis, and one or two drops lac-

topenol-blue were added to stop growth and increase the

contrast of the images. The samples were stored at 4 �C for

later analysis. Before analysis, the samples were diluted to

approximately 0.25 g dry weight per litre to get a reason-

ably amount of hyphal element on each slide. Image

capture was carried out via a CCD camera (DXM 1200C,

Nikon Corp, Japan) mounted on a microscope (Nikon

eclipse 50i, Nikon Corp., Japan). Morphological measure-

ments were carried out using the Image-Pro PLUS software

(Media Cybernetics Inc., MD, USA). Measurements of

pellet mean diameter were done on images obtained using

49 objective, and measurements of other hyphal parame-

ters were measured using 109 or 409 objective. The

equivalent diameter of approximately 50 pellets was

measured per sample. For filamentous growth (freely dis-

persed mycelium), the average length of the filaments was

determined by estimating branches in individual mycelial

trees [8]. During the batch phase, the hyphal diameter was

measured approximately 10 lm from the end of the hyphal

tip. The number of tips was measured in individual

mycelial trees. Microscopic morphology was also per-

formed by electron microscope (Quanta-200, FEI,

Netherlands).

Pellets were distinguished from clumps and individual

mycelial trees by greyness levels differences, an approach

which has been used to provide a definition of a pellet [8].

Fully entangled filaments could be easily distinguish by it

irregular shape and tissue like form from pellet and freely

dispersed mycelium.

Fluid dynamics measurements

The rheological measurements were performed in the

250 mL beaker contained 200 mL fermentation broth using

a Brookfield viscometer (Brookfield DV-E, Brookfield,

USA), fitted with a disc-spindle impeller (1#). The shear

stress (s) of the fermentation broth was characterized by

the simple power law model of Ostwald–de Waele power

law approach for non-Newtonian fluids as shown below,

where c is the shear rate.

s ¼ Kcn ð1Þ

The constants, K and n represent the consistency index and

the flow behavior index, respectively. The apparent

viscosity, ga, is given by:

ga ¼sc¼ Kcn�1 ð2Þ

Taking natural logarithms on both sides of Eq. 3:

InðgaÞ ¼ InK þ ðn� 1ÞInc ð3Þ

Because of the DV-E Brookfield viscometer could only

give out the relationship between apparent viscosity ga and

rotational speed Ni. So, a equation was used to describe the

shear rate c by Ni [23].

c ¼ KNcðnÞNi ð4Þ

where KNcðnÞ was a value which could be calculated from

the result reported by Mitschka [23]. After replaced c in

Eq. 3 by KNcðnÞ and Ni, the equation was changed to:

InðgaÞ ¼ InðK � KNcðnÞn�1Þ þ ðn� 1ÞInNi

The values of InðK � KNcðnÞn�1Þ and n were able evaluated

from a natural logarithmic plot of ga versus Ni. And K was

able get from the values of InðK � KNcðnÞn�1Þ:

Result and discussion

Effect of culture condition on the mycelial morphology

and lipase production in shake flask

In our previous studies [21], we found that culture con-

ditions, like inoculum level, rotation speed and

fermentation volume could influence the macromorphol-

ogy of R. chinensis during lipase production in shake flask

cultivation. The culture conditions and results of macro-

morphology and lipase production were listed in Table 1.

Bioprocess Biosyst Eng (2009) 32:397–405 399

123

The synthetic activity of mycelium-bound lipase changed

from 101.2 U/g to 691 U/g with the change of mycelial

morphology from dispersed mycelia to fully entangled

filaments. Inoculum is very important for the mycelial

morphology in this study, when 3.3 9 109 spores L-1

inoculum was used, dispersed mycelia (Fig. 1c) were

formed regardless the rotation speed or volume of broth

for 250 mL flask used. Fully entangled filaments (Fig. 1a)

were obtained in the application of less spore number

3.3 9 108 spores L-1 and 3.3 9 107 spores L-1. Among

the factors that determine the morphology and the general

course of fungal fermentations, the amount, type and age

of the inoculum are of prime importance [1]. Therefore,

many researchers found the inoculum have remark influ-

ence on mycelial morphology. When the inoculum level

was varied from 104 spores mL-1 to 109 spores mL-1, the

mycelial morphology change from pellet, clump to free

mycelial during citric acid production by A. niger. The

production of citric acid was affected by the change of

mycelial morphology [4]. The influence of inoculum on

pellet formation was also studied in detail by Nielsen [24]

and Tucker [25].

Rotation speed and fermentation volume also remark-

ably affected the morphology and lipase production in

shake flask. When the rotation speed was lower than

150 rpm,entangled filaments (Fig. 1b) and dispersed

mycelia (Fig. 1d) were obtained regardless the inoculum

concentration was used. Fully entangled filaments (Fig. 1a)

could be obtained while rotation speed was higher than

200 rpm. Dispersed mycelia also formed when 40 ml broth

was contained in 250 mL flask. Rotation speed and fer-

mentation volume affected the shear force in flask actually,

and the effect of shear force on mycelial morphology has

been studied extensively by many researchers [6, 26].

Therefore, Mycelial aggregation is possible caused by high

shear force in this study. This conclusion could be further

confirmed by using of baffled flask which can provide

higher shear force. Small pellet (Fig. 1d) was formed in

this flask, and lipase activity reached 368 U/g (Table 1).

Same result was reported by Du et al. [27], they found that

in the baffled flask the pellet was formed, and high anti-

biotic production was obtained in R. chinesis 12 culture.

From the above result, it was considered that mycelial

aggregation (pellet and entangled filaments) seems to be a

principal factor in the enhancement of whole-cell lipase

production by R. chinensis. The reason for the different

lipase formation which caused by the morphology changes

need to be studied more in detail.

Growth process and lipase production under two typical

mycelial morphology in shake flask

Two culture conditions were performed in shake flask to

maintain typical mycelial morphology: fully entangled fil-

aments and dispersed mycelia. For fully entangled filaments,

condition A was used: inoculum level 3 9 108 spores/L,

fermentation volume 21 mL/250 mL, rotation speed

200 rpm; For dispersed mycelia, condition B was used:

inoculum level 3 9 109 spores/L, fermentation volume

40 mL/250 mL, rotation speed 150 rpm.

Table 1 Effect of culture

condition on the mycelial

morphology and lipase

production

Conditions Macromorphology Synthetic

activity(U/g)

Inoculum level 106/30 mL Fully entangled filaments 650

107/30 mL Fully entangled filaments 691

108/30 mL Dispersed mycelia (pulp like) 109

Erlenmeyer flasks 150 rpm Entangled filaments 209

200 rpm Fully entangled filaments 691

Baffled flask 150 rpm Entangled filaments 159

200 rpm Fluffy pellets (small) 368

Broth content for 250 mL flask 20 mL Entangled filaments 293

40 mL Dispersed mycelia (pulp like) 101.2

Fig. 1 Macromorphology

under different culture

conditions for Rhizopuschinensis. a Fully entangled

filaments, b Entangled

filaments, c Dispersed mycelia

(pulp like), d Pellets

400 Bioprocess Biosyst Eng (2009) 32:397–405

123

The time courses for biomass, pH, synthetic activities

for mycelium-bound lipase in shake flask are shown in

Fig. 2 and the profiles of glucose and maltose concentra-

tion are shown in Fig. 3. By the change of morphology,

parameters during fermentation process and lipase pro-

duction characteristics are remarkable different. In both

cultures, the pH increased during the fermentation process,

but the final pH within 96 h was 6.4 for fully entangled

filaments and 6.8 for dispersed mycelia. Increased biomass

levels were obtained in the use of the higher inoculum, so

the biomass for condition A was little lower than condition

B. The most remarkable difference for the two processes

were the lipase production with synthetic activity and

maltose consumption. As Fig. 2a shows, synthetic activity

for mycelium-bound lipase production reached 600 U/g at

60 h, and then maintained at 650 U/g when fully entangled

filaments formed. However, for the dispersed mycelia, the

maximal synthetic activity reached 300 U/g at 36 h, and

then the activity decreased sharply to 60 U/g at 96 h

(Fig. 2b). The same as lipase production, maltose

consumption for fully entangled filaments was remarkably

different from dispersed mycelia. Maltose concentration

decreased from 9.5 g/L to 0.3 g/L within 36 h for fully

entangled filaments. The highest concentration of corre-

sponding hydrolysate glucose was 4 g/L at 60 h and then

decreased to 0 g/L at the end of cultivation time. On other

hand, maltose concentration decreased slowly from 9.5 g/L

to 5.5 g/L within 60 h for dispersed mycelia, and glucose

concentration was maintained at 1 g/L after 48 h (Fig. 3).

From this result, it would be considered that, maltose

consumption was relative to the lipase production. Reasons

for these differences will be discussed in the following

content.

Morphological characterization in shake flask

Not only the macroscopic morphology is important for the

level of enzyme production by filamentous fungi but also

the microscopic morphology influences the production.

Therefore, the same as two typical macromorphology, and

the micro-morphology for two conditions were also dif-

ferent for condition A and B. Fig. 4 presents the

micromorphology for the dispersed mycelia and the fully

entangled filaments obtained by optical microscope and

electron microscope. Hypha from the dispersed mycelia

was fragmentated compare with the normal mycelia of R.

chinensis, the shape of mycelia was not asymmetric, dis-

sepiments appeared and some part of hyphal elements

began to swell (Fig. 4a, c). However, hypha from the fully

entangled filaments was asymmetric, no dissepiments

formed and tips of mycelia were clear (Fig. 4b, d).

Mycelial fragmentation is common in fungal fermentations

[24, 28, 29]. In certain conditions, dissepiments would

formed in some fungal which were actually typical coe-

nocytic mycelium and vacuolation also appeared. Hypha

under this state will lose their biosynthetic activity and the

Fig. 2 Time courses of biomass, pH, synthetic activities for myce-

lium-bound lipase and hydrolytic activity for mycelium-bound lipase

in shake flask cultures inoculated under different conditions. a Fully

entangled filaments process, b Dispersed mycelia process. Darklyfilled triangle biomass, darkly filled circle synthetic activity, opensquare pH

Fig. 3 The profiles of glucose (filled marker) and maltose (openmarker) concentration in two processes: diamond fully entangled

filaments; triangle dispersed mycelia

Bioprocess Biosyst Eng (2009) 32:397–405 401

123

total number of mitochondria. Even though, the vacuola-

tion and mitochondria were not determined in this study,

but abundant inclusion body was observed in fully entan-

gled filaments. Thus, mycelia from fully entangled

filaments could exhibit high biosynthetic activity which

caused rapid maltose consumption high enzyme produc-

tion, and the situation is reverse for mycelia from dispersed

mycelia.

Parameters of micromorphology for two morphologies

were described in Fig. 5. Mean diameter of mycelia from

dispersed mycelia was little higher than the mycelia from

fully entangled filaments, and the mean length of mycelia

from dispersed mycelia was much higher than the mycelia

from fully entangled filaments. The mumber of tips for

fully entangled filaments was higher than the dispersed

mycelia. Because of the tip growth is important for protein

secretion in filamentous fungi [18, 30], some researchers

has focused on this phenomenon and reported several

compellent results [11, 12]. From these results, it would be

concluded that mycelium-bound lipase production by R.

chinensis was associated with tip growth to some extent.

High number of tips could lead to high branches, and the

high branches could cause high level of cell-to-cell inter-

actions which leads to more mycelial aggregation. In

mycelial aggregates, the high level of cell-to-cell interac-

tion and signaling resulting from short diffusional

distances, leads to a state of differentiation quantitatively

different from that of free dispersed mycelia [8, 31].

Lipase production under two typical mycelial

morphology in STR

In our preliminary experiments, we found that morphology

in stirred tank reactor was different from morphology in

shake flask. Fully entangled filaments were difficult to form

in STR, only dispersed mycelia and pellet were achieved.

In order to investigated the change of morphology on broth

rheology and lipase production in STR, two culture con-

ditions were performed as well as in shake flask. For pellet,

condition A was used: agitation speed 400 rpm, inoculum

level 3 9 108 spores/L; For dispersed mycelia, condition B

Fig. 4 Contrast of microscopic morphology for the dispersed mycelia

and the fully entangled filaments: a microscopic morphology of

dispersed mycelia, b microscopic morphology of fully entangled

filaments, c electron microscope picture of dispersed mycelia, delectron microscope picture of fully entangled filaments

Fig. 5 Time courses of mean diameter, equivalent mean length and

average number of tips for filaments during two typical processes.

Open square fully entangled filaments, darkly filled circle dispersed

mycelia

402 Bioprocess Biosyst Eng (2009) 32:397–405

123

was used: Agitation speed 200 rpm, inoculum level

3 9 109 spores/L.

The effects of inoculum and agitation on morphology in

submerged has been discussed above, and were well-

known effects which have been demonstrated in a number

of studies [4, 6, 24–26, 32, 33]. The same as these reports,

pellet formation by R. chinensis is under condition of low

inoculum and high agitation speed. Morphological deter-

mination showed that smooth pellet and hairiness pellet

were obtained simultaneously, and the diameter of these

pellets was 1,000–1,600 lm under condition A.

The time courses for biomass, pH, synthetic activities

for mycelium-bound lipase and dissolved oxygen (DO) are

shown in Fig. 6. The same as shake flask, parameters

during fermentation process and lipase production charac-

teristics were remarkable different for two morphology

process. Biomass levels for condition A was little lower

than condition B because of the inoculum was different.

Specific growth rate for condition A was 0.15/h and 0.13/h

for condition B, this result might due to the high mass

transfer in condition A. Maximum lipase activity was

270 U/g in the case of condition B at 36 h, and then

decreased to 25 U/g at the end of cultivation time.

However, the maximum lipase activity was 315 U/g in the

case of condition A at 36 h, and decreased slowly to

200 U/g at end of fermentations. These result illustrated the

relationship between the morphological change and lipase

biosynthesis in the fermentation medium, with the pellet

form of growth being associated with high lipase

production.

Morphology and broth rheology study in STR

Compare with unicellular microorganism, the broth rheol-

ogy for filamentous fungi is more complex because of the

complex morphological changes for filamentous fungi [17].

Both of the increasing biomass and morphological changes

[34, 35] would lead to non-Newtonian broths, especially

for dispersed mycelia process. Therefore, pellets were

preferred in many researches [15, 36, 37], because of the

most important advantage of the pellet morphology is the

decrease in the viscosity of the culture fluid, resulting in

improved mixing and mass transfer properties.

To investigate the relationship between morphology

change and broth rheology, offline broth rheology study

was performed for two morphologies. As shown in Fig. 7,

it was observed that broth from both processes exhibit a

typical pseudoplastic non-Newtonian behavior and follow

power law model. The viscosity of the broth increased in

the early hours of fermentation and reached to maximum at

about 60 h for pellet and 72 h for dispersed mycelia.

However, broth rheology properties for pellet and dis-

persed mycelia process were significant different. For

dispersed mycelia process, the consistency index (K) was

changed from 1 to 3.5 which indicate high viscosity, and

Fig. 6 The profiles of cell growth, DO, pH, and lipase production

under condition A and B. Condition A, agitation speed 400 rpm,

inoculum level 3 9 108 spores/L; Condition B, agitation speed

200 rpm, inoculum level 3 9 109 spores/L; darkly filled trianglesynthetic activity, open circle DO, darkly filled circle biomass, darklyfilled square pH

Fig. 7 Variations in flow behavior index (n) and consistency index

(K) of fermentation under condition A (dark square) and B (opensquare). Conditon A, agitation speed 400 rpm, inoculum level

3 9 108 spores/L; Conditon B, agitation speed 200 rpm, inoculum

level 3 9 109 spores/L

Bioprocess Biosyst Eng (2009) 32:397–405 403

123

the flow behavior index (n) was changed from 0.2 to 0.4

which indicate a typical pseudoplastic broth. Under this

condition, the mass transfer and broth rheology were

affected significantly by biomass concentration and mor-

phyology change. When the biomass was 18 g/L at

stationary phase (48 h), it would be found that broth around

the wall of fermentor was nearly immovable and the olive

oil in the fermentor could not disperse sufficiently. The oil

clump which caused by the insufficient oil dispersion

resulted in the insufficient contact between oil and cell.

Because the oil is very important for the mycelium-bound

lipase biosynthesis [21], the effect of broth rheology on oil

dispersion would affect the lipase production directly.

For pellet process, the consistency index (K) was

changed from 0.7 to 2.5 which is little lower than the

dispersed mycelia process and the flow behavior index (n)

was changed from 0.4 to 0.7. This result indicated that

viscosity of the broth is lower than dispersed mycelia

process, and non-Newtonian characteristic is also not

obviously. The mixing and mass transfer properties were

improved under this condition, and oil could disperse suf-

ficiently in the fermentation system. Corresponding high

and stable mycelium-bound lipase production was

achieved.

Moreover, DO concentration in the culture broth was

different for different morphology process because of the

different agitation speeds and broth rheology, and high DO

concentration was obtained for pellets process. These

results suggest that high DO concentration may play a key

role in the enhancement of mycelium-bound lipase pro-

duction. It was well known that, even at a high DO

concentration, oxygen transfer would be limited in the

central part of the pellet. In Hilles study [38], they found

that in the pellet growth by A. niger, DO concentration was

nearly zero at pellet core (r = 0–200 lm). Other

researchers also reported that 100 lm is a limitation dis-

tance for mycelium which with biofilm [39]. Investigations

have shown that pellet formation is necessary for the pro-

duction of some enzymes from filamentous

microorganisms, such as polygalacturonase [14, 37], phy-

tase [16], glucoamylase [8], fructosyltransferase [15], and

other proteins [33]. Based on other researchers and their

results, Papagianni concluded that such phenomena may be

related to diffusional limitations in pellets, which either

reduce the extent of catabolic repression in pellets or limit

the oxygen supply, preventing oxidative inactivation of a

specific set of enzymes [1, 8]. Thus, for fully entangled

filaments in shake flask in our study, the mycelium was

more compact than pellet and mass transfer was limited

strictly and a much higher lipase production was obtained

compare with pellet. It is interesting that an enhancement

in intracellular lipase production has been observed with

immobilized cultures or aggregated format in earlier work

[13, 40]. However, those previous works were focused on

the comparison between free and immobilized or aggre-

gated mycelium and did not include studies on the

morphological changes.

Conclusion

The present study on morphological variation during

mycelium-bound lipase production by R. chinensis both in

shake flask and fermentor suggests a direct correlation of

macromorphology and micromorphology changes with the

lipase production. Morphology change is strongly influ-

enced by inoculum level and shear stress both in shake

flask and fermentor. Low inoculum level and high shear

stress results in aggregated mycelia which can produce

more mycelium-bound lipase. Study of micromorphology

reveals that microscopic morphologies for dispersed

mycelia and aggregated mycelia are different in cell shape

and biosynthetic activity. Hypha from fully entangled fil-

aments is asymmetric, no dissepiments formed and tips of

mycelia were clear, nearly 650 U/g activity is achieved

which is ten times higher than the dispersed mycelia.

Macromorphology and broth rheology study in fermentor

indicates that pellet formation results in low broth viscos-

ity. Under this condition, the oil can disperse sufficiently in

broth which is very important for lipase inducement.

However, lipase activity in fermentor is much lower

compare to shake flask. In order to further improve the

lipase activity in fermentor, a deeper understanding of the

aggregated mycelia formation mechanism and detailed

study on effect of different culture conditions on mor-

phology change are necessary.

Acknowledgment Financial supports of National Natural Science

Foundation of China (3047006), the Program for Changjiang Scholars

and Innovative Research Team in University (IRT0532), the Ministry

of Education, PR China under Program for New Century Excellent

Talents in University (NCET-04-0498), The National High Tech-

nology Research and Development Program of China (863)

(2006AA020202) and Program for Hi-Tech Research of Jiangsu

Province (BG2006011) are gratefully acknowledged.

References

1. Papagianni M (2004) Fungal morphology and metabolite pro-

duction in submerged mycelial processes. Biotechnol Adv

22:189–259

2. Thomas CR (1992) Image analysis: putting filamentous micro-

organisms in the picture. Trends Biotechnol 10:343–348

3. Grimm LH, Kelly S, Krull R, Hempel DC (2005) Morphology

and productivity of filamentous fungi. Appl Microbiol Biotechnol

69:375–384

4. Papagianni M, Mattey M (2006) Morphological development of

Aspergillus niger in submerged citric acid fermentation as a

function of the spore inoculum level. Application of neural

404 Bioprocess Biosyst Eng (2009) 32:397–405

123

network and cluster analysis for characterization of mycelial

morphology. Microb Cell Fact 5:12

5. Patnaik PR (2000) Penicillin fermentation: mechanisms and

models for industrial-scale bioreactors. Crit Rev Biotechnol

20:1–15

6. Heydarian SM, Mirjalili N, Ison AP (1999) Effect of shear on

morphology and erythromycin production in Saccharopolysporaerythraea fermentations. Bioprocess Eng 21:31–39

7. Tamura S, Park Y, Toriyama M, Okabe M (1997) Change of

mycelial morphology in tylosin production by batch culture of

Streptomyces fradiae under various shear conditions. J Ferment

Bioeng 83:523–528

8. Papagianni M, Moo-Young M (2002) Protease secretion in glu-

coamylase producer Aspergillus niger cultures: fungal

morphology and inoculum effects. Process Biochem 37:1271–

1278

9. Jin B, van Leeuwen JH, Patel B (1999) Mycelial morphology and

fungal protein production from starch processing wastewater in

submerged cultures of Aspergillus oryzae. Process Biochem

34:335–340

10. El-Enshasy H, Hellmuth K, Rinas U (1999) Fungal morphology

in submerged cultures and its relation to glucose oxidase excre-

tion by recombinant Aspergillus niger. Appl Biochem Biotechnol

81:1–11

11. Spohr A, Carlsen M, Nielsen J, Villadsen J (1997) Morphological

characterization of recombinant strains of Aspergillus oryzaeproducing alpha-amylase during batch cultivations. Biotechnol

Lett 19:257–261

12. Haack MB, Olsson L, Hansen K, Lantz AE (2006) Change in

hyphal morphology of Aspergillus oryzae during fed-batch cul-

tivation. Appl Microbiol Biotechnol 70:482–487

13. Nakashima T, Kyotani S, Izumoto E, Fukuda H (1990) Cell

aggregation as a trigger for enhancement of intracellular lipase

production by a Rhizopus species. J Ferment Bioeng 70:85–89

14. Oncul S, Tari C, Unluturk S (2007) Effect of various process

parameters on morphology, rheology, and polygalacturonase

production by Aspergillus sojae in a batch bioreactor. Biotechnol

Prog 23:836–845

15. Lim JS, Lee JH, Kim JM, Park SW, Kim SW (2006) Effects of

morphology and rheology on neo-fructosyltransferase production

by Penicillium citrinum. Biotechnol Bioprocess Eng 11:100–104

16. Papagianni M, Nokes SE, Filer K (1999) Production of phytase

by Aspergillus niger in submerged and solid-state fermentation.

Process Biochem 35:397–402

17. Olsvisk ES, Kristiansen B (1994) Rheology of filamentous fer-

mentations. Biotechnol Adv 12:1–39

18. Peberdy JF (1994) Protein secretion in filamentous fungi-Trying

to understand a highly productive black-box. Trends Biotechnol

12:50–57

19. Metz B, Kossen NWF (1977) Growth of molds in form of pellets-

Literature-review. Biotechnol Bioeng 19:781–799

20. Xu Y, Wang D, Mu XQ, Zhao GA, Zhang KC (2002) Biosyn-

thesis of ethyl esters of short-chain fatty acids using whole-cell

lipase from Rhizopus chinensis CCTCC M201021 in non-aqueous

phase. J Mol Catal B-Enzym 18:29–37

21. Teng Y, Xu Y (2008) Culture condition improvement for whole-

cell lipase production in submerged fermentation by Rhizopuschinensis using statistical method. Bioresour Technol 99:3900–

3907

22. Dong W, Yan X, Yun T (2007) Synthetic activity enhancement of

membrane-bound lipase from Rhizopus chinensis by pretreatment

with isooctane. Bioprocess Biosys Eng 30:147–155

23. Mitschka P (1982) Simple conversion of brookfield Rvt readings

into viscosity functions. Rheol Acta 21:207–209

24. Nielsen J, Johansen CL, Jacobsen M, Krabben P, Villadsen J

(1995) Pellet formation and fragmentation in submerged cultures

of Penicillium chrysogenum and its relation to penicillin pro-

duction. Biotechnol Prog 11:93–98

25. Tucker KG, Thomas CR (1992) Mycelial morphology: the effect

of spore inoculum level. Biotechnol Lett 14:1071–1074

26. Kelly S, Grimm LH, Hengstler J, Schultheis E, Krull R, Hempel

DC (2004) Agitation effects on submerged growth and product

formation of Aspergillus niger. Bioprocess Biosyst Eng 26:315–

323

27. Du LX, Jia SJ, Lu FP (2003) Morphological changes of Rhizopuschinesis 12 in submerged culture and its relationship with anti-

biotic production. Process Biochem 38:1643–1646

28. McNeil B, Berry DR, Harvey LR, Grant A, White S (1998)Measurement of autolysis in submerged batch cultures of Peni-cillium chrysogenum. Biotechnol Bioeng 57:297–305

29. Harvey LM, McNeil B, Berry DR, White S (1998) Autolysis in

batch cultures of Penicillium chrysogenum at varying agitation

rates. Enzyme Microb Technol 22:446–458

30. Wosten HAB, Moukha SM, Sietsma JH, Wessels JGH (1991)

Localization of growth and secretion of proteins in Aspergillusniger. J Gen Microbiol 137:2017–2023

31. Braun S, Vechtlifshitz SE (1991) Mycellal morphology and

metabolite production. Trends Biotechnol. Trends Biotechnol

9:63–68

32. Amanullah A, Christensen LH, Hansen K, Nienow AW, Thomas

CR (2002) Dependence of morphology on agitation intensity in

fed-batch cultures of Aspergillus oryzae and its implications for

recombinant protein production. Biotechnol Bioeng 77:815–826

33. El-Enshasy H, Kleine J, Rinas U (2006) Agitation effects on

morphology and protein productive fractions of filamentous and

pelleted growth forms of recombinant Aspergillus niger. Process

Biochem 41:2103–2112

34. Olsvik E, Tucker KG, Thomas CR, Kristiansen B (1993) Corre-

lation of Aspergillus niger broth rheological properties with

biomass concentration and the shape of mycelial aggregates.

Biotechnol Bioeng 42:1046–1052

35. Riley GL, Tucker KG, Paul GC, Thomas CR (2000) Effect of

biomass concentration and mycelial morphology on fermentation

broth rheology. Biotechnol Bioeng 68:160–172

36. Lopez JLC, Perez JAS, Sevilla JMF, Porcel EMR, Chisti Y

(2005) Pellet morphology, culture rheology and lovastatin pro-

duction in cultures of Aspergillus terreus. J Biotechnol 116:61–77

37. Gogus N, Tari C, Oncu S, Unluturk S, Tokatli F (2006) Rela-

tionship between morphology, rheology and polygalacturonase

production by Aspergillus sojae ATCC 20235 in submerged

cultures. Biochem Eng J 32:171–178

38. Hille A, Neu TR, Hempel DC, Horn H (2005) Oxygen profiles

and biomass distribution in biopellets of Aspergillus niger. Bio-

technol Bioeng 92:614–623

39. Viniegra-Gonzalez G, Favela-Torres E (2006) Why solid-state

fermentation seems to be resistant to catabolite repression. Food

Technol Biotechnol 44:397–406

40. Nakashima T, Fukuda H, Nojima Y, Nagai S (1989) Intracellular

lipase production by Rhizopus chinensis using biomass support

particles in a circulating bed fermenter. J Ferment Bioeng 68:19–

24

Bioprocess Biosyst Eng (2009) 32:397–405 405

123