Influence of Stirrer Speed on the Morphology of Aspergillus carbonarius var (Bainier) Thom IMI 366159 During Raw Starch Digesting Amylase Production
Onyetugo C. Amadi
Bartho N. Okolo
Agitation speed had various influence on the performance of Aspergillus carbonarius during the production of raw starch digesting amylase. By means of image analysis the following parameters; mean hyphal growth unit, mean total hyphal length, mean number of tips/mycelium and mean convex perimeter (P) of clump/pellet were used to characterize the mycelia morphology. Amylase activity, protein content, biomass concentration and pH were determined. The study was aimed at investigating how agitation speed will affect mycelia morphology and culture parameters during raw starch digesting amylase production in shake flask cultures. Fungal morphology was greatly influenced by agitation, with large pellets predominating at 100 revolutions per minute (rpm) while clumped mycelia was predominant at 150 and 200 rpm. Increasing the agitation speed caused a reduction in mycelia particle sizes. Maximum Raw Starch Digesting Amylase (RSDA) activity was achieved at a shake speed of 100 rpm with minimum protein concentration. In contrast, maximum protein concentration and lower amylase enzyme activity was achieved at a shaker speed of 150 rpm. On the other hand, at the highest shaker speed of 200 rpm used in this study, highest biomass and intermediate amylase enzyme activity as well as intermediate protein concentration were observed. These results show that shaker speed will play important role in obtaining optimum results of the various different parameters (amylase activity, protein concentration and biomass production) during the production of amylase from Aspergillus carbonarius. In these experiments, shaker speed had little or no influence on pH of the media during RSDA production.
to cite this article:
Onyetugo C. Amadi and Bartho N. Okolo, 2013. Influence of Stirrer Speed on the Morphology of Aspergillus carbonarius var (Bainier) Thom IMI 366159 During Raw Starch Digesting Amylase Production. Asian Journal of Biological Sciences, 6: 84-95.
Received: November 28, 2012;
Accepted: March 13, 2013;
Published: June 04, 2013
Amylases are very important and widely used enzymes, accounting for about 30%
of world enzyme production (Van Der Maarel et al.,
2002). They play very prominent role in biotechnology with wide range application
which span over the textile industries, paper and cellulose, leather, detergents,
beverages, conversion of starch to sugars, animal chow, fermentation, medical
and pharmaceutical industries (Okolo et al., 2000;
Pandey et al., 1999; Butzen
and Haefele, 2008). Amylases are enzymes that breakdown starch to simple
sugars (Akpan et al., 1999; Okolo
et al., 2000) and they can be found in plants, fungi, yeast, bacteria
and actinomycetes (Okoli et al., 2010; Jaiswal
et al., 2011; Nwagu and Okolo 2011; Roohi
et al., 2011; Joshi, 2011; Pandey
et al., 2000), however, the bacterial and fungal source of the enzyme
are mostly applied in the industry (Pandey et al.,
1999). The efficient production of amylases depends on several physical
and chemical factors such as pH of reaction mixtures, temperature, medium composition,
moisture, agitation, incubation period as well as nature of inoculum (Smith
et al., 1990, Teodoro and Martins, 2000;
Saxena et al., 2007; EL-Tayeb
et al., 2007; Robertson et al., 2006;
Juge et al., 1998; Papagianni
et al., 1999; Singh et al., 2011).
Enzymatic bioconversion of raw starch without prior gelatinisation has gained
attention in both academia and industry because of the simplicity of the process
and the fact that energy is saved. Raw starch-digesting amylases can catalyse
the degradation of raw starch to reducing sugars and maltodextrins bypassing
the gelatinisation step. Aspergillus carbonarius var (Bainier) Thom IMI
366159 isolated from rotten cassava in the laboratory of the University of Nigeria
is able to produce large amounts of the raw starch-digesting enzyme, as well
as the ability to digest a wide range of starch from different sources (Okolo
et al., 2000). Other special features of the amylase from A.
carbonarius, from a biotechnological perspective are their broad pH tolerance
and stability, broad substrate specificity, production of maltose as the predominant
degradation product and its temperature tolerance and stability (Okolo
et al., 2000). These features differentiate the amylase of A.
carbonarius from similar enzymes earlier reported and may provide practical
advantages during large-scale industrial bioconversion of starch to value-added
It is well known that in submerged fermentations, agitation is important in
order to achieve a good mixing, mass and heat transfer. Although mycelial damage
at high stirrer speeds or power inputs can limit the acceptable range of speeds
and subsequently, the oxygen transfers capability and the volumetric productivity
of the fermentor (Amanullah et al., 1998). Agitation
speed of culture broth has a variety of effects on microorganisms which include;
damage to cell structure, morphological changes, as well as variations in growth
rate and product formation (Papagianni et al., 1999).
For each culture, optimum conditions of agitation exist that will partly depend
on the resistance of the hyphae to mechanical forces and also on their physiological
state (Mitchell and Lonsane, 1990; Paul
et al., 1994; Moo-Young et al., 1992;
Papagianni et al., 1998). Several authors (Lin
et al., 2010; Ahamed and Vermette, 2010;
Fadzilah and Mashitah, 2010; El-Enshasy
et al., 2006; Park et al., 2002; Gerlech
et al., 1998; Papagianni et al., 1998)
have demonstrated the effects of mechanical forces on the morphology of filamentous
microorganisms and the overall process productivities (Metz
et al., 1981) in studies with P. Chrysogenium showed that
the length of the mycelial particle decreased with increasing power input per
unit mass in the reactor as the increased agitation caused the hyphae to become
shorter, thicker and more highly branched. Studying the behaviour of three citric
acid producing A. niger strains, Ujcova et al.
(1980) observed that higher speed resulted in thicker, densely branched
filaments while citric acid production was optimum within a narrow range of
speeds and a drop in productivity resulted from higher stirrer speeds. The effect
of agitation on A. niger was investigated by Papagianni
et al. (1994, 1998) in a stirred tank and
tubular loop bioreactor through a series of batch and feed batch experiments.
Morphological measurements using image analysis showed that by increasing the
intensity of agitation, the size of clumps decreased, as did the length of the
filaments that arose from the core of the clumps while the diameter of the filaments
increased, in both fermenters, specific rates of citric acid formation increased
with agitation. Schugerl et al. (1998) and
Gerlech et al. (1998) investigated the effect
of different reactor types and types of shear stress (shake flask, stirred tank,
airlift bioreactors) in studies with xylanase fermentation by Aspergillus
awomari. They observed that large loose hairy pellets were formed in pneumatically
mixed reactors (airlift tower loop) while small and compact pellets were formed
in shake flask and stirred tank reactors.
Ahamed and Vermette (2010) reported that influence
of mechanical agitation using draft-tube airlift bioreactor, resulted in shorter
mycelia hyphae and larger number of tips during the production of cellulase
by Trichoderma reesei. The relationship between fungal morphology and
process productivities aroused interest from both academia and industry and
attempts have been made to manipulate morphology to achieve maximal performance.
Despite all this studies there has been no report relating the production of
amylase to the producing organism. Again the fact that RSDA is important in
industrial application it will be interesting to document the effect of agitation
on the morphology of A. carbonarius and raw starch digesting amylase
production. Hence, considering the current economic situation and with emphasis
on the use of locally available resources (bearing in mind that commercial enzymes
are very expensive), the study focused on the use of readily available organism
Aspergillus carbonarius to investigate how agitation speed will affect
mycelia morphology, amylase enzyme activity, protein content and biomass formation
during raw starch digesting amylase production in shake flask cultures. The
results obtained are presented in this study.
MATERIALS AND METHODS
Microorganism: Aspergillus carbonarius var (Bainier) Thom IMI 366159
used in the study was obtained from University of Nigeria, Nsukka culture collection.
The organism was maintained on Potato Dextrose Agar (PDA) slants subcultured
every 3 months.
Inoculum and media preparations: Aspergillus carbonarius maintained
on Potato Dextrose Agar (PDA) slants was transferred to fresh PDA plates and
incubated at 30°C for 7 days. Spores were harvested from culture plates
in sterile distilled water and spore suspensions were used as inocula at a level
of 107 spores mL-1. The fermentation medium comprised
(g L-1): Raw cassava starch (Manihot utilissima Crantz), 20;
yeast extract, 5; KH2PO4, 7; CaCl2.6H2O,
0.3 and MgSO4.7H2O, 0.3. Five hundred millilitre (500
mL) flask containing 100 mL sterile fermentation medium was inoculated with
spores and fermentation was carried out with rotary shaking, three levels of
shaker speed were examined 100, 150 and 200 rpm and 30°C for 144 h.
Analytical methods: Dry weight was determined by filtering 10 mL of
broth with pre-weighed Whatman No. 1 filter paper, washed with distilled water
three times and dried in an oven at 80°C until a constant weight was obtained.
Results were expressed as grams dry weight per litre of broth. Raw starch digesting
amylase was assayed as described by Okolo et al.
(2000). Total protein was estimated by Bradford (1976)
method using Bovine Serum Albumin (BSA) as standard.
Image analysis and processing: Image capture was carried out via a semiautomatic
image analyser Moticam 1000 digital microscope camera UK, (Motic Images Plus
2.0 mL) with a USB 2.0 cable. Mounted on a microscope (Optika, Italy) connected
to a PC.
Samples for morphological characterization were taken from 24 h twice a day
for 144 h. Samples were immediately fixed with an equal volume of fixative as
described by Packer and Thomas (1990) using 13 mL of
40% w/v formaldehyde and 5 mL glacial acetic acid added to 200 mL of 50% w/v
ethanol. The fixed samples were further diluted with fixative and dilution was
adjusted to separate the mycelia clumps on the microscope slide. Images were
captured at 640-512 pixels. A magnification of 40x and 100x was applied for
measurements on mycelia particles.
Segmentation was performed to obtain a binary image; measurements were carried
out on binary images and by adjustment of greyness levels. A filter was applied
to the segmentation to show the ends and branching points and the total length
was measured. Medium particles artefacts, debris and morphological particles
which were touching the organism and could have biased the results were removed
and in some cases computer aided manual measurement was applied to measure and
subtract false images. A total of 150 elements were analysed. These elements
included clumps (dense aggregates of entangled hyphae) as well as freely dispersed
form. Morphological parameters of interest for the freely dispersed mycelia
were mean hyphal growth unit (total length divided by number of growing tips),
mean total hyphal length and mean number of tips/mycelium. Clump/pellet morphology
was quantified in terms of mean convex perimeter (Tucker
et al., 1992).
RESULTS AND DISCUSSION
The effect of agitation speed on Aspergillus carbonarius morphology
and RSDA production in shake flask cultures was investigated at 100, 150 and
200 rpm. Image analysis of broth samples at 100 rpm revealed, a mixture consisting
mainly of pellets and a small number of entangled mycelia (clumps). At a speed
of 150 or 200 rpm, the bulk of the mycelium was in the form of clumps (aggregated
mycelial trees). Figure 1 shows the characteristic morphology
of A. carbonarius at 144 h in shake flask cultures at different shaker
speeds. At a shaker speed of 100 rpm pellets were large; hairy with a central
core, formation of large pellets was partly due to low mechanical stress. Also,
the clumps were loose with filaments that did not look fragmented (Fig.
1c and d). Small and compact pellets were formed at 150
and 200 rpm and clumps appeared more tightly packed. Papagianni
et al. (1998) investigated the effect of agitation on A. niger
morphology and citric acid production and they observed that the bulk of the
mycelium was in the form of clumps. This is however, contrary to the results
obtained in this study which showed that at a shaker speed of 100 rpm the bulk
of the mycelium was in pelleted form, though the experiment was carried out
in a shaker flask, in contrast to stirred tank used by Papagianni
et al. (1998). It is not clear at present if the difference observed
in the mycelia form or shape was caused by differences in the reactor systems
used. In this regard while in this study the shaker method was used, Papagianni
et al. (1998) used stirred tank method to obtain their results. Schugerl
et al. (1998) and Gerlech et al. (1998)
compared results obtained from shear stress and different reactor types; shake
flask, stirred tank and airlift bioreactors in xylanase production by A.
awamori., they observed that large, loose, hairy pellets were formed in
mixed reactors (airlift tower loop) while small and compact pellets formed in
shake flasks and stirred tank reactors. This is in agreement with the current
results were small and compact pellets formed in shake flask cultures and confirms
the fact that the method of agitation and reactor type will influence the shape
of mycelium. Figure 2 shows the mean hyphal growth unit, mean
number of tips/mycelium, mean total hyphal length and mean convex perimeter
of clumps/pellets of Aspergillus carbonarius under different agitation
speed. The results revealed that increasing the agitation speed caused a decrease
in the mycelial particles examined. The mean number of tips per hyphal element
(of freely dispersed form) was found to be similar at all three agitation speeds.
|| Characteristic morphology of the fungus Aspergillus carbonarius
in shake flask cultures at 144 h at different shaker speed, (a-d) Pellet/clumps
at 100 rpm, (e-h) Pellets/clumps at 150 rpm and (i-l) Pellets/clumps at
Although it is not clear why this was the case, it is possible that the rate
at which growth occurred and subsequent fragmentation of mycelia particles was
relatively similar between the different agitation speeds giving rise to similar
mean number of tips per mycelium or it may require a much higher agitation speed
(say 500-1000 rpm) to obtain a remarkable difference in mean number of tip.
Amanullah et al. (1999) reported similar values
of (7.4±1.3 and 7.7±1.6) respectively for 550-700 rpm in chemostat
cultures of recombinant Aspergillus oryzae. They proposed that the balance
between growth and fragmentation were similar and hence, gave rise to similar
mean number of tips per hypha. However, several authors have reported that hyphae
are shorter, thicker and more highly branched at high agitation speeds compared
to low speed (Dion et al., 1954; Ujcova
et al., 1980; Metz et al., 1981;
Van Suijdam and Metz, 1981). The mean hyphal growth
unit and the mean total hyphal length decreased as agitation speed increased.
|| The effect of shaker speed on morphology of Aspergillus
carbonarius, (a) Mean total hyphal length, (b) Mean hyphal growth unit,
mean number of tips/mycelium and (c) Mean convex perimeter of clumps/pellets
at 144 h of shake flask cultures at different shaker speed (100,150 and
Smith et al. (1990) and Metz
et al. (1981) in their studies with Pe. chrysogenum showed
that the length of mycelial particles decreased as agitation increased. The
clump/pellet sizes (convex perimeter) decreased with increase in agitation speed.
This observation is consistent with the results of other studies Papagianni
et al. (1998), who investigated the relationship between the morphology
of Aspergillus niger and citric acid production in a tubular loop and
a conventional stirred tank bioreactor. Morphology characterized by the parameter
P (mean convex perimeter of clumps) showed that increased agitation caused a
reduction of clump sizes. On the contrary, Maazi et al.
(1998) and Amanullah et al. (1999) reported
that entangled mycelia may occur due to longer branches that have a higher probability
of overlapping on each other. Thus, the mean projected area of branched mycelia
became larger with time hence, under these conditions, this signify that mycelia
growth dominated over mycelia fragmentation.
|| Time course of (a) Raw starch digesting amylase activity,
(b) Protein content, (c) Biomass concentrations and (d) pH, in shake flask
cultures at different shaker speed (100, 150, 200 rpm)
The results in Fig. 3 show the different effects of agitation
speed on the performance of Aspergillus carbonarius during raw starch
digesting amylase production. The results in Fig. 3 show that
the lower shaker speed of 100 rpm produced the highest RSDA activity (Fig.
3a). In contrast, a speed of 150 rpm produced the least enzyme activity.
Generally peak production of RSDA was achieved at 72 h at all shaker speed studied.
In contrast to the results found in RSDA activity, agitation speed of 150 rpm
released the highest protein content while a shaker speed of 100 rpm released
the least protein content (Fig. 3b). These results (Fig.
3a, b) confirm the well known fact that all enzymes are
protein but not all proteins are enzymes. In these results (Fig.
3a and b) agitation speed of 200 rpm produced intermediate
results observed for agitation speed of 100 and 150 rpm. It is interesting to
observe that while agitation speed of 200 rpm produced intermediate results
for RSDA and protein content when compared to agitation speed of 100 and 150
rpm, higher biomass production was achieved at agitation speed of 200 rpm (Fig.
3c).The exact reasons for these observations are not clear at present. The
results in Fig. 3d show that agitation speed did not affect
the pH of the medium during amylase production from Aspergillus carbonarius.
Many investigators have discussed effect of agitation on morphology and biosynthesis.
There are cases where growth rate and productivities were linked to morphology
or cases were no link was observed (Markel and Bronnemeier,
1985; Belmer-Beiny and Thomas, 1991; Anusha
et al., 2012). From the trends of the RSDA time courses, it is very
likely that fungal morphology played a key role in the process (Amadi
and Okolo, 2012). In the present study, the three different agitation levels
resulted in the development of distinctive morphological forms. Pellets predominated
at 100 rpm shaker speed which resulted in increased RSDA levels and this morphological
form has been associated with increased specific growth rates. Formation of
mycelial pellets is often regarded as a prerequisite for successful production
of microbial metabolites, including some fungal enzymes such as polygalacturonidase
or glucosidase and RSDA (Hermersdorfer et al., 1987;
Amadi and Okolo, 2012). But in some cases, the free
filamentous morphology is preferred for optimal metabolite formation (Schugerl
et al., 1998). In any case, Polygalacturonidase synthesis correlates
well with the pelleted morphological type of A. niger. The more compact
the pellet, the greater the polygalacturonidase synthesis, regardless the composition
of the medium (Hermersdorfer et al., 1987). In
the present study, the increased rates of RSDA production from pellets may suggest
that this morphological form is most suitable for the RSDA formation (Amadi
and Okolo, 2012). However, as studies have shown the centre of the pellets
becomes autolysed with time and does not contribute to metabolite biosynthesis
anymore (Wittler et al., 1986). Therefore, a
lower proportion of the biomass is involved in biosynthesis and this may explain
the reversing trend observed beyond 75 h of fermentation. This is not the case
with the bulk of clumped morphology obtained from 150 and 200 rpm which had
lower RSDA levels and higher biomass concentration. The high biomass concentration
could have been the reason for low volumetric enzyme productivity in terms of
quantity with the increased agitation speed (150 and 200 rpm). Lower productivities
have been reported at high agitation speeds and were attributed to cell damage
(Smith et al., 1990). Paul
et al. (1999) also reported that citric acid yield was decreased
with high agitation speed in submerged cultures of Aspergillus niger.
In this study, fragmentation is likely to have occurred at increased agitation
levels resulting in low levels of RSDA production and high biomass concentration
in 150 and 200 rpm. Usually fragmentation resulted from increased shear stress
applied to the mycelium which is characteristic of high stirrer speed (Ayazi
Shamlou et al., 1994). Fragmentation of hyphal elements and pellets
during submerged fermentation often results in growth renewal since fragments
may act as centres for new growth, enabling reseeding of the mycelia population
(Papagianni, 2004). Long and ramified hyphae increase
the number of possible interaction sites with the substrate (De
Nicolas-Santiago et al., 2006). The number of newly growing tips
from the main hyphae is an indication of active biomass accumulation in the
culture medium. Protein secretion in filamentous fungi is believed to mainly
occur at the tips of growing hyphae because these tips are more porous, therefore,
facilitating exo-enzymes to pass through the cell wall (Peberdy,
1994; Wosten et al., 1991; Wessels,
1993). Thus, factors that increase the number of active tips may improve
protein yield (Juge et al., 1998). Branched
hyphae resulted in an increase in the number of tips and yielded more proteins
into the fermentation broth (Wosten et al., 1991).
This may account for the increase in protein content and increased biomass concentration
at 150 and 200 rpm earlier observed. Tip growth and branching result in different
macroscopic appearances of the culture, ranging from single hypha elements,
dispersed mycelia, over connected networks of hyphae up to distinct biomass
particles. Previous studies (Peberdy 1994; Juge
et al., 1998; Ahamed and Vermette, 2009)
have shown that the morphology and physiology of filamentous microorganisms
in submerged cultures are dependent on culture conditions. Ahamed
and Vermette (2010) reported that in fed-batch cultures, T. reesei
were mainly found in the dispersed form which comprises unbranched, branched
and entangled mycelia. In the filamentous growth form a lower proportion of
cells become autolysed with time and this can result in increased final productivities
compared with the pelleted growth form (Papagianni et
In this study, from the view point of using locally available materials authors
have also been able to demonstrate that Aspergillus carbonarius morphology
is also affected by the agitation speed during RSDA production. At lower agitation
speed pellet morphology dominated resulting in higher RSDA activity. But increased
protein production was observed at the highest stirrer speed. In other words,
when studying enzyme stabilization it will be wise to use pellet morphology
to obtain enzyme with high activity.
Ahamed, A. and P. Vermette, 2009.
Effect of culture medium composition on Trichoderma reesei's
morphology and cellulase production. Bioresour. Technol., 100: 5979-5987.CrossRef |
Ahamed, A. and P. Vermette, 2010.
Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor. Biochem. Eng. J., 49: 379-387.CrossRef |
Akpan, I., M.O. Bankole, A.M. Adesemowo and G.O. Lantunde-Data, 1999.
Production of amylase by A. niger
in a cheap solid medium using rice bran and agricultural materials. Trop. Sci., 39: 77-79.Direct Link |
Amadi, O.C. and B.N. Okolo, 2012.
Characterisation of morphological forms of Aspergillus carbonarius
and the effect of inoculums size on raw starch digesting amylase production. Afr. J. Microbiol. Res., 6: 1934-1941.Direct Link |
Amanullah, A., L. Serrano-Carreon, B. Castro, E. Galindo and A.W. Nienow, 1998.
The influence of impeller type in pilot scale Xanthan fermentations. Biotechnol. Bioeng., 57: 95-108.CrossRef |
Anusha, N.C., M.S. Umikalsom, T.C. Ling and A.B. Ariff, 2012.
Relationship between fungal growth morphologies and ability to secrete lipase in solid state fermentation. Asian J. Biotechnol., 4: 15-29.CrossRef |
Amanullah, A., R. Blair, A.W. Nienow and C.R. Thomas, 1999.
Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae
. Biotehnol. Bioeng., 62: 434-446.CrossRef |
Ayazi Shamlou, P., H.Y. Makagiansar, A.P. Ison and M.D. Lilly, 1994.
Turbulent breakage of filamentous microorganisms in submerged cultures in mechanically stirred bioreactors. Chem. Eng. Sci., 49: 2621-2631.CrossRef |
Belmer-Beiny, M.T. and C.R. Thomas, 1991.
Morphology and clavulanic acid production of Streptomyces clavuligerus
: Effect of stirrer speed in batch fermentations. Biotech. Bioeng., 37: 456-462.CrossRef |
Bradford, M.M., 1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.CrossRef | PubMed | Direct Link |
Butzen, S. and D. Haefele, 2008.
Dry-grind ethanol production from corn. Crop Insights, 18: 10-15.
Dion, W.M., A. Carilli, G. Sermonti and E.B. Chain, 1954.
The effect of mechanical agitation on the morphology on the morphology of Penicillum chrysogenum
Thom in stirred fermenters. Rend 1st Super Sanita, 17: 187-205.
El-Enshasy, H., J. Kleine and U. Rinas, 2006.
Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger
. Process Biochem., 41: 2103-2112.CrossRef |
EL-Tayeb, O., F. Mohammad, A. Hashem and M. Aboulwafa, 2007.
Optimizationof the industrial production of bacterial alpha amylase in Egypt. IV fermenter production and characterization of the enzyme by two strains of Bacillus subtillis
and Bacillus amyloliquefacious
. Afr. J. Biotechnol., 24: 4521-4536.
Fadzilah, K. and M.D. Mashitah, 2010.
Cellulases production in palm oil mill effluent: Effect of aeration and agitation. J. Applied Sci., 10: 3307-3312.CrossRef |
Gerlech, S.R., D. Siedenberg, D. Gerlech, K. Schugerl, M.L.F. Giuseppin and J. Hunik, 1998.
Influence of reactor systems on the morphology of Aspergillus awamori
. Application of neural network and cluster analysis for characterization of fungal morphology. Process Biochem., 33: 601-615.CrossRef |
Hermersdorfer, H., A. Leuchetenberger, C.H. Wardsock and H. Ruttloff, 1987.
Influence of culture conditions on mycelial structure and polygalacturanase synthesis of Aspergillus niger
. J. Basic Microbiol., 27: 309-315.PubMed | Direct Link |
Wessels, J.G.H., 1993.
Wall growth protein excretion morphogenesis in fungi. New Phytol., 123: 397-413.CrossRef |
Joshi, B.H., 2011.
A novel thermostable alkaline α-amylase from Bacillus circulans
PN5: Biochemical characterization and production. Asian J. Biotechnol., 3: 58-67.CrossRef | Direct Link |
Juge, N., B. Syensson and G. Williamson, 1998.
Secretion, purification and characterization of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger
. Applied Microbial. Biotechnol., 49: 385-392.CrossRef | PubMed |
Maazi, A., M.N. Pons, H. Vivier, E. Latrille, G. Corrieu and T. Cossen, 1998.
Morphological characterization. Proceedings of the 2nd European Symposium on Biochemical Engineering Science, September 16-19, 1998, Porto, Portugal, pp: 1-234
Markel, H. and R. Bronnemeier, 1985.
Mechanical Stress and Microbial Production. In: Fundamentals of Biochemical Engineering, Brauer, H. (Ed.), Vol. 2, Weinheim, VCH., pp: 370-390
Metz, B., E.W. de Bruijn and J.C. van Suijdam, 1981.
Method for quantitative representation of the morphology of molds. Biotechnol. Bioeng., 23: 149-162.CrossRef |
Mitchell, D.A.T. and B.K. Lonsane, 1990.
Dennition, Characterization and Economic Evaluation. In: General Principles of Solid Substrate Fermentation, Doelle, H.W. and C. Rolz (Eds.). Rapid Publications Oxford Ltd., London, UK
Moo-Young, M., Y. Christi and D. Vlech, 1992.
Fermentative conversion of cellulosic substrates to microbial protein by Neurospora sitophila
. Biotechnol. Lett., 14: 863-868.CrossRef | Direct Link |
Jaiswal, N., O. Prakash, M. Talat, S.H. Hasan and R.K. Pandey, 2011.
Application of response surface methodology for the determination of optimum reaction conditions (Temperature and pH) for starch hydrolysis by α-amylase. Asian J. Biochem., 6: 357-365.CrossRef |
Nwagu, T.N. and B.N. Okolo, 2011.
Growth profile and Amylolytic hydrolytic activity of a thermophilic fungi Aspergillus fumigatus
isolated from soil. Asian J. Biotechnol., 3: 46-57.CrossRef | Direct Link |
Okoli, E.V., B.N. Okolo, A.N. Moneke and F.S. Ire, 2010.
Effects of cultivar and germination time on amylolytic potential, extract yield and wort fermenting properties of malting sorghum. Asian J. Biotechnol., 2: 14-26.CrossRef | Direct Link |
Okolo, B.N., F.S. Ire, L.I. Ezeogu, C.U. Anyanwu and F.J.C. Odibo, 2000.
Purification and some properties of a novel raw starch-digesting amylase from Aspergillus carbonarius
. J. Sci. Food Agric., 81: 329-336.Direct Link |
Pandey, A., S. Benjamin, C.R. Soccol, P. Nigam, N. Krieger and V.T. Soccol, 1999.
The realm of microbial lipases in biotechnology. Biotechnol. Applied Biochem., 29: 119-131.PubMed | Direct Link |
Pandey, A., P. Nigam, C.R. Soccol, V.T. Soccol, D. Singh and R. Mohan, 2000.
Advances in microbial amylases. Biotechnol. Applied Biochem., 31: 135-152.PubMed | Direct Link |
Papagianni, M., M. Mattey and B. Kriatiansen, 1994.
Morphology and citric acid production of Aspergillus niger
PMI. Biotechnol. Lett., 16: 929-934.CrossRef |
Papagianni, M., M. Mattey and B. Kriatiansen, 1998.
Citric acid production and morphology of Aspergillus niger
as function of mixing intensity in stirred tank and tubular loop bioreactor. Biochem. Eng. J., 2: 197-205.CrossRef |
Papagianni, M., M. Mattey and B. Kriatiansen, 1999.
Hyphal vacuolation and fragmentation in batch and fed-batch culture of Aspergillus niger
and its relationship to citric acid production. Process Biochem., 35: 359-366.CrossRef |
Papagianni, M., 2004.
Fungal morphology and metabolite production in submerged mycelia processes. Biotech. Adv., 22: 189-259.CrossRef | PubMed | Direct Link |
Papagianni, M., S.E. Nokes and K. Filer, 2001.
Submerged and solid-state phytase fermentation by Aspergillus niger
: Effects of agitation and medium viscosity on phytase production, fungal morphology and inoculum performance. Food Technol. Biotechnol., 39: 319-326.Direct Link |
Packer, H.L. and C.R. Thomas, 1990.
Morphological measurement on filamentous microorganism by fully automatic image analysis. Biotechnol. Bioeng., 35: 890-901.CrossRef |
Park, J.P., Y.M. Kim, S.W. Kim, H.J. Hwang and Y.J. Cho et al
Effect of agitation intensity on the exobiopolymer production and mycelial morphology in Cordyceps militaris
. Let. Applied Microbiol., 34: 433-438.
Paul, G.C., C.A. Kent and C.R. Thomas, 1994.
Hyphal vocuolation and fragmentation Inpenicillium chrysogenum
. Biotechnol. Bioeng., 44: 655-660.CrossRef |
Paul, G.C., M.A. Priede and C.R. Thomas, 1999.
Relationship between morphology and citric acid production in submerged Aspergillus niger
fermentations. Biochem. Eng. J., 3: 121-129.CrossRef |
Peberdy, J.F., 1994.
Protein secretion in filamentous fungi-trying to understand a highly productive black box. Trends Biotechnol., 12: 50-57.CrossRef |
Lin, P.J., A. Scholz and R. Krull, 2010.
Effect of volumetric power input by aeration and agitation on pellet morphology and product formation of Aspergillus niger. Biochem. Eng. J., 49: 213-220.CrossRef |
Robertson, G.H., D.W.S. Wong, C.C. Lee, K. Wagshal, M.R. Smith and W.J. Orts, 2006.
Native or raw starch digestion: A key in energy efficient biorefining of grain. J. Agric. Food Chem., 54: 353-365.CrossRef |
Roohi, K.M., I.Z. Ahmad and J.M. Arif, 2011.
Production of coldactive extracellular α-amylase by newly isolated Microbacterium foliorum
GA2 from gangotri glacier, Western Himalaya, India. Asian. J. Biotechnol., 3: 449-459.CrossRef |
Saxena, R.K., K. Dutt, L. Agarwal and P. Nayyar, 2007.
A highly thermostable and alkaline amylase from a Bacillus
sp. PN5. Bioresour. Technol., 98: 260-265.CrossRef | Direct Link |
Schugerl, K., S.R. Gerlech and D. Siedenberg, 1998.
Influence of the process parameters on the morphology and enzyme production of Aspergilli
. Adv. Biochem. Eng. Biotechnol., 60: 195-266.CrossRef |
Singh, R., V. Kapoor and V. Kumar, 2011.
Influence of carbon and nitrogen sources on the α- amylase production by a newly isolated thermotolerant Streptomyces
sp. MSC702 (MTCC 10772). Asian J. Biotechnol., 3: 540-553.CrossRef | Direct Link |
Smith, J.J., M.D. Lilly and R.I. Fox, 1990.
The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum
. Biotechnol. Bioeng., 35: 1011-1023.CrossRef |
Teodoro, C.E.D.S. and M.L.L. Martins, 2000.
Culture conditions for the production of thermostable amylase by bacillus
sp. Braz. J. Microbiol., 31: 298-309.CrossRef | Direct Link |
Tucker, K.G., T. Kelly, P. Delgrazia and C.R. Thomas, 1992.
Fully-automatic measurement of mycelial morphology by image analysis. Biotechnol. Prog., 8: 353-359.CrossRef |
Ujcova, E., Z. Fencl, M. Musilkova and L. Seichert, 1980.
Dependence of release of nucleotides from fungi on fermentor turbine speed. Biotechnol. Bioeng., 22: 237-241.CrossRef |
Van Suijdam, J.C. and B. Metz, 1981.
Influence of engineering variables upon the morphology of filamentous molds. Biotechnol. Bioeng., 23: 111-148.CrossRef |
Wittler, R., H. Baumgartl, D.W. Lubberts and K. Schugerl, 1986.
Investigations of oxygen transfer into Penicillium chrysogenum
pellets by microprobe measurements. Biotechnol. Bioeng., 28: 1024-1036.CrossRef |
Wosten, H.A.B., S.M. Moukha, J.H. Sietsma and J.G.H. Wessels, 1991.
Localization of growth and secretion of proteins in Aspergillus niger
. J. Gen. Microbiol., 137: 2017-2023.CrossRef | PubMed | Direct Link |
De Nicolas-Santiago, S., C. Regalado-Gonzalez, B. Garcia-Almendarez, F.J. Fernandez, A. Tellez-Jurado and S. Huerta-Ochoa, 2006.
Physiological, morphological and mannanase production studies on Aspergillus niger
uam-gs1 mutants. Elect. J. Biotechnol., 9: 51-60.CrossRef | Direct Link |
Van der Maarel, M.J.E.C., B. van der Veen, J.C.M. Uitdehaag, H. Leemhuis and L. Dijkhuizen, 2002.
Properties and applications of starch-converting enzymes of the α-amylase family. J. Biotechnol., 94: 137-155.CrossRef | PubMed | Direct Link |