INTRODUCTION
Lipase enzymes have achieved a prominent positioning in the global market with
continuous increase in market demand. The huge and attractive industrial applications
of lipases have moved this enzyme into the third group in volume of sales after
proteases and amylases (Pandey, 2003; Mahadik
et al., 2002; Freire et al., 1997).
Lipases are able to produce by various microorganisms, however, fungi which
is widely known as one of the best lipase sources can be easily cultured in
Solid State Fermentation (SSF) where growth and secretion of the product may
occurs on the surface of the solid support and within the support matrix.
Ample of research has been conducted to enhance lipase production by fungi
in SSF. The parameters that were evaluated include supplementation of specific
nutrients to the solid substrate, pH, temperature, moisture content and aeration.
Moreover, in the design and operation of bioreactor employing fungus, knowledge
on fungal morphology is one of the key factors need to be considered. In SF,
variation in hydrodynamic conditions may influence growth morphology of the
fungi. On the other hand, growth is restricted to the surface of the solid matrix
in SSF, where details structural information of growth morphology is difficult
to be studied. Reports on the effect of fungal growth morphology on the performance
of SF are available in the literature (Teng et al.,
2009; Haack et al., 2006; Spohr
et al., 1997; Oncu et al., 2007; Lim
et al., 2006), however, reports concerning morphological aspects
of the fungi and their relationship with metabolites production in SSF are limited.
However, at the microscopic level, there is evidence to suggest that extracellular
protein yield is positively correlated with increased numbers of actively growing
hyphal tips (Wessels, 1993; Gordon
et al., 2000; Muller et al., 2002).
It has also been suggested that more branching leads to more tips which should
result to more products (Wosten et al., 1991).
In fermentation employing fungus, productivity can be increased if the growth
morphologies could be controlled at the required characteristics (Ahamed
and Vermette, 2009). Moreover, Teng et al. (2009)
has described in the research paper that enzymes, primary and secondary metabolites
has been affected by the fungal morphology. Growth morphology of the fungi can
be divided into two types, (1) micromorphology and (2) macromorphology. Micromorphology
has direct influences on enzyme biosynthesis and secretion capacity. On the
other hand, macromorphology influences rheology and the mass transfer properties
of the culture. Micromorphology deals with the morphology of the individual
hyphal elements such as diameter and length of the hyphal elements as well as
number of tips of an individual hyphal (Teng et al.,
2009).
Fungal morphology has been established in several processes to lead to more
productive industrial processes, however, to-date a clear relationship between
the morphology and the product enhancement has not been well established as
described by Grimm et al. (2005). This is due
to the difficulties into investigating many interrelated factors that may affect
both the fungal morphology and the product formation. Hwang
et al. (2004) has suggested that quantitative structural information
of the fungal morphology would need to be experimented to obtain a complete
understanding of this relationship.
The objective of this study was to evaluate the relationship between the micro-morphology
characteristics and growth kinetics of several fungi species on extracellular
lipase production in SSF. Several types of lipase producing-fungi that have
potential for industrial processes were used as the model systems. Micromorphology
dimensions of the fungi which include spore diameter, hyphae diameter, branch
diameter, tip diameter and tip length and their relationship with the ability
to produce lipase was studied. The information generated from this study may
give better understanding on fungal growth kinetics and their relationship to
micro-morphological forms and productivity in industrial SSF.
MATERIALS AND METHODS
Microorganisms and inoculum preparation: Four fungal strains which have ability to produce lipase, were used throughout this study. Trichoderma viride SDTC EDF 002 and Aspergillus niger SDTC SRW-4 were obtained from Sime Darby Culture Collection Centre, Kuala Lumpur, Malaysia. Aspergillus terreus DSMZ 5770 and Aspergillus niger DSMZ 2466 were purchased from DSMZ Culture Collection Centre, Germany. The cultures were maintained in 20% (v/v) glycerol and preserved at -80°C. For spore production, the culture was grown in PDA Petri dish, incubated at 28±1°C for 7 days. The spores were harvested by pouring 10 mL of sterile 0.05% (v/v) Tween-20 into the Petri dish and dislodging the spores into suspension. The number of spore in suspension was determined using a Haemocytometer. For all strains, appropriate dilutions were made to obtain an inoculum with spore concentration of approximately 1x107 spores mL-1.
Solid state fermentation (SSF): Palm Kernel Cake (PKC), obtained from
Premium Vegetables Company, Malaysia and was used as the main substrate in SSF
for lipase production. These types of agro-wastes which once regarded as wastes
are now known as the major renewable natural resource for various valuable products
(Karmakar and Ray, 2010). The PKC was obtained by solvent
extraction of the crushed palm kernel. This substrate was chosen due to its
high nutrient composition (~13% made up of other chemicals like crude fat, crude
protein, fibre, ether extract, ash and hemicelluloses). PKC (30 g) was placed
in 500 mL flask and autoclaved at 121°C for 40 min. After cooling, 100%
(v/w) of sterile distilled water and 10% (v/w) of inoculum were added into the
flask. The content of the flask was mixed thoroughly and incubated at ambient
temperature (~27±1°C) under static condition for 10 days. All fermentations,
employing all four different fungal strains, were conducted concurrently in
triplicates.
Extraction of lipase from solid substrate: At the specified incubation days (0, 2, 4, 6, 8 and 10 days), 15 mL of 0.02 M sodium phosphate buffer at pH 7.0 was added to 1.5 g of the fermented sample from each flask. The mixtures were shaken at 250 rpm and 4°C in a rotary shaker for 30 min. The mixtures were then filtered using Whatman filter paper (No. 1), where the filtrates were used for enzyme assays and determination of total protein content.
Analytical procedures: Total fungal biomass was estimated using glucosamine
method as described by Swift (1973). In this method,
0.1 g of sample was added into 5 mL of 2 M HCl and heated at 95°C for 2
h. The aliquot (3 mL) was added with 1 mL of acetylacetone and heated at 95°C
for 20 min. The solution was cooled and then added with 6 mL absolute ethanol
and 1 mL of Ehrlich reagent. This mixture was incubated at 65°C for 15 min
and the absorbance was read at 530 nm.
Lipase activity was determined using olive oil-polyvinyl alcohol emulsion as
substrate (Mustranta et al., 1993). In this assay,
0.5 mL extracted sample was added into a reaction tube containing 2 mL sodium
phosphate buffer (pH 7) and 2.5 mL olive-oil emulsion. The reaction was incubated
at 37°C in water bath agitated at 200 rpm for 30 min. The reaction was terminated
by the addition of 5 mL acetone-ethanol (1:1% v/v). The mixture was then titrated
with 0.05 M NaOH using the autotitrator system (Mettler Toledo, T70) to obtain
an equivalent point (EVP). One unit of lipase activity was defined as the amount
of enzyme which liberated 1 μmol equivalent of fatty acid at 37°C,
pH 7.0 in 1 min.
Protease activity was determined by casein digestion assay (Ibrahim-Granet
and Bertrand, 1996). In this method, 0.5 mL 1% casein in 0.05 M Tris-phosphate
buffer (pH 7.8) was equilibrated at 37°C for 5 min prior to the addition
of 0.1 mL enzyme extract. The reaction mixture was incubated at 37°C, agitated
at 200 rpm for 10 min. The reaction was stopped by adding 0.5 mL of 10% trichloroacetic
acid and left to stand at room temperature for 10 min. The solution was centrifuged
at 13,000 rpm for 20 min and the absorbance of the supernatant was then read
at 280 nm using spectrophotometer (Thermo Electron Corporation, Biomate 5).
One unit of protease activity was defined as the amount of enzyme which released
acid soluble fragments equivalent to 0.001 A280 per minute at 37°C
and pH 7.8.
Total protein content was determined using Bio-Rad Protein Assay Dye Reagent Concentrate (Chemoscience). In this method, 800 μL sample was added to 200 μL of the diluted Bradford reagent (Bradford reagent-H2O, 1:4% v/v) and then vortexed for 2 min. The solution was incubated at room temperature for 5 min and the absorbance was read at 595 nm.
Evaluation of growth kinetic: Growth and product formation kinetics
were evaluated using logistic and Luedeking-Piret equations as described elsewhere
(Gong and Lun, 1996; Younesi et
al., 2005):
where, X = biomass concentration (mg g-1), t = time (h), μm = maximum specific growth rate (h-1), Xm = maximum biomass concentration (mg g-1), P = enzyme productivity (U g-1), α = growth associated product formation constant and β = non-growth associated product formation constant (1 h-1).
Microscopic imaging: The samples from SSF for the four fungal strains were withdrawn at different intervals (0, 2, 4, 6, 8 and 10 day) and examined under Scanning Electron Microscope (SEM) for imaging analysis. The fresh samples were viewed under Phillips SEM FEI Quanta 200 without any fixing or dehydration process. Micromorphology dimensions of spore diameter, hyphae diameter, branch diameter, tip diameter and tip length for each fungal sample were observed and recorded. However, hyphae length, branch length and number of tips per hyphae that has proven to increase protein production in submerged fermentation based on previous research work could not be observed from this study. This is because the fungal hyphae penetrates into the fermentation substrate and creates large complex mycelia surrounding the substrate that hinders the measurements to be taken.
Statistical analysis: Biomass content, enzyme productivity, total protein
content and micromorphology data are means from triplicates. Micromorphology
data for the zero and second day of fermentation could not be measured because
distinct fungal structures were not detected in the samples. Generalizing the
results from fungal fermentations is difficult due to their high sensitivity
toward a slight variation in starting conditions, poor reproducibility and variations
with strains (Cui et al., 1997). Therefore, statistical
analysis was performed to obtain more comprehensive and useful information using
the software package SPSS 15.0 for Windows (SPSS, Germany). The results presented
were the mean values of ten replicates and the standard deviations were used
to analyze the experimental data. Pearson correlation analysis was performed
to measure the strength of association between two variables (bivariate data)
statistically and also to evaluate whether the data are significant or not.
Scatter plots are used to visualize the patterns of association between the
bivariate data (independent and dependent variables). When the scatter plot
shows a high level of association and correlation value, a regression equation
was modeled in order to explain one variable to the other.
RESULTS
Relationship between fungal growth and enzyme formation rate: The performance and kinetic parameters values of solid state fermentation by several fungi for lipase production are shown in Table 1. A. niger SDTC SRW-4 had the highest lipase production (44.43 U g-1 DM) followed by A. niger DSMZ 2466 (42.05 U g-1 DM). The highest Xmax for all the four fungal strains was observed at day 6. Among the strains tested, the highest growth (Xmax = 0.710 mg g-1 DM), maximum specific growth rate (μmax = 0.029 h-1) and protease production (90.33 U g-1 DM) was obtained with A. terreus DSMZ 5770.
Figure 1 shows a typical time course of solid state fermentation by A. niger DSMZ 2466 for lipase production. Growth was in a lag phase up to day 2 of fermentation and exponential phase was continued until 6 days. The specific growth rate (μ) during exponential phase was 0.011 h-1 and growth entered a deceleration phase after 10 days. Lipase production increased gradually with fermentation time and the highest production rate (9.8693 U gh-1) was observed during exponential phase. Decrease in lipase production rate (2.4578 U gh-1) was observed when growth entered the deceleration phase (Table 2). The specific enzyme activity reached a maximum value (151.970 U mg-1 protein) after 10 days of fermentation. Luedeking-Piret equation (R2 = 0.8858) showed that the formation of lipase was partially growth associated, where the values of α and β were calculated as 39.665 and 1.0106, respectively.
Growth profile of T. viride STC EDF 002 was similar to A. niger DSMZ
2466. In contrary, lag phase was not observed for A. niger SDTC SRW-4
and A. terreus DSMZ 5770.
| Table 1: |
Performance and kinetic parameters values of solid state fermentation
using palm kernel cake for lipase production by several fungi |
|
| Data of each strain was selected from the highest lipase production
from a ten-day fermentation time |
| Table 2: |
Specific enzyme activity and product formation rate |
|
| * There were no distinct relationship between the product
formation and growth of fungus based on the Luedeking-Piret equation |
|
| Fig. 2: |
Scatter plots on high positive correlation bivariate data |
However, growth curve for all the fungal strains employed in this study showed
that the exponential phase peaked at 6 days, showing that all the fungal strains
had the same growth pattern on palm kernel cake and entered into a deceleration
phase thereafter. During the 6 days of exponential growth, spore swelling, elongation
of germ tubes and evolution of the total length of hyphae were observed. Subsequently,
the water content in the fermentation substrate was reduced to low levels that
caused substrate hardening. This hardening reduced and blocked the channeling
of the substrate pores which in turn, retarded the penetration of the mycelia
into the substrate to absorb the nutrients. The inability of the fungus to absorb
more nutrients after hardening of substrate caused growth to enter into the
deceleration phase. This was visually observed after 10 days of fermentation
where approximately only 80% of the substrate was covered by fungal mycelia.
In fermentation with A. niger SDTC SRW-4, the highest specific activity of lipase (152.891 U mg-1 protein) was obtained after 10 days. In this case, lipase formation was found as growth-associated based on the Luedeking-Piret equation (R2 = 0.9773). Lipase formation and fungal growth for T. viride SDTC EDF 002 and A. terreus DSMZ 5770 did not show a similar trend and could not be correlated. Nevertheless, total protease production for T. viride SDTC EDF 002 contributed linearly to the total protein content in the fermentation substrate.
Micromorphology dimensions of fungal strains grown on PKC in SSF: Both
A. niger strains of DSMZ 2466 and SRW-4 produced conidia black in colour,
globose and with the diameter of 3.8 to 4.6 μm.
| Table 3: |
Micromorphology dimensions of fungal strains grown on PKC
in SSF |
|
|
On the other hand, A. terreus DSMZ 5770 produced smaller conidia (diameter
of 2.0 to 2.6 μm) which were globose and have smooth surface while T.
viride SDTC EDF 002 produced conidia (diameter of 2.5 to 3.2 μm) which
was green in colour and grouped in sticky heads at the tips of the phialides.
Micromorphology dimensions for the spore diameter, hyphae diameter, branch diameter, tip diameter and tip length for the four fungal strains cultivated in solid state fermentation using PKC as substrate are summarized in Table 3. Data for the early stages of fermentation (0 to 2 days) could not be obtained because no distinct fungal spore structure could be identified. In addition, dimension of the branches diameter of T. viride SDTC EDF 002 and A. terreus DSMZ 5770 could not be measured because the fine branches overlapped and formed in a matrix structure.
A. niger DSMZ 2466 and A. niger SDTC SRW-4 displayed similar pattern on the morphology dimensions. In general, spore diameter and tip length were increased with fermentation time while reduction in hyphae diameter and tip length were observed. During growth in PKC, spore diameter and tip length for both fungal strains were more or less the same. However, hyphae and branch diameters for A. niger SDTC SRW-4 were comparatively larger than A. niger DSMZ 2466. Both fungal strains formed complex matrix structure within the substrate and produced long hyphae with diameter ranging from 9 to 12 μm.
Morphology dimension for T. viride SDTC EDF 002 and A. terreus DSMZ 5770 was almost similar, where the dimensions were increased with fermentation time. Spore diameter and tip length for T. viride SDTC EDF 002 were comparatively larger than those observed for A. terreus DSMZ 5770. On the other hand, hyphae and tip diameters for T. viride SDTC EDF 002 were smaller than the diameters for A. terreus DSMZ 5770.
Production of lipase by A. niger DSMZ 2466 and A. niger SDTC SRW-4 was about 10 fold higher than those obtained by A. terreus DSMZ 5770 and T. viride SDTC EDF 002. The ability of the fungi to produce lipase correlated well with the difference in their morphology dimensions.
| Table 4: |
Pearson correlation analysis for A. niger DSMZ 2466 |
|
| *Correlation is significant at the 0.05 level (2-tailed).
**Correlation is significant at the 0.01 level (2-tailed) |
Pearson correlation analysis for micromorphology dimensions and lipase production:
Table 4 shows the variables, the significant levels and Pearson
correlation for all dimensions of A. niger DSMZ 2466. Correlation measures
the linear relationship between two quantitative variables which is known as
bivariate data. Pearson correlation coefficient quantifies the strength of linear
relationship between the bivariate data. The correlation coefficient ranges
in value from -1 to 1. A value of 0 indicates no linear relationship between
the variables while a value of +1 indicates a perfect positive relationship
and -1 for a perfect negative relationship. This positive relationship among
the bivariates can be visually demonstrated in a scatter plot to show the association
between the two variables (Fig. 2). The following guidelines
could be used to interpret correlation strength namely small, medium and large
with the values of 0.10 to 0.29, 0.30 to 0.49 and 0.50 to 1.0, respectively
(Cohen, 1988).
The data which have high significant levels (p<0.05) are extracted for further analysis to ensure the data are normally distributed (Table 4). Pearson correlation for lipase activity versus protein content (R2 = 0.994) and spore diameter versus branch diameter (R2 = 0.959) have high significance levels, suggesting the occurrence of a large strength correlation between these variables. Bivariate data of spore diameter and lipase activity was an exceptional data with the significance level on par at 0.05 with a high correlation coefficient (0.950).
Spore diameter versus hyphae diameter and tip diameter versus lipase activity, protease activity and protein content showed very strong negative relationship at a high significance level (R2>0.97). These results indicate that these variables are independent. For example, size of the spore was not influenced by the thickness of the hyphae. The thickness of the tip also did not influence lipase and protease production.
Both, lipase and protease activity for A. niger SDTC SRW-4 was strongly correlated with the size of the spore and the thickness of the branch. Moreover, production of these enzymes had similar trends throughout the fermentation period. Pearson correlation showed that the thickness of the hyphae and tip did not influence lipase production. Strong negative relationship was observed between variables of hyphae and branch diameters; and spore and tip diameters. Spore diameter, branch diameter and lipase production in fermentation with A. niger SDTC SRW-4 and A. niger DSMZ 2466 were interrelated as evaluated by Pearson correlation. The bivariate significance level for branch diameter and lipase activity for A. niger DSMZ 2466 was low (>0.05). However, the correlation was high (R2>0.9) showing a positive relationship.
T. viride SDTC EDF 002 and A. terreus DSMZ 5770 showed positive correlations for tip length and tip diameter as well as size of hyphae diameter and total protein content in the fermentation substrate. Linear relationship with the tip length and tip diameter was observed for spore size of A. terreus DSMZ 5770. Even though lipase and protease production by A. terreus DSMZ 5770 shared similar pattern with A. niger SRW-4 but Pearson correlation did not show any inter-relationship between the micro-morphology dimensions with lipase production.
Regression analysis: Regression analysis creates a model that relates
a dependent (outcome or response) variable to an independent variable. The appropriate
model is subsequently used for prediction of equations. Table
5 describes the summary of the models that were generated from a curve fit
plot. Linear or quadratic models were selected based on their best regression
value that fits the curve plot. Figure 3 demonstrates an example
of a curve fit plot of A. niger DSMZ 2466 spore diameter (independent
variable) versus lipase activity (dependent variable), showing that the quadratic
curve best-fitted to the plot with R2 of 0.999.
|
| Fig. 3: |
Example of a curve fit plot from a regression analysis (data
extracted from A. niger DSMZ 2466) |
| Table 5: |
Summary of regression analysis and equation models |
|
| Only bivariate data which are significant (<0.05) and R2
more than 0.97 are tabled. aLinear model equation is Y = b0 +
(b1 * X). The series values are modeled as a linear function of time. bQuadratic
model equation is Y = b0 + (b1 * X) + (b2 * X2) |
|
Both strains of A. niger DSMZ 2466 and A. niger SDTC SRW-4 tested in this study showed good micro-morphology characteristics with larger spore sizes and hyphae diameter as compared to A. terreus DSMZ 5770. Lipase activity and the branch diameter for A. niger SDTC SRW-4 was highly correlated in a linear model (R2 = 0.971) while lipase activity and spore diameter for A. niger DSMZ 2466 was correlated in a quadratic model (R2 = 0.999). For A. niger DSMZ 2466, the total protein content was strongly influenced by the total lipase activity in the fermentation substrate. There was no relationship between the micro-morphology dimensions and production of lipase for T. viride SDTC EDF 002 and A. terreus DSMZ 5770. Nevertheless, the relationships between tip length and tip diameter as well as the tip diameter and spore diameter were existence.
DISCUSSION
Palm oil kernels are available as palm kernel press cake (PKC) which is a residue
from extraction of oil from palm kernels. The extraction can be either mechanical
or solvent extraction resulting in a residue containing about 50% carbohydrate
and 15-20% protein (Knudsen, 1997; Cervero
et al., 2010). Since PKC consisted of sugar such as mannose (30-35%)
and glucose (7-9%) in PKC (Dusterhoft et al., 1991;
Knudsen, 1997), it has potential to be used as substrate
in SSF for production of industrial enzymes. All the four fungal strains tested
in this study showed good growth in SSF using PKC as substrate, where final
biomass content ranging from 0.491 to 0.710 mg biomass g-1 DM was
achieved. All the strains grew in filamentous form and the hyphae showed ability
to elongate and form complex branches filling spaces in the substrate matrix
at a similar rate in the exponential phase. This fungus utilizes the nutrients
available through the development of penetrative hyphae into the substrate matrix
and degrades the high content of hemicelluloses and celluloses by producing
various types of hydrolytic enzymes. The predominance between the ability to
penetrate and to degrade may be related to the extent of diffusion and assimilation
of nutrients from the medium to fungus which is associated to different control
mechanisms related to growth kinetics (Cano and Bago, 2005;
Boswell, 2008).
A. niger SDTC SRW-4 and A. niger DSMZ 2466 showed high productivity
of lipase enzyme while T. viride SDTC EDF 002 and A. terreus DSMZ
5770 showed higher productivity of protease enzyme. Several other types of hydrolytic
enzymes such as xylanases, mannanases or cellulases were also produced by these
fungal strains during growth on PKC due to high content of mannan (~35.2%) (Cervero
et al., 2010).
|
| Fig. 4(a-o): |
SEM images on growth of fungal strains on PKC, (a-b) A.
Niger DSMZ 2466, (c-g) A. Niger SRW-4, (h-k) A. terreus
DSMZ 5770 and (l-o) T. viride EDF 022 |
Two stages of growth were occurred in SSF inoculated with spores; (1) swelling
of a population of conidiospores and (2) full development of branched hyphae.
Result from this study indicated that increased in mean equivalent spore diameter
was approximately linear with time. Barry et al.
(2009) has stressed that differences in spore swelling rate and germination
time would make a significant contribution to the observed variation in the
sizes. Both fungal strains, A. niger SDTC SRW-4 and A. niger DSMZ
2466, have higher spore swelling and germination rate as compared to T. viride
SDTC EDF 002 and A. terreus DSMZ 5770 which have higher ability in
producing lipase. The presence of a layer of mannose-rich glycoproteins in the
walls of A. niger germinating spores was associated with adhesion phenomena
(Brul et al., 1997). Large diameters of germinating
spores contained large amount of mannose-rich glycoproteins layer existed which
may related to higher enzyme production.
From SEM results in Fig. 4, a complex branching with fine
branches emerged from the hyphae was observed for T. viride SDTC EDF
002 and A. terreus DSMZ 5770. The formation of this matrix made the dimensions
of branch length and diameter difficult to be measured. Although, the spore
diameter and hyphae diameter of these two fungal strains were shorter as compared
to A. niger SDTC SRW-4 and A. niger DSMZ 2466, they have good
growth on PKC. The local fractal dimension within a colony of T. viride
was increased with branching frequency which can be seen from the occurrence
of ‘loops’ in the mycelium (Hitchcock et al.,
1996). In addition, good growth is imputable from the branching mechanism
that promotes exponential growth of filamentous microorganisms and together
with tip extension which determines the overall specific growth rate and the
morphology of freely dispersed hyphal elements (Pazouki
and Panda, 2000). The branching mechanism in T. viride SDTC EDF 002
and A. terreus DSMZ 5770 has no relationship with high lipase production
but may related to protease production. Results from this study showed that
large spore and branch diameter was correlated with high ability in producing
lipase, as observed in models for A. niger SDTC SRW-4 and A. niger
DSMZ 2466. A good linear relationship between sporangium diameter and enzyme
production using Pearson correlation analysis has also been reported (De
Nicolas-Santiago et al., 2006).
Hyphae and tip diameters for A. niger SDTC SRW-4 and A. niger DSMZ
2466 strains were decreased with fermentation time, most probably due to differentiation.
In the production of secondary metabolites by Streptomyces differentiation
was observed for growth on solid media (Pons et al.,
1998). Vegetative mycelia consist of thick filaments and with aging, septation
occurs and the filaments get thinner or tend to lose their cellular content
while giving rise to empty compartments. The empty parts then start to appear
at the end of the rapid growth phase and continue throughout the fermentation
where initially the empty parts appeared to the tips of the filaments. This
could also be related to changes in the membrane permeability which induces
a leakage of intracellular materials. The high degree of vacuolation may also
be viewed as a normal development change in the culture under imperfect conditions
for fungal growth, for example O2 limitation (Paul
et al., 1994).
CONCLUSION
All lipase-producing fungal strains (A. niger DSMZ 2466, A. niger SDTC SRW-4, T. viride SDTC EDF 002 and A. terreus DSMZ 5770) employed in this study showed variation in the micro-morphology characteristics towards enzyme production though the growth patterns on PKC were more or less the same. In general, hyphae and spore diameters for A. niger were larger as compared to A. terreus and T. viride which was about 7-fold and 2-fold higher, respectively. Strong positive relationship between spore and branch diameters with lipase production was observed for fermentations with A. niger. On the other hand, correlation between morphology dimensions with lipase activity was not observed for fermentations with A. terreus and T. viride. However, the tip length and tip diameter had a strong influential relationship for T. viride while the tip diameter and spore diameter showed strong positive relationship for A. terreus.
ACKNOWLEDGMENTS
This research work was financially supported by Sime Darby Technology Centre Sdn. Bhd. Ms. Anusha Nair would also like to extent appreciation to Sime Darby Technology Centre Sdn. Bhd. for her PhD scholarship.
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