Solid-State Fermentation (SSF) is a process whereby microbes of interest
will grow and utilise the moist substrate materials in the absence of
free water. Many bacteria, yeast and fungi are able to grow on solid substrate
and find application in SSF processes. However, filamentous fungi are
the most important group of microorganism for SSF processes and dominate
in research work owing to their physiological capabilities and hyphal
mode of growth. During microbial growth, secretion of hydrolytic enzymes
and production of other useful metabolites will upgrade the quality of
low nutrient value materials such as PKC. Utilization and optimization
of Fungi sp. in SSF for PKC digestibility improvement have been
extensively studied by Noraini et al. (2000) and Jaafar et al.
(2001). Previous study showed that Aspergillus niger FTCC 5003
was a suitable microbe for further investigation due to its ability in
the depolymerisation of PKC fibre (Noraini et al., 2001).
There are different types of techniques available in measuring the growth
of microorganism in SSF. Different direct biomass determination such as
matrix removal methods by using gelatine matrix, enzyme digestion and
membrane filter has been used as an attempt for recovery of fungal biomass
in SSF. However, foreign substance and non-digestible residue were found
to interfere with the measurement for methods using gelatine matrix and
enzyme digestion. The method of direct peeling off the membrane filter
for biomass recovery was reported to be able to prevent the penetration
of the fungal hyphae, Rhizopus oligosporus into the substrate (Mitchell
et al., 1989). However, this method obviously cannot be used in
actual SSF but could find application in the calibration to indirect method
of biomass determination.
Biomass is a fundamental parameter in the characterisation of microbial
growth. Its measurement is essential for kinetic studies on solid-state
fermentation. Complete recovery of fungal biomass from the substrate is
very difficult in solid-state fermentation because the fungal hyphae penetrate
into and binds tightly to the solid substrate particles. Many authors
have described indirect methods to estimate biomass in solid-state fermentations.
These indirect methods are based on metabolic measurement such as respiratory
metabolism (Saucedo-Castañeda et al., 1990; Smits et
al., 1996), extracellular enzymes (Smits et al., 1996; Mitchell
et al., 1991) and organic acids (Soccol, 1992) or specific component
measurement like protein content (Saucedo-Castañeda et al.,
1990; Favela-Torres et al., 1998), glucosamine (Ooijkaas et
al., 1998; Papagianni et al., 2001), ergosterol (Nout et
al., 1987) and nucleic acids (Bajracharya and Mudgett, 1980).
The content of the different cell components can be used to estimate
the biomass as long as the composition of the biomass is constant and
stable. Protein content is the most readily measured biomass component.
Raimbault and Alazar (1980) used soluble protein content to measure the
growth rate of A. niger on cassava meal. Mycelia biomass from solid-state
fermentation was determined indirectly from measurements of soluble proteins
contents (Favela-Torres et al., 1998). Glucosamine is a useful
compound for the estimation of fungal biomass as it is an essential and
stable component in chitin of mycelia cell walls. Although the proportion
of chitin in the mycelium will vary with age and the environmental conditions;
Desgranges et al. (1991) reported that this parameter was reliable
for solid-state culture carried out in media containing the same components,
regardless of their concentrations. Glucosamine was used as an indirect
method for biomass determination and an efficient parameter for growth
in solid-state fermentation (Papagianni et al., 2001; Krishna and
The objective of the present study was to develop correlations for cell
growth determination during SSF of PKC by A. niger FTCC 5003 based
on estimation of glucosamine and protein content.
MATERIALS AND METHODS
Aspergillus niger FTCC 5003 provided by Food Technology Centre,
MARDI, Malaysia was used throughout this study. The preserved stock culture
of A. niger FTCC 5003 was grown on the sterilised potato dextrose
agar slants and incubated at 30±2°C for three days. The spores
were collected with sterilised distilled water containing 0.01% (v/v)
Tween 80. The spores numbers were obtained via total cell count method
using haemocytometer and the spore size were fixed at 107 spores
Solid-State Fermentation on Support Material
In order to clarify that the glucosamine and protein determined solely
came from the fungus, simulated homogenous SSF on support material was
carried out in 250 mL Erlenmeyer flasks, containing 40 g of glass beads,
2% of mannose solution and 1% nitrogen source from urea. The flasks with
glass beads were sterilised at 121°C, 15 psi for 15 min prior to inoculation.
After cooling, 9.9 mL of mannose solution, 0.1 mL urea solution and 1
mL of spore suspension were added aseptically. The glass beads, inoculum,
mannose and urea solution were then shaken to thoroughly mix the system.
The flasks were incubated at 30°C for 9 days fermentation time. Samplings
were done in triplicates.
Solid-State Fermentation on Palm Kernel Cake
Solid-state fermentation was carried out in 250 mL Erlenmeyer flasks,
containing 30 g of PKC and 1% nitrogen source from urea. The flasks with
substrate were steam sterilised at 121°C, 15 psi for 15 min prior
to inoculation. The media was then allowed to cool down; appropriate amount
of sterilised distilled water and 6 mL of 3 days old spore suspension
were added. The substrate, inoculum and water were manually mixed aseptically
with sterilised spatula. The flasks were incubated at 30°C for 10
days fermentation time.
Fungal Biomass Harvesting from Solid-State Fermentation on Support
The fungal biomass from the SSF on support material was harvested
by adding 10 mL sterilised distilled water. Three milliliter of sample
suspension were withdrawn and kept at 4°C for protein and β-mannanase
Samples were collected triplicate every day for 10 days fermentation.
The fermented solid material was manually mixed under aseptic condition.
The collected samples were used for PKC dry weight determination, protein
concentration and glucosamine concentration analysis.
Fungal Dry Cell Weight (Dw) Determination for Solid-State
Fermentation on Support Material
For fungal dry cell weight analysis, the suspension after removal
of 3 mL for protein analysis was then filtered through pre-weight Whatman
filter paper No. 1. The fungal biomass was washed with sterilised distilled
water and it was repeated until the filtrate was clear. Then, the residue
was vacuum filtered through a pre-weight 0.2 μm membrane filter.
For dry cell weight measurement, the filters were dried in an oven until
constant weight and re-weighed after cooling the filters in a desiccator
as described by Ooijkaas et al. (1998).
PKC Dry Weight (Dw) Analysis for Solid-State Fermentation
on Palm Kernel Cake
Empty flask was weighed prior to incubation. Every day, the flask
containing fermented material was weighed to obtain total wet weight (ww)
by calculating the difference between weight of flask with fermented material
and empty flask weight. Approximately 40 g of fermented material from
each sample (in triplicate) were dried in oven until constant dry weight
was achieved. The PKC dry weight (DW) is defined using Eq.
Thirty milliliter of sterilised distilled water was added into the
flask containing approximately 3 g (ww) of wet sample. Cool extraction
was carried out by orbital shaking at 140 rpm, 4°C for 24 h. Ten milliliter
of slurry was centrifuged at 5000 rpm for 10 min. The filtered supernatant
was then digested with 1 N NaOH at 1:1 ratio, where the digestion was
carried out for cell breakage and protein solubilisation at 100°C
for 15 min. After cooling, the soluble protein concentration was determined
by Lowry method (Lowry et al., 1951) using bovine serum albumin
Five milligram of dry sample was hydrolysed with 5 mL of 6 M HCl in
glass vial with screw cap at 100°C for 4 h. After the acid hydrolysis,
the sample was dried under vacuum at 70°C in a rotary evaporator.
The dried residue was dissolved in 5 mL deionised water and the solution
was then analysed using HPLC by sugar sp0810 column (Shodex, 8 mm ID 300
mm L). Deionised water was used as mobile phase with a flow rate of 1.0
mL min-1 and column temperature was maintained at 70°C.
Peak was detected with a reflective index detector (Jasco, RI-1530) and
commercial glucosamine (Sigma, G4875) was used as standard.
Endo-1,4-β-D-Mannanase Activity Assays
Beta-mannanase activity was measured by method described by Michael
(2000) using azo-carob galactomannan (2%) diluted in 2 M sodium acetate
buffer, pH 4.5 as substrate.
The correlation coefficient was used to determine the relationship
between two properties in present study. The equation for the correlation
coefficient is shown in Eq. 2:
RESULTS AND DISCUSSION
An initial study of simulated homogenous SSF using glass bead
as support materials has been carried out as to obtain information of
microbial growth and complete recovery of fungal biomass during SSF on
PKC. The complete recovery of fungal biomass was also aimed to find application
for biomass estimation from indirect method of biomass determination during
SSF on PKC.
Solid-state fermentation using support material had several potential
applications in scientific studies and industrial process. Due to its
less complicated product and biomass recovery, SSF on support material
offers an advantage for easier recovery of biomass from the support material
with fewer impurities compared with the natural substrate (Ooijkaas et
al., 1998). Solid-state fermentation on support material impregnated
with defined media has been carried out. The fungal dry weight, protein
and glucosamine content in SSF on support material were determined in
order to obtain a suitable relationship for biomass estimation of Aspergillus
niger FTCC 5003.
Figure 1 shows the relationship between fungal dry
cell weight and protein concentration, while the relationship for glucosamine
content is shown in Fig. 2. For both cases, the evidence of a straight
line relationship were observed and the statistical analysis for the relationship
is shown in Table 1. Based on statistical analysis, strong correlation
values have been observed between fungal dry cell weight and protein concentration
(0.997) and between fungal dry cell weight and glucosamine concentration
||Relationship between fungal dry weight and protein concentration
||Relationship between fungal dry weight and glucosamine concentration
Both protein and glucosamine content were well correlated to the fungal
dry cell weight in SSF on support material, indicating that protein and
glucosamine were suitable calibration methods for indirect biomass estimation
in systems that have similar conditions such as SSF on PKC. Therefore, Eq.
) were used in the indirect biomass
estimation for growth of Aspergillus niger
FTCC 5003 in SSF using
PKC as substrate.
The typical growth pattern of Aspergillus niger FTCC 5003 in SSF
on PKC which estimated fungal dry cell weight was based on protein and
glucosamine are shown in Fig. 3 and 4, respectively.
It were observed that the protein and glucosamine concentration increasing
as the fermentation time increased for both fungal dry weights.
||Statistical analysis for fungal dry cell weight in relation to protein
concentration and glucosamine concentration
|Experimental unit, n = 54
||Aspergillus niger FTCC 5003 growth profile in
SSF on PKC which estimated fungal dry weight was based on protein
fungal dry weight, ()
PKC dry weight and ()
Estimated maximum fungal dry weight at day 10 was 1.297 and 0.632 g based
on protein and glucosamine concentration, respectively. It shown that β-mannanase
as part of the metabolite in the system increased gradually while the PKC
dry weight as substrate in the system decreased proportionally with fermentation
Table 2 shows the statistical analysis for PKC dry weight in relation
to β-mannanase activity and estimated fungal dry cell weight based
on the protein and glucosamine concentration. Based on statistical analysis,
correlation value of 0.970, was observed between palm kernel cake and
β-mannanase activity; on the other hand, estimated PKC and fungal
dry cell weight based on protein concentration showed a correlation value
of -0.967. This suggested that the growth of Aspergillus niger
FTCC 5003 in this system was well described by both estimated fungal dry
cell weight based on protein concentration and β-mannanase activity.
The statistical analysis showed that the correlation value between PKC
and estimated fungal dry cell weight based on glucosamine concentration
was -0.299, indicating that the estimated fungal dry cell weight based
on glucosamine concentration did not reflect the growth of Aspergillus
niger FTCC 5003 in this system. Ooijkaas et al. (1998) reported
that glucosamine content was not suitable to be used as biomass indicator
for their study because the content of mycelia increases during fungal
development was cause by the resistance of chitin to breakdown after fungal
death. The chitin accumulated in the empty ghost hyphae and caused increase
of glucosamine content that resulted in the inaccuracy of glucosamine
for biomass estimation that do not reflect the growth of Coniothyrium
minitans in solid-state fermentation.
||Aspergillus niger FTCC 5003 growth profile in
SSF on PKC which estimated fungal dry weight was based on glucosamine
fungal dry weight, ()
PKC dry weight and ()
||Statistical analysis for palm kernel cake dry weight in relation
to β-mannanase activity and estimated fungal dry cell weight
based on protein and glucosamine concentration
|Experimental unit, n = 120
The relationships between PKC and estimated fungal dry cell weight based
on protein concentration and between PKC and beta-mannanase activity well
described the growth pattern of Aspergillus niger
FTCC 5003 in this
system. Kinetic study was then performed to establish a model that able
to describe and simulate the Aspergillus niger
under various conditions. The estimated fungal dry cell weight based on
glucosamine concentration was less suitable to described the growth of Aspergillus
FTCC 5003 in SSF using PKC as substrate but the result obtained
were still be used in the kinetic and modelling study as comparison parameter
to estimate fungal dry cell weight based on protein concentration.
Correlations between protein and glucosamine contents and the dry cell
weight of A. niger FTCC 5003 have been developed. These correlations
were successfully used in the estimation of growth profile of A. niger
FTCC 5003 during SSF using PKC. However, the correlation based on protein
content gave better accuracy than glucosamine content.
The authors would like to thank the National Biotechnology Directorate,
Malaysia for their financial support and the Livestock Research Centre,
MARDI Serdang for their technical assistance.