Abstract: Solid-State Fermentation (SSF) of Aspergillus niger FTCC 5003 on Palm Kernel Cake (PKC) is a practical approach to upgrade PKC into value added product. Present study was conducted on Aspergillus niger FTCC 5003 growth profile and models that are able to describe the growth in SSF using PKC substrate. Due to the difficulties of separating cell biomass quantitatively from the substrate for SSF systems, indirect method for measurement of cell growth during SSF of PKC by Aspergillus niger FTCC 5003 was studied based on the estimation of glucosamine and protein content. Preliminary relationships between glucosamine and protein contents to fungal dry cell weight (Dw) were developed using simulated homogenous SSF data using glass beads as support materials. Both glucosamine and protein contents were well correlated to the fungal dry cell weight in SSF on support materials for protein and glucosamine, respectively. The equations obtained were used for the estimation of cell biomass profile during SSF of PKC from the data of glucosamine and protein as growth indicator study. The estimated fungal dry cell weight based on protein concentration and β-mannanase activity as metabolic activity for microbial growth were well correlated to PKC dry weight which, indicating that both were suitable marker in describing the growth of A. niger FTCC 5003 in this system. In contrast, estimated fungal dry cell weight based on glucosamine concentration was not suitable to describe the growth of A. niger FTCC 5003.
INTRODUCTION
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 Nokes, 2001).
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
Microorganism
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
mL-1.
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
Material
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
analysis.
Sampling
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.
Analytical Procedures
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.
1:
(1) |
Protein Assay
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
as standard.
Glucosamine Assay
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.
Statistical Analysis
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:
(2) |
Where:
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
(0.962), respectively.
| Fig. 1: | Relationship between fungal dry weight and protein concentration |
| Fig. 2: | Relationship between fungal dry weight and glucosamine concentration |
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.
| Table 1: | Statistical analysis for fungal dry cell weight in relation to protein concentration and glucosamine concentration |
| Experimental unit, n = 54 | |
| Fig. 3: | Aspergillus niger FTCC 5003 growth profile in
SSF on PKC which estimated fungal dry weight was based on protein
concentration ( |
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.
| Fig. 4: | Aspergillus niger FTCC 5003 growth profile in
SSF on PKC which estimated fungal dry weight was based on glucosamine
concentration ( |
| Table 2: | 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 | |
CONCLUSIONS
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.
ACKNOWLEDGMENTS
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.