Abstract: Trichoderma sp. KUPM0001 showed good growth during solid substrate fermentation (SSF) of sago pith residue known as hampas, supplemented with 10% (v/w) of mineral salts solution containing 0.5% (w/v) (83.3 mM) urea as nitrogen source and an initial moisture content of 80% (v/w). Mycelium suspension of 10% (v/w) density was used as initial inoculum and SSF was carried out at 25±2°C in static condition over a period of 120 h. The parameters optimized included the initial moisture content of the substrate, mineral salts solution, urea concentration, inoculum density, incubation temperature and incubation time. Without optimized condition, the maximum reducing sugar obtained was 24 mg mL-1 compared to 46 mg mL-1 substrate during optimized SSF after 96 h incubation. The optimum parameters obtained were 80% (v/w) of initial moisture; 10% (v/w) of inoculums size; 1.0% of urea in 20% (w/v) of mineral solution and incubated at 30±2°C. The enzyme activities using optimized condition gave maximum α-amylase, glucoamylase, carboxymethyl cellulase, filter paperase and β-glucosidase of 3.19, 2.22, 1.66, 1.11 and 1.48 U mL-1, respectively.
INTRODUCTION
In Malaysia, the use of sago starch has been increasing and it is presently being used for the production of glucose. Sago starch represents an alternative cheap carbon source for fermentation processes that is attractive out of both economic and geographical considerations (Abd-Aziz, 2002). Production of fermentable sugars from the hydrolysis of starches normally carried out by an enzymatic process that involves two reaction steps, liquefaction and saccharification that requires the use of an expensive temperature control system and a complex mixing device.
The sago starch processing industry produces three major types of by-products viz, bark of sago trunk, fibrous pith residue, commonly known as hampas and wastewater. Sago hampas (the fibrous pith residue) obtained after starch extraction from the rasped sago pith consists of about 66% starch, 15% crude fiber and 1% crude protein on a dry weight basis. On dry basis, sago hampas contains 60- 70% starch, trapped within its fibers (Robertson et al., 2006). The fibers have to be degraded first to release the starch. Bacillus subtilis has been reported to be successful in saccharifying starch within the fibers in cassava solid wastes by using α-amylase and glucoamylase. The resultant glucose-syrup was used as a feedstock for ethanol production. Therefore, sago hampas, which have similar constituents, may be amenable to the same treatment in microbial degradation.
In the utilization of agricultural materials by SSF, process parameter, such as initial moisture content, mineral salts solution, inoculum density, urea concentration, incubation temperature and time are important to enhance the successful of SSF. Therefore the emphasis of this study was to convert a locally-available agro-waste material, hampas to soluble sugars by isolated fungal Trichoderma sp. KUPM0001 and optimization of parameters via solid substrate fermentation.
MATERIALS AND METHODS
Microorganism
The locally isolated fungus, Trichoderma sp. KUPM0001
was isolated from the decayed sago hampas obtained from the collection
ramp of the sago mill. Pure cultures were maintained on potato dextrose
agar (PDA-Difco) slants and stored at 4 ± 2 °C.
Substrate
Sago hampas was collected from Hup Guan Sago factory in Johor
Darul Takzim, Malaysia. The substrate was air-dried and sieved through
a 1.0 mm sieve and stored at room temperature prior to use.
Inoculum Preparation
Trichoderma sp. KUPM0001 was grown on Potato Dextrose Agar
(PDA) plate for seven days at 30 ± 2 °C. The spore suspension
was harvested using 10 mL of sterile distilled water containing 0.1% (v/v)
Tween 80 and then inoculated into 50 mL Potato Dextrose Broth (PDB) in
250 mL Erlenmeyer flask. The inoculated flasks were incubated at 200 rpm
at 30 °C for 48 h. The vegetative inoculum was aseptically blended
at low speed for 10 sec in a Waring blender. A 10% (v/w) of this blended
mycelium culture was used as inoculum in each SSF unless otherwise stated.
Solid Substrate Fermentation (SSF)
Solid substrate fermentation cultures were developed in 250 mL Erlenmeyer
flask containing 5 g of sago hampas. The substrate was autoclaved
at 121 °C, 15 psi for 20 min. Prior to cooling, 1 mL of mineral salts
solution containing 0.2% (w/v) KH2PO4, 0.05% (w/v)
MgSO4.7H2O and filtered sterilized 1.0% (w/v) urea
w/v) as nitrogen supplement was added. The content of the flask with a
moisture content of 60% was then thoroughly mixed with sterile spatula
and allowed to stand for 1 h. Each flask was aseptically inoculated with
10% (v/w) of 48 h old mycelial suspension of Trichoderma sp. KUPM001.
To increase aeration, the content of the flask was gently mixed using
sterile L stick every 24 h. The cultures were incubated at 30 ±
2 °C in a static condition for 5 days (120 h) unless otherwise stated.
Experiments were done in triplicate.
Optimization of Reducing Sugars Production
The Effect of Initial Moisture Content
To investigate the influence of the initial moisture content on the
sugar production the fermentation was carried out at various moisture
levels (60, 65, 70, 75 and 80%) contents. The moisture content (v/w) was
adjusted with a total volume inclusive of distilled water, mineral salts
solution and inoculum suspension. Fermentation was carried out at 30 ±
2 °C for 120 h. The optimum initial moisture content was maintained
for subsequent experiments.
The Effect of Mineral Salts Solution
Mineral salts solution consisted of 0.2% (w/v) KH2PO4,
0.05% (w/v) MgSO4.7H2O and filter sterilized urea
(1.0% w/v) as nitrogen supplement was used. The different volume of salts
solution to weight of substrate (v/w: 10, 20 and 30%) was assessed. Fermentation
was carried out at 30 ± 2 °C for 120 h. The optimum volume
of the mineral salts solution for solid substrate obtained was maintained
for subsequent experiments.
The Effect of Varying Concentrations of Urea
Urea supplementation at different levels of (w/v: 0.5, 1.0 and 2.0%)
in the substrate on sugar production was studied. The optimum level of
the urea concentration required for solid substrate was used for subsequent
experiments.
The Effect of Inoculum Density
The effect of various inoculum levels (v/w: 10, 20 and 30%) on reducing
sugar production was studied. The optimum inoculum level obtained was
then fixed for subsequent experiments.
The Effect of Incubation Temperature
The fermentation was carried out at various temperatures such as 25,
30, 35, 40 and 45 °C and the effect of the varying temperature on
reducing sugar production was studied. The optimum incubation temperature
obtained was fixed for subsequent experiments.
The Effect of Incubation Time
Different incubation periods (0, 12, 24, 36, 48, 60, 72, 84, 96, 108
and 120 h) were investigated for their effect on reducing sugar production.
The cultivation was carried out at 30 ± 2 °C for 120 h keeping
other conditions at their optimum levels. The optimum incubation period
achieved by in this experiment was then fixed for subsequent experiments.
Extraction Procedure
Extraction was done by adding 50 mL of 0.01M, phosphate buffer (pH
7.0) to the contents of each flask. The solid culture was broken down
into smaller particles and then homogenized at 8000 rpm for 3 min at room
temperature. The culture slurry was centrifuged at 8000 rpm at 4 °C
for 20 min (Conti et al., 2001). The supernatant was filtered through
0.45 μm filter and kept in 1.5 mL microcentrifuge tubes at -20 °C
for 24 h prior to assay for reducing sugar and enzymes. Assays were performed
in triplicates and the results for all the values were expressed as a
mean of triplicate values.
Sample Assays
The extracellular soluble protein was quantified using the dye-binding
method with crystalline bovine albumin as standard (Bradford, 1976). Total
nitrogen was determined by Kjeldahl method. The dinitrosalicylic acid
(DNS) method was employed for reducing sugar determination (Miller, 1959).
The absorbance was then translated into glucose equivalent using a standard
graph obtained by plotting glucose against absorbance.
Enzyme Assays
Alpha-amylase activity was assayed by incubating the crude enzyme
with 1.0 mL (1% w/v) cooked soluble starch along with 0.01 M phosphate
buffer, pH 6.9 for exactly 6 min at 37 °C (Bernfeld, 1955). The amount
of reducing sugar was determined using the DNS method and a standard of
pure maltose was used. Glucoamylase activity was assayed by incubating
the crude enzyme with 500 μL of 1% (w/v) raw soluble starch in 0.1
M sodium citrate buffer (pH 4.0) at 60 °C for 1 h. Reducing sugar
produced was determined by DNS method (Bradford, 1976) and glucose was
used as standard. Carboxymethyl cellulase (CMCase) activity was determined
by adding 1.8 mL substrate solution and 0.2 mL of enzyme sample and the
resulting solution was incubated at 40 °C for 30 min in a water bath
with moderate shaking (Mandels et al., 1974). Filter paper hydrolysis
activity was measured by adding 0.2 mL of supernatant and 1.8 mL of 0.05 M sodium citrate
buffer (pH 4.8) into test tubes containing 25 mg Whatman No. 1 filter
paper strips (1.0x3.0 cm). The substrate used for the determination of
ß-D-Glucosidase activity was p-nitrophenol- β-D-glucopyranoside
(Sigma).
Statistical Analysis
Analysis of variance was done with data of 96 h incubation when maximum
reducing sugar productivity was recorded and the fermentation profile
of SSF after optimization was compared with the fermentation profile before
optimization.
RESULTS AND DISCUSSION
Solid Substrate Fermentation (SSF)
Significant growth of Trichoderma sp. KUPM0001 was observed
on sago hampas. The first sign of growth was seen after 12 h of
inoculation. Visual observation every 24 h revealed a spreading white
mycelia web on the surface of the substrate and covered it entirely by
time. As the culture grew older the colour of the mycelia change from
white to dark green and by 96 to 120 h of solid substrate fermentation
complete colonization by the fungus was observed. The initial pH of the
supplemented culture on 0 h was 5.54 (Fig. 1a). There
was not much variation of the pH in the crude culture extract during the
fermentation period, the pH ranging from 5.54 to 5.60. The slight decreased
in pH of the substrate may be correlated directly with the decomposition
activity of the fungus. In the present experiment, it was found that pH
range between 5 and 7 to be suitable for the growth of Trichoderma
sp. KUPM001 on sago hampas.
The concentration of soluble protein in 0 h sample was 0.54 mg mL-1
and the extractable protein reached 1.97 mg mL-1 after the
first 48 h of fermentation (Fig. 1b). The increased
in protein was due to the secretion of extracellular enzymes responsible
for the degradation of hampas. The soluble protein content can
be used to relate the growth of fungus. The soluble protein content was
used as an indirect assessment of fungal biomass and changes in soluble
protein correlated with the morphogenesis of the fungus.
| Fig. 1a: | Profiles of pH during SSF of sago hampas by Trichoderma sp. KUPM0001 |
| Fig. 1b: | Profiles of soluble protein during SSF of sago hampas by Trichoderma sp. KUPM0001 |
| Fig. 2: | Effect of different percentage of initial moisture content of solid substrate medium on reducing sugar production by Trichoderma sp. KUPM0001 via SSF |
Effect of Initial Moisture Content on Reducing Sugar Production
The effect of initial moisture content of the substrate on reducing
sugar production is presented in Fig. 2. In the present
study, the highest reducing sugar production obtained was 27 mg mL-1
at 80% (v/w) initial moisture content. During the course of fermentation,
the content of the flask was gently mixed using sterile L stick at 24
h intervals to increase the aeration and to evenly distribute water availability
to the fungus. A decrease in reducing sugar production was observed when
the moisture level was set at levels higher or lower than the 80%. The
results showed that the levels of reducing sugar increased significantly
(p<0.05) with incubation time. Higher substrate moisture in SSF resulted
in suboptimal product formation due to reduced mass transfer such as diffusion
of solutes and gas to cell during fermentation while, lower moisture level
minimized heat exchange, oxygen transfer and low availability of nutrients
to the culture (Rahardjo et al., 2005). These conditions affected
microbial activity and resulted in decreased productivity.
Effect of Mineral Salts Solution on Reducing Sugar Production
The effect of mineral salts solution on reducing sugar production
was studied by altering the volume of salts solution to the weight of
the substrate. In this study, the results indicated a positive relationship
between levels of salts solution used and reducing sugar production from
sago hampas using Trichoderma sp. KUPM0001 via SSF. On lower
and further enhancement in mineral solution level inversely influenced
the production of reducing sugar significantly. The maximum sugar obtained
was 29 mg mL-1 (Fig. 3), significantly (p<0.01)
when mineral salts solution concentration was at 20% (v/w), compared to
lower and higher levels whereby the productions were much lower. The high
and low concentration of salts had a significant effect on the metabolic
activities of the organism. Basically, the addition of nutrients to the
solid medium improved the growth of organism and thus the production of
the desired products. Supplementation of sago hampas with minerals
also stimulated the production of relevant enzymes by Trichoderma
sp. KUPM0001 and as the result good production of reducing sugar was obtained.
| Fig. 3: | Effect of different concentration of mineral salt solution on reducing sugar production by Trichoderma sp. KUPM0001 via SSF |
| Fig. 4: | Effect of different concentration of urea on reducing sugar production by Trichoderma sp. KUPM0001 via SSF |
Effect of Urea Concentration on Reducing Sugar Production
Based on the results obtained, among the different concentration levels
of urea tested, 1.0% (w/v) gave significant increased (p<0.01) on reducing
sugar production (38 mg mL-1) compared to 0.5, 1.5 and 2.0%
(Fig. 4). In this study, supplementation of 1.0% (w/v)
urea was 48.32% of the total nitrogen measured in the hampas. Urea
has been reported to stimulate fungal growth when cultures were supplemented
up to 40-50% (v/v) of the total nitrogen in the substrate. Furthermore,
from ecological investigation it was known that the degradation of lignocellulose
containing substrates was enhanced by supplementation of nitrogen. Addition
of nitrogen sources to the substrate enhanced the consumption of the easily
degradable components especially cellulose, leading to a reduction of
in vitro digestibility. It was shown that addition of urea to the
solid medium, enhanced the production of reducing sugar. The use of urea
as inorganic nitrogen source also supported good fungal growth and protein
production, which was attributed to the increase in enzyme protein. However,
further increase in the concentration levels led to a sharp decline in
sugar yield. This may explain the decrease in levels of reducing sugar
when higher concentrations of urea were used.
| Fig. 5: | Effect of different inoculum densities on reducing sugar production by Trichoderma sp. KUPM0001 via SSF |
Effect of Inoculum Density on Reducing Sugar Production
To evaluate the effect of inoculum level on reducing sugar production,
different cell density (10%, 20 and 30% v/w) were added. Fermentations
were carried out for 120 h and results are shown in Fig.
5. A 10% (v/w) of inoculum density gave maximum reducing sugar (41
mg mL-1) which was significantly higher (p<0.01) when compared
to higher inoculum densities. It has been shown that inoculum-to-substrate
ratio (v/w) had a significant impact on enzymes production in SFF hence
effecting the production of reducing sugar. This was because at higher
inoculum levels, biomass production was increased leading to poor product
formation (Nutan et al., 2002). Further with the increase in inoculum
levels, the production of enzymes declined due to the exhaustion of nutrients
in the fermentation mash. In addition, free excess liquid present in an
unabsorbed form would pose an additional diffusion barrier together with
that imposed by the solid nature of the substrate leadings to decrease
in cell growth and enzymes production.
Effect of Incubation Temperature on Reducing Sugar Production
Different incubation temperatures (25, 30, 35, 40 and 45 °C) were
used on reducing sugar production. The fermentation was carried out for
120 h. The results are presented in Fig. 6. Maximum
reducing sugar production (37 mg mL-1) was obtained at 30 °C.
A decreased in the production was observed when the incubation temperature
was higher or lower than the optimum incubation temperature. Higher temperatures
were found to have adverse effect on the metabolic activities of the microorganisms
and it has been reported by various researchers that the metabolic activities
of the microorganisms become slow at lower than the optimum temperature.
Incubation temperature and its control in SSF process is crucial as the
heat evolved during SSF processes is accumulated in the medium due to
poor heat dissipation in solid medium. This phenomenon resulted in reduced
microbial activity, thereby decreasing the product yield. The significance
of temperature in development of biological process was such that it could
determine the protein denaturation, enzyme inhibition, promotion or suppression
of the production of a particular metabolite, cell viability and death.
At higher temperatures, the heat may destroy the catalytic activities
of the enzymes and the growth was inhibited.
| Fig. 6: | Effect of incubation temperature ( °C) on reducing sugar production by Trichoderma sp. KUPM0001 via SSF |
| Fig. 7: | Effect of different incubation time on reducing sugar production by Trichoderma sp. KUPM0001 via SSF |
Effect of Incubation Time on Reducing Sugar Production
The reducing sugar production showed growth relatedness as the incubation
period progressed and maximum sugar was obtained (46 mg mL-1)
after 96 h (Fig. 7). During the first 24 h of fermentation,
Trichoderma sp. KUPM0001 was at its log phase, which extended up
to further 96 h followed by the period when it was gradually decreased.
The pre-germinated mycelial inoculum ensured the rate of production was
very rapid and incubation beyond the optimum time showed rapid decline
in sugar levels (25 mg mL-1) at 120 h. A prolonged fermentation
time beyond this period did not increase significantly the production
of reducing sugar. The production of reducing sugar was very much associated
with the enzyme production, which was highest when the fungus was at its
peak log phase. The growth-associated production of amylases in a recombinant
A. oryzae proved that the specific amylases production was closely
coupled to the growth of the fungus. This suggests that the growth of
the mycelium is crucial for high production of extra-cellular protein.
Incubation beyond the optimum time was undesireable as this resulted in
decreased of reducing sugar production which indicated that the fungus
was at its stationary phase from 96 to 120 h of incubation.
Solid Substrate Fermentation with Optimum Conditions
Solid substrate fermentation was conducted and optimal parameters
evaluated were applied such as 80% moisture content; 20% (v/w) mineral
salts solution; 1.0% (w/v) urea concentration; 10% (v/w) inoculum density
at 30 °C for 120 h. Reducing sugar, enzymes activities, soluble protein
and pH involved were determined. In the present study, it was shown that
as the enzymes (amylases) increased, the reducing sugar yield increased
proportionately. The maximum alpha-amylase produced was at 72 h (3.19
U mL-1) for the optimum production of reducing sugar at 46
mg mL-1 (Fig. 8). The correlation between
the alpha-amylase and reducing sugar yield was positively correlated (r
= 0.767; p = 0.006) and significant at p<0.01 (2-tailed). However,
maximum production of glucoamylase was at 72 h (2.24 U mL-1).
The correlation between the glucoamyalse and reducing sugar yield was
positively correlated (r = 0.730; p = 0.011) and significant at p<0.05
(2-tailed). The correlation between the amylases was significant p = 0.01
(2-tailed) level of significant (r = 0.928; p = 0.001).
| Fig. 8: | Relationship between reducing sugar yield and amylases activities at optimized parameters; 80% moisture content; 20% (v/w) mineral salt solution; 1% (w/v) urea concentration; 10% (v/w) inoculum density and incubated at 30 °C,120 h via SSF |
| Fig. 9: | Relationship between reducing sugar yield and cellulases activities at optimized parameters; 80% moisture content; 20% (v/w) mineral salts solution; 1% (w/v) urea concentration; 10% (v/w inoculum density at 30 °C, 120 h via SSF |
Fungi are in direct contact with their nutrients in the environment. Smaller molecules (simple sugars and amino acids) in the solution in the watery film surrounding the hyphae can be directly absorbed by the hyphae. Larger insoluble polymers such as cellulose, starch and proteins must undergo a preliminary digestion before they can be used. Molecules that are too large to be absorbed by the fungus are attacked by extracellular enzyme. The ability to utilize large molecules ultimately depends on the ability of the fungus to digest them, which in turn depends on the enzymes with which the fungus is equipped.
There are two major classes of starch degrading enzymes identified in the fungi: α-amylase and glucoamylase. A single species may secrete both. The amylases activity generally increased as fermentation proceeded. Amylases activities are known to be regulates by catabolite repression.
Extracellular enzyme was commonly controlled by catabolite repression, in the presence of rapidly metabolized carbon source, generally glucose, which is little or no synthesis of the enzymes. Some authors have suggested that the absence of catabolite repression in SSF system is due to several factors collectively, including the slow and low processes of diffusion in SSF cultures due to the low water activity. However, all SSF system described as resistant to catabolite repression were developed using wheat bran as substrate and in this present study sago hampas acts the same way. During the active growth, cellulose is often cell or substrate bound. They only appear in medium as the growth rate slow down. This was probably associated with a decrease on the area of substrate available for adsorption of enzymes and/or cells. Hence at the end of growth enzymes are desorbed from residual substrate and released from the surface on non-growing cells only if catabolite repressor was present in the medium.
Cellulases may not be produced until the repressors are mostly utilised and the growth rate has slowed down, i.e. late in the growth of the culture. Data of comparable enzymes activities were shown in Fig. 9. In the present study there were no significant difference (p = 0.314), (p = 0.799) and (p = 0.712) in CMCase, FPase and BGDase activity, respectively. Like cellulases produced from other sources, cellulases from KUPM001 are known to be catabolite sensitive. The CMCase activity of Trichoderma sp. KUPM0001 on sago hampas may be inhibited by high reducing sugar concentration in the culture.
There was no catabolic repression was observed in sago hampas during solid substrate fermentation and maximum production of reducing sugar and the relevant enzymes was obtained from sago hampas by fermentation by KUPM001 after 96 h of cultivation. However, prolonged incubation time after the optimal period caused a reduction in the production of both amylases and cellulases. After 96 h of incubation, the reducing sugars levels decreased and this may be due to the consumption of fungal cells.
The starchy fibrous residue hampas from sago starch processing may be a potential renewable resource for the production of reducing sugars via SSF. The sugars can then be a cheap feedstock for bioethanol production. The results of this study indicate the potential and further scale-up studies are warranted.
CONCLUSION
Production of reducing sugars by Trichoderma sp. KUPM0001 during solid substrate fermentation of sago starch processing waste hampas were successfully optimized. The optimum parameters obtained were 80% (v/w) of initial moisture; 10% (v/w) of inoculums size; 1.0% of urea in 20% (w/v) of mineral solution and incubated at 30 h ± 2 °C which gave the enzyme activities for α-amylase, glucoamylase, carboxymethyl cellulase, filter paperase and ß-glucosidase of 3.19, 2.22, 1.66 , 1.11 and 1.48 U mL-1, respectively. It was suggested that sago hampas is a suitable substrate for conversion to reducing sugar by local fungal. The recovered sugar could be further utilized as fermentation feedstock or converted into bioethanol.
ACKNOWLEDGMENTS
The authors wish to thank the National Biotechnology Directorate Malaysia for their financial support of the project and Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia and Institute of Biological Sciences, University of Malaya for support of this study.