Maximizing Glucose Production From Palm Kernel Cake (Pkc) From Which Residual Oil Was Removed Supercritically Via Solid State Fermentation (Ssf) Method Using Trichoderma Reesi Isolate Pro-A1
R Nik Nourulaini N. Ab, B Moftah, T Lah, A Nawi, M Kadir, M Aliyu-Paiko
animal feeds, glucose production, palm kernel cake, residual oil extraction, solid state fermentation, supercritical fluid extraction, trichoderma sp.
R Nik Nourulaini N. Ab, B Moftah, T Lah, A Nawi, M Kadir, M Aliyu-Paiko. Maximizing Glucose Production From Palm Kernel Cake (Pkc) From Which Residual Oil Was Removed Supercritically Via Solid State Fermentation (Ssf) Method Using Trichoderma Reesi Isolate Pro-A1. The Internet Journal of Microbiology. 2012 Volume 10 Number 1.
The present trial was carried out to evaluate the effects of incubation temperature, pH and time during solid state fermentation (SSF) of PKC substrates using
The agricultural industry in Malaysia generates large quantities of wastes, which has been estimated at approximately 5 million tons per annum and this is expected to increase by two-fold or more by the year 2010 (Pang et al. 2006). One notable feature of these generated wastes however, is that majority originate from the palm industry including; oil palm trunks and fronds, palm kernel and palm kernel cake (PKC), among many other materials.
PKC is a waste product generated after crushing the palm kernel, to extract the oil from the fruit using the screw-press extraction (expeller) technique (Alimon 2004; Akpan,
The use of large quantities of cell wall components like cellulose, mannan and ligno-cellulose in agricultural wastes available in the environment as raw materials for solid state fermentation (SSF) processes for use in animal feedstock continue to receive global attention (Alimon 2004; Soltan, 2009; Wallace et al., 2010). The potential of PKC as a feed ingredient for ruminant animals in the livestock feeds industry after fungi fermentation is well documented (Akpan
From residual oil-free PKC, the lignocellulose could be converted to fermentable sugars, after they have been broken down, to be used as carbon sources by several microorganisms. This is because fermentable sugars are known to be produced by fungi in their natural habitats via SSF processes (Ibrahim, 2007). Among the wood-degrading fungi however,
Supercritical fluid extraction (SFE) was shown to be a powerful extraction method for the removal of oils from fibers (Lau et al., 2006). In the SFE technique, supercritical carbon dioxide (SC-CO2) is employed as the solvent and has replaced other traditional solvents in the food industries because of its characteristic advantages of being non-toxic, non-flammable and its availability at reasonably low costs (Nik Norulaini et al., 2004). Therefore, the principal objective of the present study was to evaluate the effects of temperature, pH and time used in SSF of Oily and Oil-less PKC substrates, using
Isolation and Identification of Trichoderma Isolate Pro-A1, the Potential Producer of Cellulase
The solid agar media used for this experiment comprised of 163 mL of V8 mixed vegetable juice added to 87 mL of distilled water and 3 g of Nutrient Agar and mixed thoroughly, using a glass rod. The pH of this mixture was determined to be 5.00 at room temperature, using a bench top pH meter (Hach sensION3 CO, USA) fitted with a gel-filled platinum electrode. The solution was heated to boil at 121 C for 15 min and then allowed to cool to ambient temperature. After cooling, the media was poured into petri dishes. Commercial
Sampling and Preparation of substrates (PKC)
Freshly produced PKC was obtained from a local palm kernel mill in Pinang, Malaysia. The fresh samples were divided into two equal portions; one portion was immediately packed and stored in a refrigerator maintained at 4 C until used later and was labeled as Oily PKC treatment. The other portion was defatted using SFE method, with SC-CO2as the extraction solvent as explained in the subsequent section. Residual hexane from this extraction procedure was then removed and the defatted PKC was stored at 4 C until ready for use; where the treatment was labeled Oil-less PKC. Both PKC treatments were dried in an oven maintained at 60 C for 24 hours before use.
Dried PKC substrates were properly ground and screened through a sieve shaker, to obtain substrate particles of three mesh sizes (250µM, 500µM and 1mM), which were used for subsequent evaluations. Substrates of mesh size 250µM were chosen for use in subsequent evaluation.
Supercritical Fluid Extraction (SFE), using C0₂ as the extraction solvent
Initial study was conducted using the equipment of Supercritical system; SFX 220 extraction system (ISCO, Lincoln, NE, USA). This consisted of carbon dioxide cylinder, chiller (B-L;730, Yih Der, Taiwan), C0₂ syringe pump (ISCO, Model 100DX), modifier syringe pump (ISCO, Model 100 DX), extraction chamber (ISCO, SFX 220), extraction cartridge, controller (ISCO, SFX 200) and Restrictor Temperature Controller (ISCO) (Nik Norulaini, 2004a). From PKC samples with particle size 250 μM, residual oil was super critically removed, at a pressure range of between 27 and 41 MPa, a temperature range of 50 C to 80 C and with the substrate flow rate set at 1.5 mL/min. The substrate yielding the highest residual oil after the SFE extraction was used in subsequent evaluation (as Oil-less treatment).
Solid State Fermentation (SSF) of Oily and Oil-less PKC, using Trichoderma Isolate Pro-A1
Solid state fermentation of substrates using the fungus was performed on 5 g PKC and PK as the solid substrates (Oily and Oil-less treatments), with the addition of 2 mL of Mandel’s medium. The Mandel's medium was prepared containing the following components (g/L); urea, 0.3; peptone, 0.75; yeast extract, 0.25; (NH4)2SO4, 1.4; KH2PO4, 2.0; CaCl2, 0.3; MgSO4.7H2O, 0.3 and trace elements (mg/L): FeSO4.7H2O, 5; MnSO4. 4H2O, 1.6; ZnSO4.7H2O, 1.4 and CoCl2.6H2O, 20.0 (Mandels
Optimization of pH, Incubation Temperature and Incubation time
The SSF was conducted at 3 different pH which included; 3.0, 5.0 and 7.0. As for the incubation temperature, the tests were also performed at 3 different temperatures as follows; 20 C, 30 C and 40 C. The differences in the incubation times were measured at 4 different time intervals of 4 hours, 8 hours, 12 hours and 16 hours, accordingly. All experimental procedure was maintained as same, except the variations in the pH, temperature and time as mentioned earlier. Experiments were carried out in triplicates.
Measurement of Glucose Concentration
To measure the concentration of glucose produced in the culture medium, a standard curve was plotted, using standard glucose solution (Sigma MO, USA). Approximately 10 mL containing 10 mg/mL of glucose stock solution was prepared. From this stock solution, dilutions were made with distil water to prepare concentrations of 20, 40, 60, 80 and 100 μg/mL of glucose solutions, following standard laboratory protocols. From each of these concentrations, 0.5 mL of glucose solution was measured out into separate vials with a pipette. To each solution in a vial, 0.5 mL of 5% phenol and 0.5 mL of concentrated H2SO4were added. The vials and contents were allowed to stand for 10 min for reactions to take place, after which they were shaken to allow the solutions to mix. Finally, the mixed solutions in the vials were left for 30 min for color development and their absorbance was read in a spectrophotometer at 490 nm. The result was used to plot a standard curve.
Approximately 0.5 mL of the sample solution was also measured with a pipette into a vial. To this 0.5mL of 5% phenol was added, followed by the addition of 0.5 mL of concentrated H2SO4and the solution also left to stand for 10 minutes for the reaction to take place. Subsequently the vials were shaken to mix the solutions and the solutions were also left for 30 minutes for the color to develop and the absorbance of the colored mixture was read at 490 nm in a spectrophotometer. All the procedure was carried out in triplicates and results are reported as mean ± SD of 3 determinations.
Statistical mean of data for the effects of pH, temperature and time on the concentration of glucose produced in the different culture media were compared using 1 and 2 way ANOVA in the SPSS statistical software package; where all values of P<0.01 were considered significant.
As could be observed in the SEM micrographs shown in Figure 1,
Result of incubation temperature, pH and time of SSF on glucose concentration produced by
In comparison, result of incubation temperature, pH and time of SSF on glucose concentration produced by
Two-way ANOVA showing the interactions of incubation Temperature, pH and time during SSF of the Oily PKC substrate (Table 1) or that for Oil-less PKC substrate (Table 2) all clearly show the significant (P<0.01) interactions of incubation temperature and time, temperature and pH, time and pH and temperature, time and pH, respectively suggesting the effects of all the variables tested.
Temperature is a very important factor in the growth of fungi, particularly
In a similar development, pH plays a critical role in the growth of the fungus. According to Lorant (2008),
Recently, the emergence of solid state fermentation (SSF) as a promising technology for the development of several bioprocesses and products, including the production of therapeutic enzymes on a commercial scale cannot be over-emphasized (Pandey et al., 1999). The primary advantage of SSF is the fact that many metabolites could be produced at very high concentrations. From analysis of the literature with regards to enzymatic hydrolysis, it was revealed that high cellulase activity per unit volume of fermentation broth is the most important factor in obtaining sugars (such as glucose as fermentation products) in concentrations of 20 to 30%, from hydrolysis of cellulose for ethanol production from cellulosic materials (Chahal, 1982). Usually, glucose production by
The large scale availability of PKC, which according to estimates of Atasie & Akinhanmi (2009) stood at over 4 million tons in 2002, has been used as a feed ingredient for ruminant animals in the livestock feeds industry (Akpan