Introduction

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, et al., 2005; Soltan, 2009). The global production of this by-product has consistently increased in volume with the expansion of the oil palm industry in many countries in Asia (particularly Malaysia and Indonesia) and Africa in recent years. The global production of PKC in the year 2002 was estimated at over 4 million metric tons (Atasie and Akinhanmi, 2009). The proximate composition of PKC suggests that it could be classified as an energy feed, as it is richly composed of nutrients containing (on the average); 59.9% carbohydrate, 10.2% fat, 2% sugar and 391 kcal of energy. It is also an important source of protein (18.6% crude protein, consisting of high quality amino acids), contains crude dietary fiber of 37% and average residual oil content of 10% (Alimon 2004). The analysis of PKC showed that over 60% is cell wall components, with the fiber content composed of mainly insoluble, mannose-based polysaccharides (Alimon 2004). Jaafar and Javis (1994) also noted that PKC cell wall consists of 58% mannan, 12% cellulose and 4% xylan.

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 et al., 2005). However, the major existing problem in the use of PKC for such purposes remains the residual oil content after extraction, where little is known about the process involved in the removal of this residual oil from the PKC matrixes. The residual oil in the cake is partly responsible for PKC’s characteristic unpleasant taste and odor, due to incidents of mould and rancidity during storage, thus hindering its use (Pearson 1985).

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, Trichoderma reesei has been reported as one of the most successful and extensively studied cellulase systems (Claeyssens and Tomme, 1989; Goyal et al., 1991; Teeri et al., 1998). Cellulolytic enzymes from a filamentous fungus, Trichoderma sp. has also been a subject of intensive research because this organism secretes large amounts of enzymes required for the complete hydrolysis of crystalline cellulose (Kubicek, 1992; Teeri et al., 1992). To maximize the use of PKC as animal and human feed components therefore, alternative methods are needed to remove the residual oils, with supercritical fluid extraction being one such method currently receiving global attention.

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 Trichoderma Isolate Pro-A on the potential of maximizing glucose production from the substrates before and after the residual oil is extracted via SFE. This paper describes the physico-chemical parameters explored, to produce glucose from the PKC substrates.

Methodology

Isolation and Identification of Trichoderma Isolate Pro-A1, the Potential Producer of Cellulase

Sampling and Preparation of substrates (PKC)

Supercritical Fluid Extraction (SFE), using C0₂ as the extraction solvent

Solid State Fermentation (SSF) of Oily and Oil-less PKC, using Trichoderma Isolate Pro-A1

Optimization of pH, Incubation Temperature and Incubation time

Measurement of Glucose Concentration

Statistical Analysis

Results

As could be observed in the SEM micrographs shown in Figure 1, Trichoderma reesei colonies covered the surface of the Oily and Oil-less PKC substrate, when incubated for 5 days at 30°C, with 8 hours of lighting per day, although the conidiophores were thicker on the Oil-less substrates. This indicates that the culture conditions were favorable for the growth of the fungi, although the substrates containing less oil seem to support better growth. The particle size of 250 μm for the PKC substrates was selected based on preliminary experiment carried out earlier, which showed this particle size to yield significantly the highest amount of residual oil during SFE.

Figure 1

Figure 1: SEM micrographs of grown on substrates (A) Oily PKC and (B) Oil-less PKC, 5 days after incubation at 30°C with 8 hours of lighting per day. Notice the codia and coniophores of the colonies on the surface of the culture media.

Result of incubation temperature, pH and time of SSF on glucose concentration produced by T. reesei colonies in the Oily PKC substrates is shown in Figure 2. Increase of incubation temperature from 20 to 30°C led to a significant (P<0.01) increase in glucose concentration from ~16.5 to over 18.5 μg/mL, while further increase in temperature to 40°C led to a significant decline in glucose concentration to levels recorded at 20°C (below 15 μg/mL). At pH 3, glucose concentration was recorded at ~16.3 μg/mL. Increasing the incubation pH to 5 also led to significant increase in glucose concentration to ~18 μg/mL; whereas a further increase in incubation pH manifested in a decline in the glucose concentration back to 16 μg/mL. On the other hand, changing the incubation time from 4 to 8 and further to 12 hours led to no significantly (P>0.01) observed changes in the glucose concentration in the media. However, further increase in incubation time from 12 to 16 hours led to a sharp and significant increase in glucose concentration from ~16.4 to 18 μg/mL.

Figure 2

Figure 2: The effects of incubation Temperature, Time and pH of SSF of Oily PKC substrate with on glucose concentration (μg/mL).

In comparison, result of incubation temperature, pH and time of SSF on glucose concentration produced by T. reesei colonies on the Oil-less PKC substrates is shown in Figure 3. The trends observed in the concentration of glucose in the media following increase in incubation temperature and pH were similar to those noted in the Oily PKC substrate. However, the concentration of glucose was significantly higher compared to that in the Oily PKC substrate, at each of the temperatures and pHs tested. In the case of the incubation times tested however, glucose concentration in the media was noticed to slightly (but insignificantly) decline when the time was increased from 4 to 8 hr. Further increase in the incubation time from 8 to 12 and then to 16 hr, led to significantly (P<0.01) higher glucose concentrations, respectively.

Figure 3

Figure 3: The effects of incubation Temperature, Time and pH of SSF of Oil-less PKC substrate with on glucose concentration (μg/mL).

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.

Figure 4

Table 1: Types and sources of error, degree of freedom and significance of variation (P-values) of the effects incubation Temperature, Time and pH and their interactions during SSF of PKC substrate with on glucose concentration (μg/mL)

Figure 5

Table 2: Types and sources of error, degree of freedom and significance of variation (P-values) of the effects incubation Temperature, Time and pH and their interactions during SSF of PKC substrate with on glucose concentration (μg/mL)

Discussions

Trichoderma reesei colonies were noticed to have completely covered the surface of the PKC substrates after 5 days of incubation at 30°C (the best temperature among those tested, which yielded the highest concentration of glucose in the Oil-less substrate), with 8 hours of lighting per day. This was a good indication, suggesting that the culture conditions provided at that temperature favored the growth of the fungi. In Singhania et al. (2006), the use of Trichoderma reesei strains (NRRL 11460) on lingo-cellulose substrates was also reported, using SSF and shown that the temperature favorable for the growth of the fungi was at 28°C, for the highest cellulose enzyme activity to produce cellulose. However, in a study which reported the physiological characteristics of Trichoderma spp. isolated from oyster mushrooms (Pleurotus spp.), Choi et al. (2003) demonstrated that the optimal growth temperature of Trichoderma spp. was between 27 and 30°C, consistent with the conditions observed in the present study. This supports that the choice of 30°C as one of the incubation temperatures tested in this experiment was within the range acceptable for optimal growth of the fungi.

Temperature is a very important factor in the growth of fungi, particularly Trichoderma spp. Studies of the influence of different temperatures on the mycelial growth of Trichoderma revealed that the growth of Trichoderma was favored by higher temperatures, reaching a maximum at 30°C, whereas no growth was observed at 15°C (Lorant, 2008), but growing at wider range between 20-28°C (Woo et al. 2004). In describing the effects of temperature on growth patterns of Trichoderma, Denesh et al. (2000) reported that at temperature of 25°C, growth patterns of T. longibrachiatum, T. harzianum, T. virens and Trichoderma sp. collected from mushroom farms showed significant differences in their type of growth and sporulation patterns between various species and isolates. However, Choi et al. (2003) reported that the optimal growth temperature of Trichoderma spp. was between 27 and 30°C. Trichoderma species are asexual, soil-inhabiting filamentous fungi with teleomorphs. According to Chen and Moy (2004), a fluctuation in such important parameters which are ideal for mushroom cultivation, such as the sources of carbon and nitrogen, high relative humidity, warm temperatures and the absence of light during most of spawn run, are ideal conditions in the environment for the green moulds as well, which can easily lead to their contamination.

In a similar development, pH plays a critical role in the growth of the fungus. According to Lorant (2008), Trichoderma prefer acidic-neutral conditions (pH 5-7) for optimal growth. The mycotoxins produced by Trichoderma have been reported and identified as gliotoxin, viridin, trichodermin and peptide type (Kim, 1985). Furthermore, Trichoderma species secrete hydrolytic enzymes including chitinases, β-glucanases and cellulases which are kind of mycotoxins and they lyse the fungal cell walls and are speculated to play key roles in the myco-parasitic activity of this fungus (Goltapeh and Danesh, 2000). Certain species of Trichoderma are well known to possess the ability of antagonizing a series of plant pathogenic fungi (Papavizas 1985). Their proposed mechanisms of antagonism include mycoparasitism by the action of cell-wall degrading enzymes, antibiosis by the production of antibiotics, competition for space and nutrients through rhizosphere competence, facilitation of seed germination and growth of the plants via releasing important minerals and trace elements from soil and induction of the defense responses in plants (Herrera-Estrella and Chet 2003; Howell 2003; Benítez et al. 2004).

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 Trichderma occurs via acidic or enzymatic hydrolysis of starch present in the substrates which the fungus acts upon. In America and many other parts of the world, glucose is produced mainly by using enzymatic hydrolysis of grains starch (Van de Veen et al., 2005). Similarly, Silva and colleague (2010) used two microorganisms extracted from the soil; Aspergillus niger and Strepotmyces sp., to produce glucoamilase and glucoisomerase enzymes, respectively, through the conversion of cassava starch to glucose and fructose syrup, further justifying the use of micro-organisms for sugar production.

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 et al., 2005). This great advantage of PKC holds a great potential when properly harnessed, for the commercial production of glucose and other sugars through SSF method (Ab Rashid et al. 2011), as was also demonstrated in this study, after the removal of the residual oil via SFE technique. When properly harnessed, the menace created by the accumulation of PKC wastes could be converted to cash, through the combination of a cheap residual oil extraction technique (SFE) and an efficient glucose production method (SSF) using Trichderma, respectively, as was demonstrated in the present experiment.

Acknowledgement

The fund for this research was provided by the Universiti Sains Malaysia Fundamental Research Grant Scheme ( FRGS) No. 203/PJJAUA/6711112, which is gratefully acknowledged. The authors thank Puan Jamilah at Electron Microscopy Unit of the School of Biological Sciences, USM for the technical assistance during the processing of the SEM micrographs.