The oil palm sector is one of the major plantation industries in Malaysia. Palm kernel cake is a byproduct of extracted palm kernel oil. Mostly palm kernel cake is wasted or is mixed with other nutrients and used as animal feed, especially for ruminant animals. Recently, palm kernel cake has been identified as an important ingredient for the formulation of animal feed, and it is also exported especially to Europe, South Korea, and Japan. It can barely be consumed by nonruminant (monogastric) animals owing to the high percentages of hemicellulose and cellulose contents. Palm kernel cake must undergo suitable pretreatment in order to decrease the percentage of hemicellulose and cellulose. One of the methods employed in this study is fermentation with microorganisms, particularly fungi, to partially degrade the hemicellulose and cellulose content. This work focused on the production of enzymes by Aspergillus niger and profiling using palm kernel cake as carbon source.
The effects of phenolic monomers (i.e. rho-coumaric acid, ferulic acid, rho-hydroxybenzaldehyde and vanillin) on the enzymes and fermentation activities of Neocallimastix frontalis B9 grown in ball-milled filter paper and guinea grass media were studied. The enzymes studied were carboxymethylcellulase (CMCase), filterpaperase (FPase), xylanase and beta-glucosidase. At 96 h of incubation, N. frontalis grown in ball-milled filter paper medium produced comparable xylanase and CMCase activities (0.41, 0.5 micromol/min/mg protein) while in guinea grass medium, N. frontalis produced higher xylanase activity than that of CMCase activity (2.35, 0.05 micromol/min/mg protein). The other enzymes activities were low. When N. frontalis was grown in ball-milled filter paper medium, only acetic acid was produced. However, when grown in guinea grass medium, the major end-product was acetate, but propionic, butyric and isovaleric were also produced in lesser amount. Vanillin showed the least inhibitory effects to enzyme activities of N. frontalis B9 grown in both ball-milled filter paper and guinea grass media. For total volatile fatty acid production, all phenolic monomers showed inhibitory effects, but rho-coumaric and ferulic acids were the stronger inhibitors than rho-hydroxybenzaldehyde and vanillin.
A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.
White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20-30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02-26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.
Acetone-butanol-ethanol (ABE) production from renewable resources has been widely reported. In this study, Clostridium butyricum EB6 was employed for ABE fermentation using fermentable sugar derived from treated oil palm empty fruit bunch (OPEFB). A higher amount of ABE (2.61 g/l) was produced in a fermentation using treated OPEFB as the substrate when compared to a glucose based medium that produced 0.24 g/l at pH 5.5. ABE production was increased to 3.47 g/l with a yield of 0.24 g/g at pH 6.0. The fermentation using limited nitrogen concentration of 3 g/l improved the ABE yield by 64%. The study showed that OPEFB has the potential to be applied for renewable ABE production by C. butyricum EB6.
The importance of bioethanol currently has increased tremendously as it can reduce the total dependency on fossil-fuels, especially gasoline, in the transportation sector. In this study, Ceiba pentandra (kapok fiber) was introduced as a new resource for bioethanol production. The results of chemical composition analysis showed that the cellulose (alpha- and beta-) contents were 50.7%. The glucose composition of the fiber was 59.8%. The high glucose content indicated that kapok fiber is a potential substrate for bioethanol production. However, without a pretreatment, the kapok fiber only yielded 0.8% of reducing sugar by enzymatic hydrolysis. Thus, it is necessary to pre-treat the kapok fiber prior to hydrolysis. Taking into account environmentally friendliness, only simple pretreatments with minimum chemical or energy consumption was considered. It was interesting to see that by adopting merely water, acid and alkaline pretreatments, the yield of reducing sugar was increased to 39.1%, 85.2% and >100%, respectively.
Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum.
Mushroom industries generate a virtually in-exhaustible supply of a co-product called spent mushroom substrate (SMS). This is the unutilised substrate and the mushroom mycelium left after harvesting of mushrooms. As the mushroom industry is steadily growing, the volume of SMS generated annually is increasing. In recent years, the mushroom industry has faced challenges in storing and disposing the SMS. The obvious solution is to explore new applications of SMS. There has been considerable discussion recently about the potentials of using SMS for production of value-added products. One of them is production of lignocellulosic enzymes such as laccase, xylanase, lignin peroxidase, cellulase and hemicellulase. This paper reviews scientific research and practical applications of SMS as a readily available and cheap source of enzymes for bioremediation, animal feed and energy feedstock.
The response surface method was applied in this study to improve cellulase production from oil palm empty fruit bunch (OPEFB) by Botryosphaeria rhodina. An experimental design based on a two-level factorial was employed to screen the significant environmental factors for cellulase production. The locally isolated fungus Botryosphaeria rhodina was cultivated on OPEFB under solid-state fermentation (SSF). From the analysis of variance (ANOVA), the initial moisture content, amount of substrate, and initial pH of nutrient supplied in the SSF system significantly influenced cellulase production. Then the optimization of the variables was done using the response surface method according to central composite design (CCD). Botryosphaeria rhodina exhibited its best performance with a high predicted value of FPase enzyme production (17.95 U/g) when the initial moisture content was at 24.32%, initial pH of nutrient was 5.96, and 3.98 g of substrate was present. The statistical optimization from actual experiment resulted in a significant increment of FPase production from 3.26 to 17.91 U/g (5.49-fold). High cellulase production at low moisture content is a very rare condition for fungi cultured in solid-state fermentation.
Various pretreatments on Ceiba pentandra (L.) Gaertn. (kapok) fiber prior to enzymatic hydrolysis for sugar production were optimized in this study. The optimum conditions for water, acid, and alkaline pretreatments were 170°C for 45 min, 120°C for 45 min in 1.0% (v/v) H2SO4 solution and 120°C for 60 min in 2.0% (v/v) NaOH solution, respectively. Among the three pretreatments, the alkaline pretreatment achieved the highest total glucose yield (glucose yield calculated based on the untreated fiber) (38.5%), followed by the water (35.0%) and acid (32.8%) pretreatments. As a result, the relative effectiveness of the pretreatment methods for kapok fiber was verified as alkali>water>acid at the condition stated.
Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2% NaOH with autoclave, which was composed of 59.7% cellulose, 21.6% hemicellulose, and 12.3% lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1% of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5% of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33% and 19.11%, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.
This study examined the potential of untreated and alkali-pretreated sugarcane bagasse (SCB) in cellulase, reducing sugar (RS) and fungal biomass production via solid state fermentation (SSF) using Pycnoporus sanguineus. The impact of the composition, structure and cellulase adsorption ability of SCB on the production of cellulase, RS and fungal biomass was investigated. From the morphological and compositional analyses, untreated SCB has relatively more structural changes with a higher percentage of depolymerisation on the cellulose, hemicellulose and lignin content compared to alkali-pretreated SCB. Thus, untreated SCB favoured the production of cellulase and fungal biomass whereas alkali-pretreated SCB yielded a higher amount of RS. The composition and morphology of untreated SCB did not encourage RS production and this suggested that RS produced during SSF might be consumed in a faster rate by the more abundantly grown fungus. Besides that, alkali-pretreated SCB with higher cellulase adsorption ability could have adsorbed the cellulase produced and resulted in a lower cellulase titre. In short, the production of specific bioproducts via SSF is dependent on the structure and composition of the substrate applied.
A recombinant Trichoderma reesei cellulase was used for the ultrasound-mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4-11.8 W cm(-2) sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis-Menten kinetics. The Michaelis-Menten parameter of the maximum reaction rate Vmax was enhanced by sonication relative to controls, but the value of the saturation constant Km was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm(-2) . Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm(-2) power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose.
This research was carried out to evaluate the effects of flavone, myricetin, naringin, catechin, rutin, quercetin, and kaempferol at the concentration of 4.5% of the substrate (dry matter basis) on the rumen microbial activity in vitro. Mixture of guinea grass and concentrate (60 : 40) was used as the substrate. The results showed that all the flavonoids except naringin and quercetin significantly (P < 0.05) decreased the dry matter degradability. The gas production significantly (P < 0.05) decreased by flavone, myricetin, and kaempferol, whereas naringin, rutin, and quercetin significantly (P < 0.05) increased the gas production. The flavonoids suppressed methane production significantly (P < 0.05). The total VFA concentration significantly (P < 0.05) decreased in the presence of flavone, myricetin, and kaempferol. All flavonoids except naringin and quercetin significantly (P < 0.05) reduced the carboxymethyl cellulase, filter paperase, xylanase, and β -glucosidase activities, purine content, and the efficiency of microbial protein synthesis. Flavone, myricetin, catechin, rutin, and kaempferol significantly (P < 0.05) reduced the population of rumen microbes. Total populations of protozoa and methanogens were significantly (P < 0.05) suppressed by naringin and quercetin. The results of this research demonstrated that naringin and quercetin at the concentration of 4.5% of the substrate (dry matter basis) were potential metabolites to suppress methane production without any negative effects on rumen microbial fermentation.
Rubberwood (Hevea brasiliensis), a potential raw material for bioethanol production due to its high cellulose content, was used as a novel feedstock for enzymatic hydrolysis and bioethanol production using biological pretreatment. To improve ethanol production, rubberwood was pretreated with white rot fungus Ceriporiopsis subvermispora to increase fermentation efficiency. The effects of particle size of rubberwood (1 mm, 0.5 mm, and 0.25 mm) and pretreatment time on the biological pretreatment were first determined by chemical analysis and X-ray diffraction and their best condition obtained with 1 mm particle size and 90 days pretreatment. Further morphological study on rubberwood with 1 mm particle size pretreated by fungus was performed by FT-IR spectra analysis and SEM observation and the result indicated the ability of this fungus for pretreatment. A study on enzymatic hydrolysis resulted in an increased sugar yield of 27.67% as compared with untreated rubberwood (2.88%). The maximum ethanol concentration and yield were 17.9 g/L and 53% yield, respectively, after 120 hours. The results obtained demonstrate that rubberwood pretreated by C. subvermispora can be used as an alternative material for the enzymatic hydrolysis and bioethanol production.
Ionic liquids (ILs) have been used as an alternative green solvent for lignocelluloses pretreatment. However, being a salt, ILs exhibit an inhibitory effect on cellulases activity, thus making the subsequent saccharification inefficient. The aim of the present study is to produce salt-tolerant cellulases, with the rationale that the enzyme also tolerant to the presence of ILs. The enzyme was produced from a locally isolated halophilic strain and was characterized and assessed for its tolerance to different types of ionic liquids. The results showed that halophilic cellulases produced from Aspergillus terreus UniMAP AA-6 exhibited higher tolerance to ILs and enhanced thermo stability in the presence of high saline conditions.
Hydrothermal pretreatment of oil palm mesocarp fiber was conducted in tube reactor at treatment severity ranges of log Ro = 3.66-4.83 and partial removal of hemicellulose with migration of lignin was obtained. Concerning maximal recovery of glucose and xylose, 1.5% NaOH was impregnated in the system and subsequent ball milling treatment was employed to improve the conversion yield. The effects of combined hydrothermal and ball milling pretreatments were evaluated by chemical composition changes by using FT-IR, WAXD and morphological alterations by SEM. The successful of pretreatments were assessed by the degree of enzymatic digestibility of treated samples. The highest xylose and glucose yields obtained were 63.2% and 97.3% respectively at cellulase loadings of 10 FPU/g-substrate which is the highest conversion from OPMF ever reported.
Effective optimization of microalgae-to-bioethanol process systems hinges on an in-depth characterization of key process parameters relevant to the overall bioprocess engineering. One of the such important variables is the biomass particle size distribution and the effects on saccharification levels and bioethanol titres. This study examined the effects of three different microalgal biomass particle size ranges, 35 μm ≤ x ≤ 90 μm, 125 μm ≤ x ≤ 180 μm, and 295 μm ≤ x ≤ 425 μm, on the degree of enzymatic hydrolysis and bioethanol production. Two scenarios were investigated: single enzyme hydrolysis (cellulase) and double enzyme hydrolysis (cellulase and cellobiase). The glucose yield from biomass in the smallest particle size range (35 μm ≤ x ≤ 90 μm) was the highest, 134.73 mg glucose/g algae, while the yield from biomass in the larger particle size range (295 μm ≤ x ≤ 425 μm) was 75.45 mg glucose/g algae. A similar trend was observed for bioethanol yield, with the highest yield of 0.47 g EtOH/g glucose obtained from biomass in the smallest particle size range. The results have shown that the microalgal biomass particle size has a significant effect on enzymatic hydrolysis and bioethanol yield.
Eco-friendly pretreatment methods for lignocellulosic biomass are being developed as alternatives to chemical based methods. Superheated steam (SHS), hot compressed water (HCW) and wet disk milling (WDM) were used individually and with combination to partially remove hemicellulose and alter the lignin composition of recalcitrant structure of oil palm mesocarp fiber (OPMF). The efficiency of the pretreatment methods was evaluated based on the chemical compositions altered, SEM analysis, power consumption and degree of enzymatic digestibility. Hemicellulose removal (94.8%) was more pronounced under HCW compared to SHS, due to maximal contact of water and production of acetic acid which enhanced further degradation of hemicellulose. Subsequent treatment with WDM resulted in defibrillation of OPMF and expansion of the specific surface area thus increasing the conversion of cellulose to glucose. The highest glucose yield was 98.1% (g/g-substrate) when pretreated with HCW (200 °C, 20 min) and WDM which only consumed 9.6 MJ/kg of OPMF.