Lower concentration of glucose was often obtained from enzymatic hydrolysis process of agricultural residue due to complexity of the biomass structure and properties. High substrate load feed into the hydrolysis system might solve this problem but has several other drawbacks such as low rate of reaction. In the present study, we have attempted to enhance glucose recovery from agricultural waste, namely, "sago hampas," through three cycles of enzymatic hydrolysis process. The substrate load at 7% (w/v) was seen to be suitable for the hydrolysis process with respect to the gelatinization reaction as well as sufficient mixture of the suspension for saccharification process. However, this study was focused on hydrolyzing starch of sago hampas, and thus to enhance concentration of glucose from 7% substrate load would be impossible. Thus, an alternative method termed as cycles I, II, and III which involved reusing the hydrolysate for subsequent enzymatic hydrolysis process was introduced. Greater improvement of glucose concentration (138.45 g/L) and better conversion yield (52.72%) were achieved with the completion of three cycles of hydrolysis. In comparison, cycle I and cycle II had glucose concentration of 27.79 g/L and 73.00 g/L, respectively. The glucose obtained was subsequently tested as substrate for bioethanol production using commercial baker's yeast. The fermentation process produced 40.30 g/L of ethanol after 16 h, which was equivalent to 93.29% of theoretical yield based on total glucose existing in fermentation media.
Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.
To isolate a bacterial strain capable of biotransforming ferulic acid, a major component of lignin, into vanillin and vanillic acid by a rapid colorimetric screening method.
The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg β-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg β-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.
This study presents the effect of carbon to nitrogen ratio (C/N) (mol/mol) on the cell growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulation by Comamonas sp. EB172 in 2 L fermenters using volatile fatty acids (VFA) as the carbon source. This VFA was supplemented with ammonium sulphate and yeast extract in the feeding solution to achieve C/N (mol/mol) 5, 15, 25, and 34.4, respectively. By extrapolating the C/N and the source of nitrogen, the properties of the polymers can be regulated. The number average molecular weight (M n ) of P(3HB-co-3HV) copolymer reached the highest at 838 × 10(3) Da with polydispersity index (PDI) value of 1.8, when the culture broth was supplemented with yeast extract (C/N 34.4). Tensile strength and Young's modulus of the copolymer containing 6-8 mol% 3HV were in the ranges of 13-14.4 MPa and 0.26-0.34 GPa, respectively, comparable to those of polyethylene (PE). Thus, Comamonas sp. EB172 has shown promising bacterial isolates producing polyhydroxyalkanoates from renewable carbon materials.
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.
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.
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.
Bioconverting glycerol into various valuable products is one of glycerol's promising applications due to its high availability at low cost and the existence of many glycerol-utilizing microorganisms. Bioethanol and biohydrogen, which are types of renewable fuels, are two examples of bioconverted products. The objectives of this study were to evaluate ethanol production from different media by local microorganism isolates and compare the ethanol fermentation profile of the selected strains to use of glucose or glycerol as sole carbon sources. The ethanol fermentations by six isolates were evaluated after a preliminary screening process. Strain named SS1 produced the highest ethanol yield of 1.0 mol: 1.0 mol glycerol and was identified as Escherichia coli SS1 Also, this isolated strain showed a higher affinity to glycerol than glucose for bioethanol production.
Dimethyl adipate (DMA) was synthesized by immobilized Candida antarctica lipase B-catalyzed esterification of adipic acid and methanol. To optimize the reaction conditions of ester production, response surface methodology was applied, and the effects of four factors namely, time, temperature, enzyme concentration, and molar ratio of substrates on product synthesis were determined. A statistical model predicted that the maximum conversion yield would be 97.6%, at the optimal conditions of 58.5 degrees C, 54.0 mg enzyme, 358.0 min, and 12:1 molar ratio of methanol to adipic acid. The R(2) (0.9769) shows a high correlation between predicted and experimental values. The kinetics of the reaction was also investigated in this study. The reaction was found to obey the ping-pong bi-bi mechanism with methanol inhibition. The kinetic parameters were determined and used to simulate the experimental results. A good quality of fit was observed between the simulated and experimental initial rates.
Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-D-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200 rpm and 0.33 vvm with 4.1 U/l per hour and 5.97 U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74 mg of dissolved oxygen per gram of fungal biomass per hour at the kappaL a of 8.34 per hour.
Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.
Ten filamentous fungi adapted to domestic wastewater sludge (DWS) were further studied to evaluate their potential in terms of adaptation to higher sludge supplemented growing media and phytopathogenicity (induction of diseases to plants) to three germinating crop (Corn: Zea mays, Mung bean: Phaseolus aureus and Mustard: Brassica napus) seeds. The performances of the fungi in seed germination were evaluated based on percent germination index (GI) and infected/spotted seeds on direct fungal biomass (FBM) and fungal metabolite (FM). Significantly the highest biomass production was achieved with RW-P1 512 and Penicillium corylophilum (WW-P1003) at the highest (25%) sludge supplemented growing media that implied its excellent potentiality of adaptation and multiplication to domestic wastewater sludge. Significantly encouraging results of percent GI and spotted/infected seedlings were observed in FM than FBM by all fungi except the strain Aspergillus niger. A. niger gave the poorest percent of GI (24.30, 26.98 and 00.00%) and the highest percent of infected/spotted seeds (70, 100, and 100%) using FBM for corn, mung bean and mustard, respectively. On the other hand, comparatively the highest percent of GI (107.99, 106.25 and 117.67%) and the lowest percent of spotted/infected seedlings (3.3, 3.3 and 3.3%) were achieved with the isolate RW-P1 512 using FM. In FBM, the superior results of percent GI (86.61, 95.92 and 83.87%) and spotted/infected seedlings (3.3, 63.3 and 43.3%) were obtained by A. versicolor. Several crop seeds were responded differently for different fungal treatments. Hundred percent infected/spotted seeds in FM were recorded only for mustard with Trichoderma family that implied its strong sensitiveness to its metabolites.
Twenty seven filamentous fungal strains representing five genera; Aspergillus, Penicillium, Trichoderma, Myriodontium and Pleurotus were isolated from four sources; domestic wastewater sludge cake (SC) from IWK (Indah Water Konsortium) wastewater treatment plant, palm oil mill effluent compost from Sri Ulu palm Oil Processing Mill, compost of plant debris, and fungal fruiting bodies from a rotten wood stump. Thirty-three strains/isolates were tested for their ability to convert domestic wastewater sludge into compost by assessing biomass production and growth rate on sludge enriched media. The strains/isolates Aspergillus niger, SS-T2008, WW-P1003 and RW-P1 512 produced the highest dry biomass at higher sludge supplemented culture media from their respective group (Aspergillus, Trichoderma, Penicillium and Basidiomycetes, respectively). This implied these strains are better adapted for growth at higher sludge rich substances, and subsequently may be efficient in bioconversion/biodegradation of sludge. The fungi isolated from ecological closely related sources were more amendable to adaptation in a sludge rich culture media.
Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process.
The combination of superheated steam (SHS) with ligninolytic enzyme laccase pretreatment together with size reduction was conducted in order to enhance the enzymatic hydrolysis of oil palm biomass into glucose. The oil palm empty fruit bunch (OPEFB) and oil palm mesocarp fiber (OPMF) were pretreated with SHS and ground using a hammer mill to sizes of 2, 1, 0.5 and 0.25 mm before pretreatment using laccase to remove lignin. This study showed that reduction of size from raw to 0.25 mm plays important role in lignin degradation by laccase that removed 38.7% and 39.6% of the lignin from OPEFB and OPMF, respectively. The subsequent saccharification process of these pretreated OPEFB and OPMF generates glucose yields of 71.5% and 63.0%, which represent a 4.6 and 4.8-fold increase, respectively, as compared to untreated samples. This study showed that the combination of SHS with laccase pretreatment together with size reduction could enhance the glucose yield.
Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60 degrees C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.
This study was conducted in order to optimise simultaneous saccharification and fermentation (SSF) for biobutanol production from a pretreated oil palm empty fruit bunch (OPEFB) by Clostridium acetobutylicum ATCC 824. Temperature, initial pH, cellulase loading and substrate concentration were screened using one factor at a time (OFAT) and further statistically optimised by central composite design (CCD) using the response surface methodology (RSM) approach. Approximately 2.47 g/L of biobutanol concentration and 0.10 g/g of biobutanol yield were obtained after being screened through OFAT with 29.55% increment (1.42 fold). The optimised conditions for SSF after CCD were: temperature of 35 °C, initial pH of 5.5, cellulase loading of 15 FPU/g-substrate and substrate concentration of 5% (w/v). This optimisation study resulted in 55.95% increment (2.14 fold) of biobutanol concentration equivalent to 3.97 g/L and biobutanol yield of 0.16 g/g. The model and optimisation design obtained from this study are important for further improvement of biobutanol production, especially in consolidated bioprocessing technology.
Total polar compounds (TPC) and free fatty acids (FFA) are important indicators in evaluating the quality of frying oil. Conventional methods to determine TPC and FFA are often time consuming, involved laboratory analyses which required skilled personnel and used substantial amount of harmful solvent. In this study, dielectric spectroscopy technique was used to investigate the relation between dielectric property of refined, bleached and deodorized palm olein (RBDPO) during deep frying with TPC and FFA. In total, 150 batches of French fries were intermittently fried at 185 ± 5 °C for 7 hr a day over 5 consecutive days. A total of 30 frying oil samples were collected. The dielectric property of frying oil samples were measured using impedance analyzer with frequencies ranging from 100 Hz to 10 MHz. The TPC of frying oil samples were measured with a Testo 270, while the FFA analysis was done using Malaysian Palm Oil Board (MPOB) test method. Results showed that dielectric constant, TPC and FFA of RBDPO increased as the frying time increased. Dielectric constant increased from 3.09 to 3.17, while TPC and FFA increased from 9.96 to 19.52 and from 0.08% to 0.36%, respectively. Partial least square (PLS) analysis produced good prediction of TPC and FFA with the application of genetic algorithm (GA). Model developed for prediction of TPC and FFA yielded highly significant correlation with R2 of 0.91 and 0.95, respectively and both had root mean square error in cross-validation (RMSECV) of 1.06%. This study demonstrates the potential of dielectric spectroscopy in monitoring palm olein degradation during frying. PRACTICAL APPLICATION: The application of dielectric spectroscopy to detect degradation of palm olein during frying was studied. The dielectric property of palm olein during frying has successfully correlated with TPC and FFA. The model developed in this study could be used for the development of a sensing system for palm olein degradation monitoring.
β-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for β-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding β-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 β-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site's narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 β-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 β-glucosidases following relevant in-silico analyses.