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Filamentous fungi in Indah Water Konsortium (IWK) sewage treatment plant for biological treatment of domestic wastewater sludge

Fakhrul-Razi A, Alam MZ, Idris A, Abd-Aziz S, Molla AH

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002 Mar;37(3):309-20.
PMID: 11929070

A study was carried out to isolate and identify filamentous fungi for the treatment of domestic wastewater sludge by enhancing biodegradability, settleability and dewaterability of treated sludge using liquid state bioconversion process. A total of 70 strains of filamentous fungi were isolated from three different sources (wastewater, sewage sludge and leachate) of IWK's (Indah Water Konsortium) sewage treatment plant, Malaysia. The isolated strains were purified by conventional techniques and identified by microscopic examination. The strains isolated belonged to the genera of Penicillium, Aspergillus, Trichoderma, Spicaria and Hyaloflorae The distribution of observed isolated fungi were 41% in sewage sludge followed by 39% in wastewater and 20% in leachate. The predominant fungus was Penicillium (39 strains). The second and third most common isolates were Aspergillus (14 strains) and Trichoderma (12 strains). The other isolates were Spicaria (3 strains) and Hyaloflorae (2 strains). Three strains (WWZP1003, LZP3001, LZP3005) of Penicillium (P. corylophilum, P. waksmanii, and P. citrinum respectively), 2 strains (WWZA1006 and SS2017) of Aspergillus (A. terrues and A. flavus respectively) and one strain (SSZT2008) of Trichoderma (T. harzianum) were tentatively identified up to species level and finally verified by CABI Bioscience Identification Services, UK.
Sago starch and its utilisation

Abd-Aziz S

J Biosci Bioeng, 2002;94(6):526-9.
PMID: 16233345

The importance and development of industrial biotechnology processing has led to the utilisation of microbial enzymes in various applications. One of the important enzymes is amylase, which hydrolyses starch to glucose. 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. Production of fermentable sugars from the hydrolysis of starches is normally carried out by an enzymatic processes that involves two reaction steps, liquefaction and saccharification, each of which has different temperature and pH optima with respect to the maximum reaction rate. This method of starch hydrolysis requires the use of an expensive temperature control system and a complex mixing device. Our laboratory has investigated the possibility of using amylolytic enzyme-producing microorganisms in the continuous single-step biological hydrolysis of sago flour for the production of a generic fermentation medium. The ability of a novel DNA-recombinated yeast, Saccharomyces cerevisiae strain YKU 107 (expressing alpha-amylase production) to hydrolyse gelatinised sago starch production has been studied with the aim of further utilizing sago starch to obtain value-added products.
A rapid colorimetric screening method for vanillic acid and vanillin-producing bacterial strains

Zamzuri NA, Abd-Aziz S, Rahim RA, Phang LY, Alitheen NB, Maeda T

J Appl Microbiol, 2014 Apr;116(4):903-10.
PMID: 24314059 DOI: 10.1111/jam.12410

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.
Fulltext Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli

Nik-Pa NIM, Sobri MFM, Abd-Aziz S, Ibrahim MF, Kamal Bahrin E, Mohammed Alitheen NB, et al.

Int J Mol Sci, 2020 May 30;21(11).
PMID: 32486212 DOI: 10.3390/ijms21113919

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
In-Silico Characterization of Glycosyl Hydrolase Family 1 β-Glucosidase from Trichoderma asperellum UPM1

Mohamad Sobri MF, Abd-Aziz S, Abu Bakar FD, Ramli N

Int J Mol Sci, 2020 Jun 04;21(11).
PMID: 32512945 DOI: 10.3390/ijms21114035

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.
Fulltext Immunomodulatory effects of Newcastle disease virus AF2240 strain on human peripheral blood mononuclear cells

Lam HY, Yusoff K, Yeap SK, Subramani T, Abd-Aziz S, Omar AR, et al.

Int J Med Sci, 2014;11(12):1240-7.
PMID: 25317070 DOI: 10.7150/ijms.8170

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.
Fulltext Systematic Review of Brain-Eating Amoeba: A Decade Update

Ahmad Zamzuri M'I, Abd Majid FN, Mihat M, Ibrahim SS, Ismail M, Abd Aziz S, et al.

Int J Environ Res Public Health, 2023 Feb 09;20(4).
PMID: 36833715 DOI: 10.3390/ijerph20043021

INTRODUCTION: Primary amoebic meningoencephalitis (PAM) is a rare but lethal infection of the brain caused by a eukaryote called Naegleria fowleri (N. fowleri). The aim of this review is to consolidate the recently published case reports of N. fowleri infection by describing its epidemiology and clinical features with the goal of ultimately disseminating this information to healthcare personnel.
METHODS: A comprehensive literature search was carried out using PubMed, Web of Science, Scopus, and OVID databases until 31 December 2022 by two independent reviewers. All studies from the year 2013 were extracted, and quality assessments were carried out meticulously prior to their inclusion in the final analysis.
RESULTS: A total of 21 studies were selected for qualitative analyses out of the 461 studies extracted. The cases were distributed globally, and 72.7% of the cases succumbed to mortality. The youngest case was an 11-day-old boy, while the eldest was a 75-year-old. Significant exposure to freshwater either from recreational activities or from a habit of irrigating the nostrils preceded onset. The symptoms at early presentation included fever, headache, and vomiting, while late sequalae showed neurological manifestation. An accurate diagnosis remains a challenge, as the symptoms mimic bacterial meningitis. Confirmatory tests include the direct visualisation of the amoeba or the use of the polymerase chain reaction method.
CONCLUSIONS: N. fowleri infection is rare but leads to PAM. Its occurrence is worldwide with a significant risk of fatality. The suggested probable case definition based on the findings is the acute onset of fever, headache, and vomiting with meningeal symptoms following exposure to freshwater within the previous 14 days. Continuous health promotion and health education activities for the public can help to improve knowledge and awareness prior to engagement in freshwater activities.
Fulltext Lard classification from other animal fats using Dielectric Spectroscopy technique

Amat Sairin, M., Abd Aziz, S., Tan, C.P., Mustafa, S., Abd Gani, S.S., Rokhani, F.Z.

International Food Research Journal, 2019;26(3):773-782.
MyJurnal

Lard adulteration in processed foods is a major public concern as it involves religion and
health. Most lard discriminating works require huge lab-based equipment and complex sample
preparation. The objective of the present work was to assess the feasibility of dielectric
spectroscopy as a method for classification of fats from different animal sources, in particular,
lard. The dielectric spectra of each animal fat were measured in the radio frequency of 100
Hz – 100 kHz at 45°C to 55°C. The fatty acid composition of each fat was studied by using
data from gas chromatography mass spectrometry (GCMS) to explain the dielectric behaviour
of each fat. The principal component analysis (PCA) and artificial neural network (ANN)
were used to classify different animal fats based on their dielectric spectra. It was found that
lard showed the highest dielectric constant spectra among other animal fats, and was mainly
affected by the composition of C16 and C18 fatty acids. PCA classification plot showed clear
performance in classifying different animal fats. Finally, ANN classification showed different
animal fats were classified into their respective groups effectively at high accuracy of 85%.
Dielectric spectroscopy, in combination with quantitative analysis, was concluded to provide
rapid method to discriminate lard from other animal fats.
Fulltext Fluorescence spectroscopy for analysing deterioration of palm olein in batch deep-fat frying

Ibrahim, N.U.A., Abd Aziz, S., Zude, M.

International Food Research Journal, 2016;23(101):45-50.
MyJurnal

Palm olein has been commercially used as frying medium in batch deep-fat frying. During
frying, the oil usually deteriorates due to the exposure to high temperature. In this study, a
fluorescence spectroscopy technique was applied to monitor the deterioration of refined,
bleached, and deodorized palm olein (RBDPO) in batch deep-fat frying. 22.5 kg of French fries
were used as the frying material. In 30 batches, the french fries were intermittently fried at 185
± 5°C for eight hours a day over five consecutive days capturing 40 hours. The fluorescence
intensity of the RBDPO was recorded with excitation at 390 nm and resulting emission of 465
nm. The fluorescence intensity of the RBDPO over five days of frying decreased considering
the wavelength range of emission 430-640 nm and excitation 360-430 nm. The decreased in
intensity of fluorescence emission and excitation spectra were inversely correlated with the FFA
content of the oil samples. This study demonstrates the potential of fluorescence spectroscopy
in monitoring the deterioration of RBDPO during batch deep-fat frying.
Fulltext Reduction of the acidity and peroxide numbers of tengkawang butter (Shorea stenoptera) using thermal and acid activated bentonites

Darmawan MA, Muhammad BZ, Harahap AFP, Ramadhan MYA, Sahlan M, Haryuni, et al.

Heliyon, 2020 Dec;6(12):e05742.
PMID: 33364505 DOI: 10.1016/j.heliyon.2020.e05742

Tengkawang fat (Shorea stenoptera), from an indigenous plant of the Kalimantan forest, has excellent potential as an alternative source of vegetable fat because it has a high level of fatty acids composition. Activated natural bentonite can be used as a bleaching agent to improve the quality of tengkawang fat. This research aims to reduce the acidity, peroxide number values and identify the physicochemical properties (fatty acid composition, nutrients, and thermal) of tengkawang butter. Initially, tengkawang samples from Nanga Yen and Sintang were pre-treated using the degumming process with 1% phosphoric acid and the neutralization process with a 1 M NaOH 10% w/w solution. The results show that the acidity (mg NaOH/g) of the tengkawang fat samples was reduced from 11.00 to 3.36 when using bentonite activated at 200 °C. The bentonite activated with 0.5 M HCl reduced the acidity to 3.61. The peroxide number (meq O2/kg) of the tengkawang fat samples was reduced from 9.45 to 4.84 and 3.47 by bleaching with thermal-activated and acid-activated bentonites, respectively. Peroxide value correlates with β-carotene content. The smaller of the β-carotene content, the smaller the peroxide value. The acidity, peroxide number, and iodine number values from tengkawang fat after treatment adhere to the SNI 2903: 2016 standard. The main content of fatty acids in tengkawang fat is palmitic acid, stearic acid, and oleic acid. These results show that both products are suitable for the food industry in terms of the acid and peroxide numbers. The application of this research results will assist local people in increasing the economic value of the product from tengkawang plant, which is an indigenous plant from Kalimantan.
Pineapple (Ananas comosus): A comprehensive review of nutritional values, volatile compounds, health benefits, and potential food products

Mohd Ali M, Hashim N, Abd Aziz S, Lasekan O

Food Res Int, 2020 11;137:109675.
PMID: 33233252 DOI: 10.1016/j.foodres.2020.109675

Pineapple (Ananas comosus) is a tropical fruit that is highly relished for its unique aroma and sweet taste. It is renowned as a flavourful fruit since it contains a number of volatile compounds in small amounts and complex mixtures. Pineapple is also a rich source of minerals and vitamins that offer a number of health benefits. Ranked third behind banana and citrus, the demand for pineapple has greatly increased within the international market. The growth of the pineapple industry in the utilisation of pineapple food-based processing products as well as waste processing has progressed rapidly worldwide. This review discusses the nutritional values, physicochemical composition and volatile compounds, as well as health benefits of pineapples. Pineapple contains considerable amounts of bioactive compounds, dietary fiber, minerals, and nutrients. In addition, pineapple has been proven to have various health benefits including anti-inflammatory, antioxidant activity, monitoring nervous system function, and healing bowel movement. The potential of food products and waste processing of pineapples are also highlighted. The future perspectives and challenges with regard to the potential uses of pineapple are critically addressed. From the review, it is proven that pineapples have various health benefits and are a potential breakthrough in the agricultural and food industries.
Innovative approaches for amino acid production via consolidated bioprocessing of agricultural biomass

Chu PH, Jenol MA, Phang LY, Ibrahim MF, Purkan P, Hadi S, et al.

Environ Sci Pollut Res Int, 2024 May 06.
PMID: 38710845 DOI: 10.1007/s11356-024-33534-0

Agricultural plantations in Indonesia and Malaysia yield substantial waste, necessitating proper disposal to address environmental concerns. Yet, these wastes, rich in starch and lignocellulosic content, offer an opportunity for value-added product development, particularly amino acid production. Traditional methods often rely on costly commercial enzymes to convert biomass into fermentable sugars for amino acid production. An alternative, consolidated bioprocessing, enables the direct conversion of agricultural biomass into amino acids using selected microorganisms. This review provides a comprehensive assessment of the potential of agricultural biomass in Indonesia and Malaysia for amino acid production through consolidated bioprocessing. It explores suitable microorganisms and presents a case study on using Bacillus subtilis ATCC 6051 to produce 9.56 mg/mL of amino acids directly from pineapple plant stems. These findings contribute to the advancement of sustainable amino acid production methods using agricultural biomass especially in Indonesia and Malaysia through consolidated bioprocessing, reducing waste and enhancing environmental sustainability.
Fulltext Bioconversion of glycerol for bioethanol production using isolated Escherichia coli ss1

Suhaimi SN, Phang LY, Maeda T, Abd-Aziz S, Wakisaka M, Shirai Y, et al.

Braz J Microbiol, 2012 Apr;43(2):506-16.
PMID: 24031858 DOI: 10.1590/S1517-83822012000200011

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.
ACB2015: Biotechnology and bioeconomy for sustainable future

Abd-Aziz S, Faizal Ibrahim M

Biotechnol J, 2016 Jun;11(6):729-31.
PMID: 27273842 DOI: 10.1002/biot.201600156
Lipase-catalyzed dimethyl adipate synthesis: response surface modeling and kinetics

Chaibakhsh N, Rahman MB, Basri M, Salleh AB, Abd-Aziz S

Biotechnol J, 2010 Aug;5(8):848-55.
PMID: 20632329 DOI: 10.1002/biot.201000063

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.
A potential resource for bioconversion of domestic wastewater sludge

Molla AH, Fakhru'l-Razi A, Abd-Aziz S, Hanafi MM, Roychoudhury PK, Alam MZ

Bioresour Technol, 2002 Dec;85(3):263-72.
PMID: 12365494

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.
Enhanced volatile fatty acid production from sago hampas by Clostridium beijerinckii SR1 for bioelectricity generation using microbial fuel cells

Jenol MA, Ibrahim MF, Kamal Bahrin E, Abd-Aziz S

Bioprocess Biosyst Eng, 2020 Nov;43(11):2027-2038.
PMID: 32572569 DOI: 10.1007/s00449-020-02391-9

Sago hampas is a starch-based biomass from sago processing industries consisted of 58% remaining starch. This study has demonstrated the bioconversion of sago hampas to volatile fatty acids (VFAs) by Clostridium beijerinckii SR1 via anaerobic digestion. Higher total VFAs were obtained from sago hampas (5.04 g/L and 0.287 g/g) as compared to commercial starch (5.94 g/L and 0.318 g/g). The physical factors have been investigated for the enhancement of VFAs production using one-factor-at-a-time (OFAT). The optimum condition; 3% substrate concentration, 3 g/L of yeast extract concentration and 2 g/L of ammonium nitrate enhanced the production of VFAs by 52.6%, resulted the total VFAs produced is 7.69 g/L with the VFAs yield of 0.451 g/g. VFAs hydrolysate produced successfully generated 273.4 mV of open voltage circuit and 61.5 mW/m2 of power density in microbial fuel cells. It was suggested that sago hampas provide as an alternative carbon feedstock for bioelectricity generation.
Fulltext Improved properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Comamonas sp. EB172 utilizing volatile fatty acids by regulating the nitrogen source

Zakaria MR, Ariffin H, Abd-Aziz S, Hassan MA, Shirai Y

Biomed Res Int, 2013;2013:237806.
PMID: 24106698 DOI: 10.1155/2013/237806

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.
Fulltext Corrigendum to "Safety and Clinical Usage of Newcastle Disease Virus in Cancer Therapy"

Lam HY, Yeap SK, Pirozyan MR, Omar AR, Yusoff K, Abd-Aziz S, et al.

Biomed Res Int, 2017;2017:4529437.
PMID: 29333441 DOI: 10.1155/2017/4529437

[This corrects the article DOI: 10.1155/2011/718710.].
Fulltext Recovery of glucose from residual starch of sago hampas for bioethanol production

Awg-Adeni DS, Bujang KB, Hassan MA, Abd-Aziz S

Biomed Res Int, 2013;2013:935852.
PMID: 23509813 DOI: 10.1155/2013/935852

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.

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