Optimization of Cellulase Production by Chaetomium Globosum Strain 414 Using Oil Palm Empty Fruit Bunch Fibre as Substrate

The production of three major components of cellulase (FPase, CMCase and 13-glucosidase) by Chaetomium globoslim strain 414 was studied in a shake flask experiment. The effects of physical and chemical treatments on the oil palm empty fruit bunch (OPEFB) fibre for subsequent use as substrate for cellulase production were investigated. The effects of different types and concentrations of nitrogen sources on cellulase production were also examined. The optimized medium composition obtained from the shake flask experiment was used for cellulase production in a 2L stirred tank ferrnenter (impeller tip speed = 1.64 mls) where the effect of different levels of dissolved oxygen tension (DOT) at a fixed agitation speed on cellulase production was investigated. The experimental data obtained from batch fermentations in a shake flask and the fermenter using the optimized medium were analysed to form the basis for a kinetic model ofthe process. The partially purified cellulase preparation from this fungus was used for the saccharification of OPEFB fibre. The effect of different methods of treatment of OPEFB fibre on the rate and degree of hydrolysis was investigated.The use of2-mm OPEFB fibre increased cellulase production about two fold compared to 10-mm fibre. Chemical treatment significantly increased the cellulose and reduced the lignin contents. Cellulase activities, obtained from fermentation using OPEFB fibre treated with 0.5% RN03 followed by autoclaving were about three times higher than those obtained in fermentation using pure celluloses. The cellulase of C. globosum strain 414 contained a high proportion of l3-glucosidase with the ratio of specific activity of l3-glucosidase to FPase of about 8. Peptone gave the highest cellulase production followed by yeast extract, urea, KN03 and (NH4)2S04' A good agreement between the calculated data and the experimental data for both cell growth and cellulase production were observed, suggesting that the proposed model based on logistic and Luedeking-Piret equations is sufficient to describe the growth of C. globosum strain 414 and cellulase production. The maximum activities of FPase, CMCase and l3-glucosidase obtained from fermentation with 50% DOT were 2.5, 59.5 and 12.8 U/m), and these gave the overall productivities of20.8, 495 and 53.3 U/L.h, respectively. Cellulase production in stirred tank fermenter were significantly higher than that obtained in shake flask. The yield and overall productivity of the saccharification of the autoclaved OPEFB fibre treated with 2% NaOH were 0.7600 g reducing sugar/g OPEFB and 0.0178 g reducing sugar/g cellulose.h, respectively

Characteristics of methanogens and methanotrophs in rice fields: a review

Methane is the second most important greenhouse gas after carbon dioxide (CO2) with a global warming potential 25 times more than CO2. Rice fields are one of the main anthropogenic sources for methane and responsible for approximately 15-20% of the annual global methane efflux. Methanogens and methanotrophs are two microbial communities which contribute to the biogeochemical methane cycle in soil by producing and oxidizing methane, respectively. In fact, the total methane emission from rice soil is the balance between methanogen and methanotroph activities. Methanogenic archaea are more active in highly reduced conditions and anoxic soils. However, methanotrophs are more active in oxic soils. These microorganisms have been studied frequently in different soils from natural wetlands to rice fields. This article has mainly focused on the characteristics of methanogens and methanotrophs in a rice soil ecosystem with the objective of deriving solutions the high level of methane emissions from paddy fields

Production and characterization of cellulases by Bacillus pumilus EB3

Cellulase production from bacteria can be an advantage as the enzyme production rate is normally higher due to bacterial high growth rate. Screening of bacteria, optimisation of fermentation conditions and selection of substrates are important for the successful production of cellulase. This study is conducted to produce cellulase from our local isolate Bacillus pumilus EB3, using carboxymethyl cellulose (CMC) as substrate. Following that, cellulase produced from Bacillus pumilus EB3 was purified using ion exchange chromatography with anion exchanger (HiTrap QXL) for characterisation of the cellulase. Cellulase was successfully produced in 2L stirred tank reactor (STR) with the productivity of 0.53, 3.08 and 1.78 U/L.h and the maximum enzyme activity of 0.011, 0.079 and 0.038 U/mL for FPase, CMCase and β-glucosidase respectively. Purification of cellulase from Bacillus pumilus EB3 using ion exchange chromatography showed that 98.7% of total CMCase was recovered. Protein separation was however based on subtractive separation where the contaminants were bound to the column instead of CMCase. Characterisation of the enzyme found that CMCase from Bacillus pumilus EB3 has a molecular weight range from 30-65 kDa and was optimally active at pH 6.0 and temperature 60℃. The CMCase also retained its activity over a wide pH range (pH 5.0–9.0) and temperature range (30-70℃).Seminar on Engineering and Technology (SET2006), September 4-5, 2006, Putrajaya, Malaysi

Saccharification of pretreated oil palm empty fruit bunch fiber using cellulase of Chaetomium globosum

The effectiveness of different chemical and physical pretreatments to alter cellulose structure and to reduce hemicellullose and lignin contents in oil palm empty fruit bunch (OPEFB) fibers for subsequent use as substrate for enzymatic saccharification was studied. The saccharification was carried out using concentrated cellulase preparation from culture broth of Chaetomium globosum strain 414 containing 10 U/mL FPase, 285 U/mL CMCase, and 60 U/mL β-glucosidase. The use of 0.5% NaOH to treat OPEFB by soaking at 30 °C for 4 h gave the highest rate and degree of hydrolysis followed by 0.5% HNO3, HCl, EDA, and EDTA. Autoclaving the chemically treated OPEFB fiber at 121 °C for 5 min improved the hydrolysis by 2-fold. The improvement in hydrolysis was related to a decrease in the hemicellulose and lignin contents and an increase in the cellulose content. The qualitative hydrolysis yield for autoclaved OPEFB fiber treated with 2% NaOH was 85.9%. During saccharification of OPEFB using cellulase of C. globosum, the amount of glucose produced was higher while the amount of cellobiose produced was lower than those obtained with commercial cellulase of Trichoderma viride

Improvement of Aspergillus flavus Link S44-1 using random mutational method for kojic acid production

AIM: The aim of this study was to improve the kojic acid producing strain, A. flavus Link S44-1, by random mutation methods. METHODS: Random induced mutation method was applied to A. flavus Link S44-1 with N-Methyl-N’ nitoro-N nitrosoguandine (NTG), ultraviolet (UV), and γ irradiation for improvement of its ability in producing kojic acid. The changes in the activity of cell bound enzyme responsible for kojic acid synthesis after the mutation were evaluated. RESULTS: The improved mutants (A. flavus NTG-MTDC-22, A. flavus UV-MTDC-12, and A. flavus G-MTDC-8) were capable of producing kojic acid up to final concentrations of 46, 42, and 49 g/L in fed-batch fermentation respectively. These which were 2.3 to 2.7 fold higher than the parent strain. In the mutated strains, the activities of enzymes related to kojic acid pathway such as glucose dehydrogenase and gluconate dehydrogenase detected during the fermentation were significantly higher than the parent strain. Spores of the mutants were stable and have consistent ability in producing kojic acid after prolonged storage in glycerol at -20 °C. CONCLUSION: The mutant A. flavus and the simple method used to improve the strains have potential applications in the development of fermentation process for industrial production of kojic acid, where the demand for this organic acid has increased enormously with its increasing applications in various industries

Enhanced cellulase production by a novel thermophilic Bacillus licheniformis 2D55: characterization and application in lignocellulosic saccharification

Effects of nutritional and physicochemical factors were investigated for cellulase production by the newly isolated thermophilic strain Bacillus licheniformis 2D55 (Accession No. KT799651). The optimum cellulase production in shake flask fermentation was attained at 60 °C, pH 3.5, 180 rpm, and in a medium containing untreated sugarcane bagasse and pre-treated rice husk at 7% (w/v), urea, 1 g/L, peptone, 11.0 g/L, Mg(SO4)2, 0.40 g/L, CaCl2, 0.03 g/L, Tween 80, 0.2% (w/v), and 3% inoculum. The highest carboxymethyl cellulase (CMCase), filtre paperase (FPase), and β-glucosidase produced under the optimized conditions were 29.4 U/mL, 12.9 U/mL, and 0.06 U/mL, respectively, after 18 h of fermentation. Optimization of the parameters increased the CMCase, FPase, and β-glucosidase activities by 77.4-fold, 44.5-fold, and 10-fold, respectively. The crude enzyme was highly active and stable over broad temperature (50 to 80 °C) and pH (3.5 to 10.0) ranges with optimum temperature at 65 °C and 80 ºC for CMCase and FPase, respectively. The optimum pH for CMCase and FPase was 7.5 and 6.0, respectively. Saccharification of sugar cane bagasse and rice husk by crude cellulase resulted in perspective yields of 0.348 and 0.301 g g-1 dry substrate of reducing sugars. These results suggest prospects of thermostable cellulase from B. licheniformis 2D55 in application for bio-sugar production and other industrial bioprocess applications involving high temperatures

Production and characterisation of cellulase from solid state fermentation of rice straw by Trichoderma harzianum SNRS3

Research on production and the use of cellulase and xylanase by commercial microbial strains is widely reported. However, research on production of cellulase and xylanase by local isolates of Trichoderma harzianum known as potential cellulase producers is still very limited. T. harzianum SNRS3 was used for cellulase and xylanase production from rice straw under solid state fermentation. Our study revealed that unlike Trichoderma sp. that is normally associated with low amounts of β-glucosidase, insufficient to perform an efficient hydrolysis, T. harzianum SNRS3 could be considered as a potential β-glucosidase producer, but not an efficient xylanase producer. As a result of storage of the crude cellulase at room temperature, β-glucosidase activity only decreased to above 80% of its original activity at the end of the 3rd week of storage. The crude cellulase produced by T. harzianum SNRS3 could be industrially applied as the enzyme is still highly active at 60°C and over a wide range of acidic pH

Oil Palm Frond Juice as Future Fermentation Substrate: A Feasibility Study

Oil palm frond (OPF) juice is a potential industrial fermentation substrate as it has high sugars content and the OPF are readily available daily. However, maximum sugars yield and storage stability of the OPF juice are yet to be determined. This study was conducted to determine the effect of physical pretreatment and storage duration of OPF petiole on sugars yield. Storage stability of OPF juice at different storing conditions was also investigated. It was found that OPF petiole squeezed by hydraulic pressing machine gave the highest sugars recovery at almost 40 g/kg, accounting for a recovery yield of 88%. Storage of OPF petiole up to 72 hrs prior to squeezing reduced the free sugars by 11 g/kg. Concentrated OPF juice with 95% water removal had the best storage stability at both 4 and , when it was stored for 10 days. Moreover, concentrated OPF syrup prepared by thermal processing did not give any Maillard effect on microbial growth. Based on our results, OPF juice meets all the criteria as a good fermentation substrate as it is renewable, consistently available, and easy to be obtained, it does not inhibit microbial growth and product formation, and it contains no impurities

Production of bacterial endoglucanase from pretreated oil palm empty fruit bunch by bacillus pumilus EB3

In this study, endoglucanase was produced from oil palm empty fruit bunch (OPEFB) by a locally isolated aerobic bacterium, Bacillus pumilus EB3. The effects of the fermentation parameters such as initial pH, temperature, and nitrogen source on the endoglucanase production were studied using carboxymethyl cellulose (CMC) as the carbon source. Endoglucanase from B. pumilus EB3 was maximally secreted at 37°C, initial pH 7.0 with 10 g/l of CMC as carbon source, and 2 g/l of yeast extract as organic nitrogen source. The activity recorded during the fermentation was 0.076 U/ml. The productivity of the enzyme increased twofold when 2 g/l of yeast extract was used as the organic nitrogen supplement as compared to the non-supplemented medium. An interesting finding from this study is that pretreated OPEFB medium showed comparable results to CMC medium in terms of enzyme production with an activity of 0.063 U/ml. As OPEFB is an abundant solid waste at palm oil mills, it has the potential of acting as a substrate in cellulase production

Identification and characterization of actinomycetes for biological control of bacterial wilt of Ralstonia solanacearum isolated from tomato

Five actinomycetes which showed antimicrobial activity towards Ralstonia solanacearum were identified using specific polymerase chain reaction (PCR) of 16S rDNA gene. Strain C1 and Strain G10 were identified as Streptomyces aureofaciens and S. roseoflavus respectively. All actinomycetes were then characterized using antimicrobial and extracellular enzyme activity, metabolic and restriction fragment length polymorphism (RFLP) profiles. Strain A3 showed positive reaction to three bacteria namely Xanthomonas campestris, Staphylococcus aureus and Listeria monocytogenes. Strain C1 and Strain I15 showed positive reaction towards S. aureus and X. campestris respectively. Strains A3, C1 and I15 were able to metabolize xylan and cellulose, while Strain G10 and Strain L8 were able to use all substrates (xylan, mannan and cellulose) as carbon sources. All the Streptomyces strains were positive towards more than 25 carbon sources and can be differentiated into five distinct strains. These results were consistent and confirmed with DNA analysis of RFLP profiles. The specific amplification of 16S rDNA PCR restriction profiles for the strains using three restriction endonucleases, resulted two restriction profiles produced from the digested 16S rDNA product using HaeIII (H1–H2) and HinfI (Hf1–Hf2), while PstI produced three restriction profiles (P1–P3). No profiles were produced from restriction endonuclases of XbaI, SpeI and BamHI