Azlin Suhaida Azmi

Ragi tapai and Saccharomyces cerevisiae as potential coculture in viscous fermentation medium for ethanol production

Although the delayed-type hypersensitivity (DTH) skin reaction to tuberculin is used worldwide for tuberculosis (TB) detection, it has poor diagnostic specificity due to the presence of common antigens in tuberculin shared by many mycobacterial species. The problem is noticed, especially in countries where the Bacillus Calmette-Gue´rin (BCG) vaccination is widely practiced. Thus, a new skin test antigen specific for the diagnosis of Mycobacterium tuberculosis (MTB) infection is urgently needed. CFP-10, a mycobacterial secretary protein that is absent in Mycobacterium bovis BCG and most other mycobacterial species including Mycobacterium avium, Mycobacterium intracellulare, has been shown to elicit cellular immune responses in MTB infected individuals and can be a good candidate for MTB specific diagnosis. We prepared recombinant MTB CFP-10, rCFP-10, and its utility as specific antigen for TB diagnosis was evaluated by skin testing in guinea pigs sensitized with M . tuberculosis, M. bovis, and M. bovis BCG. Our results show that the purified MTB rCFP-10 antigen elicits a positive skin response only in the guinea pigs sensitized with M. tuberculosis and M. bovis , and not in the animals sensitized with M. bovis BCG vaccine. The data presented in this study supports further testing of the use rCFP-10 as the specific antigen in the skin test for the diagnosis of MTB infection in humans. Key words : Recombinant CFP-10 protein, skin test, delayed-type hypersensitivity, tuberculosis infection, Mycobacterium tuberculosis, Mycobacterium bovis, Bacillus Calmette-Gue´rin.

Single-Step Bioconversion of Unhydrolyzed Cassava Starch in the Production of Bioethanol and Its Value-Added Products

The global economic recession that began in 2008 and continued into 2009 had a profound impact on world income (as measured by GDP) and energy use. Since then the price of the energy supply by conventional crude oil and natural gas production has been fluctuating for years which has resulted in the need to explore for other alternative energy sources. One of the fastest-growing alternative energy sources is bioethanol, a renewable energy which can reduce imported oil and refined gasoline, thus creates energy security and varies energy portfolio. Global biofuel demand is projected to grow 133% by 2020 (Kosmala, 2010). However, the biofuel supply is estimated deficit by more than 32 billion liters over the same period and the deficit is worse for ethanol than biodiesel. Ethanol may serve socially desirable goals but its production cost is still remained as an issue. Extensive research has been carried out to obtain low cost raw material, efficient fermentative enzyme and organism, and optimum operating conditions for fermentation process. In addition to that, researchers have been trying to capitalize certain features of the plant equipment and facilities to increase the throughput of ethanol and other high value by products as well as to apply suitable biorefinery for the product recovery. At the same time, effort has been made to reduce utilities costs in water usage, cooling or heating, and also consumables usage via minimizing the effluent production. Aimed to provide an alternative means for ethanol production, this book chapter introduces a single-step or direct bioconversion production in a single reactor using starch fermenting or co-culture microbes. This process not only eliminates the use of enzymes to reduce the production cost but also yield added value by-products via co-culture of strains. Before further elaboration on this single-step fermentation, we will visit the conventional process, the substrate preparation and microbe used. By this way a clear picture of the differences between conventional process and the proposed single-step fermentation with the advantages and disadvantages of both processes will be discussed.

Prediction of significant factors in the production of ethanol by ragi tapai co-culture using Taguchi methodology

Ethanol production by co-culture of ragi tapai and Saccharomyces cerevisiae from unhydrolyzed cassava starch without addition of enzymes was conducted in a 2 L batch fermentor. Taguchi’s method with orthogonal array of L8 was applied in design of experiment (DOE) and the results were analyzed using MINITAB v14 software. Seven factors: Nitrogen-phosphorus-potassium (NPK), urea, fermentation temperature, ragi tapai concentration, S. cerevisiae concentration, agitation and co-culturing time were varied at two levels for each factor. The significant factors for the production of ethanol were determined by setting S/N ratio to “larger-is-better” for high yield ethanol and “smaller-is-better” for low yield of ethanol byproducts. The optimum values obtained for each factor were similar to each other. Both have optimum factors of: Urea at 0.8 w/w%, dry ragi tapai and S. cerevisiae concentrations each at 10 w/w%, co-culturing time at 3 h gap, NPK at 0.09 w/w% and agitation speed at 100 rpm. The fermentation temperature for high ethanol yield was 35°C, whereas for the byproducts was 30°C. From the validation experiment conducted at 30°C in 10 L fermentor, the ethanol concentration obtained was 68.00 g/L, while all byproducts concentrations were below 9.00 g/L at the end of the fermentation. Key words: Co-culture fermentation, ethanol, Taguchi’s experimental design, ragi tapai, Saccharomyces cerevisiae.

Pre-treatment of Malaysian agricultural wastes toward biofuel production

Various renewable energy technologies are under considerable interest due to the projected depletion of our primary sources of energy and global warming associated with their utilizations. One of the alternatives under focus is renewable fuels produced from agricultural wastes. Malaysia, being one of the largest producers of palm oil, generates abundant agricultural wastes such as fibers, shells, fronds, and trunks with the potential to be converted to biofuels. However, prior to conversion of these materials to useful products, pre-treatment of biomass is essential as it influences the energy utilization in the conversion process and feedstock quality. This chapter focuses on pre-treatment technology of palm-based agriculture waste prior to conversion to solid, liquid, and gas fuel. Pre-treatment methods can be classified into physical, thermal, biological, and chemicals or any combination of these methods. Selecting the most suitable pre-treatment method could be very challenging due to complexities of biomass properties. Physical treatment involves grinding and sieving of biomass into various particle sizes whereas thermal treatment consists of pyrolysis and torrefaction processes. Additionally biological and chemical treatment using enzymes and chemicals to derive lignin from biomass are also discussed.

Enhanced Flexibility of Biodegradable Polylactic Acid/Starch Blends Using Epoxidized Palm Oil as Plasticizer

The brittleness of polylactic acid (PLA) has always limited its usage, although it has good mechanical strength. In this study, flexibility of PLA/starch (PSt) blend was enhanced using epoxidized palm oil (EPO) as the green plasticizer. The PLA/starch/EPO (PSE) blends were prepared while using the solution casting method by fixing the content of starch and varying ratio of EPO. The thermal properties, such as glass transition temperature (Tg), melting temperature (Tm), and crystallization temperature (Tcc) were decreased by increasing the amount of EPO into PSt, indicating that EPO increases the chain mobility. Thermogravimetric analysis (TGA) showed that thermal degradation resistance of PSE was higher when compared to PSt. The mechanical testing revealed that EPO at all contents improved the mechanical properties, such as increment of the elongation-at-break and impact strength. Whereas, dynamic mechanical analysis showed that the addition of filler into PLA decreased the storage modulus of PLA. The carbonyl group of the aliphatic ester remained the same in the PSE blends. The morphological study verified the ductility of PSE blends surface when compared to the brittle surface of PSt. As for the soil burial tests, EPO accelerated the degradation of blends. From these results, it can be concluded that EPO improved the flexibility of PLA blends.

PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

A new bacterial producing L-asparaginase was successfully isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before being further purified by chromatographic method. HiTrap DEAE-Sepharose Fast Flow ion exchange chromatography followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60 oC. Kajian penyelidikan ini telah berjaya menghasilkan enzim L-aspraginase daripada bakteria-bakteria baru yang diambil dari Kolam Air Panas, Sungai Klah, Perak, Malaysia dan ia dikenal pasti sebagai Bacillus sp. Teknik isolasi bakteria ini adalah teknik terbaik dalam menghasilkan enzim L-asparaginase berbanding teknik-teknik lain. Penghasilan enzim ini dibuat menerusi proses fermentasi. Kultur bakteria yang diperolehi diempar dan diikuti presipitasi menggunakan ammonium sulfat sebelum proses penulenan seterusnya dilakukan menggunakan kaedah kromatografi. Kolum penukaran ion jenis HiTrap DEAE-Sepharose Fast Flow diikuti dengan pemisahan oleh gel Superose 12 telah digunakan untuk mendapatkan enzim L-asparaginase yang tulen. Hasil kajian mendapati enzim tulen yang diperolehi mempunyai aktiviti spesifik sebanyak 10.11 U/mg daripada 50.07% enzim yang dihasilkan dan berjaya mencapai 2.21 kali ganda tulen dari enzim tanpa proses penulenan. Enzim tulen yang diperolehi didapati dalam bentuk “dimer“ dengan berat molekular sebanyak 65 kDa yang ditentukan menerusi SDS-PAGE. Enzim ini juga menunjukkan aktiviti yang tinggi pada pH 9 dan suhu 60 oC.

Economic and Environmental Evaluation of Recombinant Enzyme Production

Economic and environmental evaluation on three different throughputs and comparison between these throughputs were presented. Comparisons are based on simulation of 10, 100 and 1000 kg/batch of recBromelain using SuperPro Designer v8.5. The economic analysis shows that 100 kg/batch gives the most competitive price and maintaining a sizeable profit gross margin of 28.21 %. A positive ROI of 18.41 % and minimal payback time of 5.43 years is predicted from 100 kg/batch of production. Environment analysis showed that total waste and energy consumption increased as production size increased. This simulation information will assist to predict the cost and environment impact at different size of productions.

Scaling-Up Recombinant Enzyme Fermentation

Bioreactor or fermenter is the heart of a process. Scaling up a bioreactor and maintaining the same optimized process in a recombinant enzyme production in a higher scale is an engineering challenge. Scale-up is about mixing and mass transfer, a combination of art and science. It is more to methodological approach with some combination of rules of thumb, experience and/or trial and error. This chapter presented several bioreactor or fermenter modes and types before encompasses four different scale-up strategies. The scale-up strategies related to mass transfer involving constants scale-up of; (i)volumetric transfer coefficient (KLa), (ii) power input per liquid volume (P/V), (iii) impeller tip speed (Vi), and (iv) mixing time (t m) are described and discussed.