Research ProposalProduction cellulases by isolated indigenous bacteria

NAME: HIKMAL FIZAR BIN JAMALUDINMATRIC NUMBER: SEQ 110003SUPERVISOR: DR. KHANOM SIMARANIDATELINE: 26th SEPTEMBER 2014

TitleProduction of cellulases by isolated indigenous bacteriaBackgroundCellulose, a biological compound that consists of D-glucose units linked together to form a linear chain via -1,4-glycosidic linkages is the major constituents of plant biomass (Salmon and Hudson, 1997). In addition, cellulose is also one of the major component in the aspect of waste material from the agricultural industry. Therefore, many studies had approach this waste material and manipulate it by utilizing the cellulose from it to become one of the energy resource and feed (Balachadrababu et al., 2012). The main enzyme that responsible of degrading the cellulose is cellulase. Cellulolytic enzyme system consists of three major components. They are endoglucanases, exoglucanases and -glucosidases. Cellulases are responsible for the hydrolysis of the -1,4-glycosidic linkages in the cellulose (Bayer et al., 1998). Cellulases have many beneficial applications as they can be use in starch processing, alcoholic beverage, pulp bleaching and textile industry (Sreeja et al., 2013).For many decades, cellulolytic bacteria had been isolated and characterized for obtaining the effective cellulases producing bacteria from variety of sources such as gut of living organisms, soil, organic matters, feces of ruminants and composts (Ann, 2008). Nowadays, many studies had focused on obtaining cellulolytic bacteria that have the ability to produce more efficient cellulases which being isolated through the fermentation technique (Irfan et al., 2012) .In this study, the isolated strain of bacteria were introduced into a modified synthetic medium that used bagasse and pre-treated bagasse as a carbon source. Therefore, the cellulase production by having cellulose from bagasse and pre-treated bagasse as a substrate will be analyzed by using standard enzyme assays in order to explore its potential.

Project objectives1. To study the production of cellulase from the isolated bacteria using the synthetic medium (bagasse and pre-treated bagasse) and carboxymethylcellulose broth.2. To determine the potential use of bagasse and pre-treated bagasse as a carbon source for bacterial growth and cellulase production3. To familiarize with the techniques in enzyme assays such as filter paper assay and carboxymethylcellulose assay.

HypothesisBagasse and pre-treated bagasse that act as a carbon source have the potential to promote bacterial growth and produce cellulase.Proposed methodology Inoculum preparation1. The inoculum will be prepare by transferring two loopful of bacteria colonies the nutrient broth. Then, the flasks containing the bacteria and nutrient broth will be incubate at 32oC room with 100rpm agitation for 24hrs.2. Next, the grown cell will be transfer into a 50ml centrifuge tube and undergo a centrifugation at 10000rpm for 10 minutes in a 4oC room3. Next the supernatant are going to discard and the pellet will be resuspend and adjusted in sterile 0.08% (v/v) phosphate buffer solution until the suspension of the cell reaches 0.8 at 600nm.Pre-treatment of bagasse1. Firstly, the bagasse obtained will be wash with tap water in order to remove any dusts and unwanted materials2. Next, the washed bagasse will be dried at 50oC for 24 hours3. The dried bagasse are going to grind and sieve with siever 4. Later, the sieved bagasse will be soaked in 1% (v/v) of ash solution by adding 5g sieved bagasse to 250ml of ash solution in a beaker for an hour. Next, the bagasse are going to wash repeatedly with distilled water until the filtrate reach the condition of pH75. Lastly, the bagasse will let to dry at 5oC overnightBatch fermentation1. 10% (v/v) of inoculum of isolated bacteria will be transfer into 250 ml of conical flask of modified synthetic medium. Then, the flask was incubated in 32oC room with 100rpm agitation for 24 hours2. After the incubation completed, 20ml of broth were collected in the sterile centrifuge tubes. The tube then centrifuged at 13000 rpm for 15 min. 3. Next, the supernatant will be collected and stored in 4oC room for the enzyme assays.4. The procedures above are going to repeat using pre-treated bagasse as a carbon source.5. For the batch fermentation using the CMC broth, the period of incubation were extended till 48 hours.Enzyme assays1. Four types of assays were used to measure the catalases activity of the bacteria. They are Filter Paper Enzyme Assay, Carboxymethylcellulose assay, -glucosidase assay and Bradford Protein Assay according to standard methods. (Wood& Bhat, 1988) Expected outcomes1. It is expected that having bagasse and pre-treated bagasse as a carbon source have the potential to promote bacteria growth and cellulase production.Research Gantt chart20132014

Semester 1Semester 2

OctNovDecJanFebMarAprMayJune

Proposal write up, literature review, collection of bagasse sample and the preparation of sieved bagasse

Cellulose production

Analyse the enzymes

Analytical and statistical analysis

Thesis write up and viva

ReferencesBayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 8(5), 548-557. Irfan, M., Safdar, A., Syed, Q., & Nadeem, M. (2012). Isolation and screening of cellulolytic bacteria from soil and optimization of cellulose production and activity. Turkish Journal of Biochemistry, 37(3). Khianngam S, Pootaeng-on Y. , T., T., & S, T. (2014). Screening and identification of cellulose producing bacteria isolated from oil palm meal. Journal of Applied Pharmaceutical Science, 4(4). Kuhad, R. C., Gupta, R., & Singh, A. (2011). Microbial cellulases and their industrial applications. Enzyme research, 2011. Ozioko, & C, P. (2013). Review Article: Cellulases, their substrates, activity and assay methods. The Experiment, 12(2).