Progressive Report

Production cellulases by isolated indigenous bacteria

Name:Hikmal Fizar bin JamaludinMatric Number:SEQ 110003Supervisor :Dr. Khanom SimaraniDateline :29th December 2014

Contents1.Introduction32.Aim43.Review of relevant literature44.Materials and methods75.Results and Discussion116.Appendices147.References17

Introduction

Sugarcane bagasseSugarcane bagasse is a plentiful lignocellulosic waste typically found in tropical countries that involved in sugarcane-processing industry such as Brazil, India, and Cuba (Samantha & Khakifrooz, 2011). The sugarcane stalk consists of two parts, an inner pith containing most of the sucrose and an outer rind with lignocellulosic fibers. During sugar processing, the sugarcane stalk is crushed to extract sucrose (Boophthy, 2004). This procedure yields a large volume of residue, the bagasse which contains both crushed rind and pith fibers.Bagasse, the sugarcane residue after the sugar extraction is one of the most available agricultural by product that have various kind of applications. They are used as sources of animal feed, pulp, paper and boards (Banerjee & Pandey, 2002). In addition, the sugarcane bagasse, SCB is also have a great potential in production of bioethanol (Sanchez & Cardona, 2008). Generally, SCB consists of cellulose, hemicellulose and lignin. The table below shows the percentage composition of SCB.Percentage composition of sugarcane bagasse 9%)Reference

CelluloseHemicelluloseLignin

43.633.518.1Sun et. al. , 2004

502525Pandey et. al., 2000

40-4530-3520-30Peng et. al. , 2009

Other than that, the SCB also can be also be applied to produce soluble sugars. The formation of simple sugars from cellulose and hemicellulose of SCB require a series of combination action of cellulolysis such as exoglucanase, endoglucanase and -glucosidase that derived from the cellulolytic microorganisms. In this study, the known indigenous bacteria of sugarcane environment was tested on the synthetic and carboxymethylcellulose (CMC) medium in order to analyze the production of simple sugar that produced. In addition, the ATCC strain is also used and tested in order to analyze the cellulase activity between the known and ATCC strain in same media.Aim

The purpose of this study is to determine the production of cellulase from the isolated bacteria and ATCC strain by using the synthetic and CMC medium. In addition, we are also determine the potential use of bagasse as a carbon source for bacterial growth and cellulase production.

Review of relevant literature

1.1 Lignocellulosic materialLignocellulose is the major structural component of woody plants such as grass and represents a major source of renewable organic matter (Howard et. al, 2004). Lignocellulose consists of lignin, hemicellulose and cellulose. Large amounts of lignocellulosic waste are generated through forestry and agricultural practices, paper-pulp industries, timber industries and many other agro-industries (Malherbe & Cloete, 2002). The table below summarized the source and the potential uses of lignocellulosic material.Lignocellulosic materialResiduesCompeting use

Grain harvesting-Wheat, rice, oats, barley and cornStraw, husks, stalks, cobsAnimal feed, burnt as fuel, compost and soil conditioner

Processed grains-corn, wheat, rice and soybeanWastewater, branAnimal feed

Fruit and vegetable harvestingSeeds, peels, husks, stones, rejected whole fruit and juiceAnimal and fish feed, some seeds for oil extraction

Fruit and vegetable processingSeeds, peels, wastewater, husks, shells, stones and rejected whole fruit and juice

Sugar cane other sugar productsbagasseBurnt as fuel

Oils and oilseed plants-Nuts, cotton seeds, olives and soybeanShells, husks, lint, fiber, sludge, press cake and wastewaterAnimal feed, fertilizer, burnt fuel

Animal wasteManure, other wasteSoil conditioners

Forestry paper and pulp-harvesting of logsWood residuals, bark, leaves, etcSoil conditioner, burnt

Saw and plywood wasteWoodchips, wood shavings, saw dustPulp and paper industries and fiber board

Pulp and paper millsFiber waste and sulfite liquorReused in pulp and board industry as fuel

Lignocellulose waste from communitiesOld newspaper, paper, cardboard, old boards, disused furnitureSmall percentage recycle, others burnt

grassUnutilized grassburnt

Table 3.1.1: Types of lignocellulosic materials and their current uses (Malherbe & Cloete, 2003)

1.2 Cellulose

Cellulose is the substance that makes up most of a plants cell wall and account for the most abundant organic compound on Earth. Chemically, cellulose is a linear natural polymer of anhydroglucose units that linked at the one and four carbon atoms by -glycosidic bonds. The fact of the chemical structure of the cellulose is prove by the presence of three hydroxyl groups with difference in aspect of acidity or reactivity, secondary OH at carbon number 2 and 3 and primary OH at the C-6 position respectively by the formation of strong various inter and intramolecular hydrogen bonds (Kadla & Gilbert, 2000). In plant, the cellulose is organized into fibrils which are surrounded by a matrix of lignin and hemicelluloses.

1.3 HemicelluloseHemicellulose, a biological compound that contributes second most common polysaccharides in nature. It represents about 20-35% of lignocellulosic biomass (Saha, 2003). Hemicelluloses are heterogeneous polymers of pentoses, hexoses and sugar acids. The composition of hemicellulose can be categorized into two species, hardwood and softwood hemicellulose. The difference between this two species can be observed by the abundance of the composition within it. The hardwood hemicellulose are rich of xylans while softwood hemicellulose are rich in glucomannans (McMillan, 1994). Hemicelluloses have lower degree of polymerization (DP), that is only 50-300 with side groups on the chain molecule and are essentially amorphous (Perez et. al., 2002).

1.4 Lignin

Lignin is the most abundant aromatic polymer in nature. It present in the cellular cell wall, conferring structural support, impermeability and resistance against microbial attack and oxidative stress. Lignin is an amorphous heteropolymer, hydrophobic and optically inactive. It consists of phenylpropane units joined together by different types of linkage. The polymer is synthesized by the generation of free radical, which are released in the peroxidase-medated dehydration of three phenyl propionic alcohols (Sjostrom, 1993).

1.5 Pre-treatment of bagasse

The main purposes of having the pre-treatment of bagasse are to improve its digestibility and easy access for microbial attack by removing the core and noncore lignin fractions (Alan & Smith, 1988 and Doran et. al.,1994). They are several physical and chemical methods that applied for the pre-treatment of SCB which include steam explosion, gamma radiation, treatment with alkali, hydrogen peroxide and solvents. The pre-treatment with alkali such as sodium hydroxide is the most effective and economical way to treat the SCB. A study by Rodriguez-Vazguez et. al. in 1992 that treated bagasse with NaOH solution in a dry pre-treatment ( dry pretreatment refers to low or no free liquid was present) shows that maximum digestibility of 75% from the biomass production. Bravo et. al. (1994) that also used alkali pre-treatment at 3 liquid:1 solid ratio revealed that the treatment significantly enhanced fungal growth compared to nontreated bagasse. Du-Toit et. al. (1984) compared the pre-treatments of bagasse with dilute alkali and acid for the determination of the monosaccharides present In bagasse hemicellulose. In their study, the pentosan fraction of the bagasse was successfully hydrolyzed and extracted with 5% (m/v)HCL. The treatment with alkali resulted in 39.8% solubilization of bagasse and about 72% of the available hemicellulose be extracted.

Materials and methods

1.6 SamplingThe sugarcane bagasse are collected from the sugarcane juice stall at the area of Pantai Dalam, Kuala Lumpur. The sugarcane bagasse, SCB next are washed with tap water and dried under sunlight for a day. Later, the bagasse were cut into smaller pieces (about 1-2 cm in size) and oven dried in the incubator at 60C temperature overnight.1.7 Pre-treatment of bagasseThe dried bagasse is grinded by using the mechanical grinder in order to obtain the powder form of bagasse. Later, the grinded bagasse was sieved using siever (1mm of pore size) to obtain fine powder of bagasse. Next, the bagasse is soaked in 1% (w/v) ash solution by adding 5g of bagasse sample to 250 ml of 1% (w/v) ash solution in a beaker for an hour. After that, the bagasse sample were washed with distilled water until the pH of the filtrate reach pH 7 in order to remove the alkalinity in the sample. Next, the sample was then dried at 50C overnight and kept in plastic bag prior to use.1.8 The production of cellulolytic enzyme1.8.1 Preparation of inoculumFirstly, two loopful of pure colony from Bacillus aerius strain 5.2 were inoculated into 50 ml nutrient broth in 250 ml of conical flask. Next, the flask is incubated overnight (25C, 100 rpm). Later, 50 ml of grown cell were transferred into sterile Falcon tube and centrifuged (10000 rpm, 4C) for 10 minutes. The cell pellet was resuspend with sterile 0.08% (v/v) phosphate saline buffer until the optical density of the cell suspension reached at 0.8A at 600nm.1.8.2 Batch fermentation10% (v/v) of inoculum was transferred into two different of 500 ml of conical flasks that contain 250 ml of synthetic medium and CMC medium respectively. Then, the flasks are incubated (32C, 100rpm) for 48 hours.1.8.3 Collection of samplesThe samples were collected for both CMC and synthetic medium at different intervals (4, 8, 12, 24, 36,48h). Next, the samples were centrifuged (10000rpm) for 15 minutes. The supernatant was collected and stored in 4C room for further assay.

1.9 Enzyme assay1.9.1 Filter paper Enzyme Assay (FPase)The FPase activity was assayed by adding 0.2mL of enzyme sample into test tube that contained 1.8mL of 0.05M sodium citrate buffer (pH 4.8) and Whatman filter paper (1.0x 3.0 cm). Next, the mixture was homogenized using vortex mixer and incubated in water bath 40C) for 60 minutes. Later, 3ml of DNS reagent was added into the tubes and incubated at boiling water bath (100C) for 3 minutes to stop the enzymatic and DNS reaction. The reducing sugars liberated were determined by using DNS reagent (Wood & Bhat, 1988). After that, 1 ml of Rochelle salt was added and the absorbance was read using spectrophotometer at 575nm. The glucose standard curve was used to determine the amount of reducing sugar that being released. One unit of FPase activity was defined as 1mole of glucose liberated per mL enzyme per minute calculated as shown in Equation 1.0Calculation:Reducing sugar (mole) = Where:OD= Absorbance of the sample at 575nmM= Slope of glucose standard curveGMR= Glucose molecular weightGMR of glucose= 180

FPase activity ( U/mL) = ...(Eq 1.0)

FPase activity is defined as 1 mol reducing sugar released per mL of enzyme per min.

1.9.2 Carboxymethylcellulose Assay (CMCase)The CMCase activity was determined by measuring the reducing sugar produced by using 1.0 % (v/v) CMC as a substrate. 1.8 ml of 1.0 % (v/v) CMC solution was added in the test tube that contained 0.2 ml of sample. The tube then incubated at 40C water bath for 60 minutes. Next, 3.0 ml of DNS reagent was added and the suspension was mixed using vortex mixer. Later, the tubes was incubated in boiling water bath (100C) for 15 minutes in order to stop the enzymatic and DNS reaction. Next, 1ml of Rochelle salt was added and the absorbance was read using spectrophotometer at 575nm (Wood & Bhat, 1988).The reducing sugar liberated from enzymatic reaction were determined using DNS reagent (Wood and Bhat, 1988) and calculated using Equation 2Calculation:Reducing sugar (mole) = Where:OD= Absorbance of the sample at 575 nm M= Slope of glucose standard curveGMR= Glucose molecular weightGMR of glucose= 180CMCase activity ( U/mL) = ...(Eq 2.0)

CMCase activity is defined as 1 mol reducing sugar released per mL of enzyme per min.

Results and Discussion

1.10 Pre-treatment of sugarcane bagasseIn this process, the weight of bagasse before and after the pre-treatment were measured to determine the amount of weight loss during the treatment. The result are as follow. Table 5.1 The weight of pre-treatment bagasse (before and after the pre-treatment)SampleWeight of bagasse (g)Mean standard deviation

Before pre-treatment (W1)After pretreatment (W2)Weight loss, W3=(W1-W2)

152.52.52.410.27

252.452.55

352.422.58

452.992.01

Total weight2010.369.64

Therefore, 48.2% of initial weight of bagasse was lost during the pre-treatment of the bagasse

1.11 Glucose concentration in the different medium

The amount of the glucose before addition of the inoculum was analyzed using DNS assay. The DNS assay was carried out by adding 1ml of medium and 9ml of 0.05M sodium citrate buffer in the test tubes. Next, the tube was incubated at 40C water bath for 60 minutes. Later, the DNS reagent was added and the tube was incubated in boiling water bath (100C) for 3 minutes. 3 ml of Rochelle salt was then added and the absorbance was read using spectrophotometer at 575nm. The glucose standard curve was used as standard comparison. The results are as follow:

Table 5.2 Glucose concentration in three different media and controlMediumAbsorbance (575nm)Concentration of glucose,x={y/0.5272} (mg/ml)

MeanStandard deviation

1212

CMC0.0850.1180.1610.2240.1930.0443

Pre-treated bagasse + autoclaved0.1170.0920.2220.1750.1980.0335

Untreated bagasse + autoclaved0.4050.3860.7680.7320.7500.0255

Control0.1910.2390.3620.4530.4080.0644

From the result above, the untreated bagasse shows the highest concentration of glucose that it 0.750 mg/ml.

1.12 FPase titer produced by Bacillus aerius strain 5.2 in different culture media

Figure 5.1: Fpase activity of Bacillus aerius strain 5.2The purpose of having the FPase assay is to determine the total cellulase activity and glucose obtained from the cellulolysis process. In diagram above, the highest cellulase activity recorded by Bacillus aerius strain 5.2 in pre-treated bagasse is 0.187 U/ml after 8 hours of incubation. This is due to the highest enzyme-substrate reaction that occurred at that particular time. For the Fpase activity using untreated bagasse and CMC, the highest cellulase activity recorded are 0.132 U/ml and 0.132 U/ml respectively. However, the Fpase activity recorded for these three synthetic media show no significance difference after 24 hours of incubation. 1.13 CMCase titer produced by Bacillus aerius strain 5.2 in different culture mediaFigure 5.3.2: Fpase activity of Bacillus aerius strain 5.2The purpose of CMCase test is to measure and determine the endoglucanase activity by using carboxymethylcellulose solution as a substrate. In the diagram above, the highest endoglucanase activity recorded for Bacillus aerius strain 5.2 is in untreated bagasse that is 0.287 U/ml after 4 hours of incubation. However, the highest cellulase activity recorded for both CMC and pre-treated bagasse are 0.147 U/ml (after 36 hours) and 0.111 U/ml (after 12 hours of incubation).

Appendices

1.14 Glucose standard curve

The determination of FPase and CMCase activity in the culture medium are obtained by measuring the amount of reducing sugar liberated in a given period of time. The determination of reducing sugar is using thus using the gradient of the glucose standard curve. The procedure of glucose standard curve eventuated by preparation of glucose stock concentration. 0.25g of anhydrous glucose was diluted with 250ml of distilled water to make 0.1mg/ml glucose stock concentration. Next, the glucose serial dilution is prepared as table below. All tubes were incubated at 40C water bath for 60 minutes. Later, 3.0 ml of DNS reagent was added and further incubation in 100C boiling water bath for 5 minutes to stop the reaction. After that, let all the tubes to cool down under room temperature before vortex it using vortex mixer. Lastly, read the absorbance of the solution by using spectrophotometer under 575nm wavelength.Test tubeVolume of glucose stock (ml)Volume of distilled water (ml)Glucose concentration (mg/ml)

A0.005.000.00

B0.504.500.50

C1.004.001.00

D1.503.501.50

E2.003.002.00

F2.502.502.50

G3.002.003.00

H3.501.503.50

I4.001.004.00

J4.500.504.50

K5.000.005.00

Data for the glucose standard curveTable 6.1.1: Data of Glucose standard curveTest tubeVolume of glucose stock (ml)Volume of distilled water (ml)Glucose concentration (mg/ml)Absorbance (575nm)Total absorbance (575nm)Average absorbance (575nm)

12

A0.005.000.000.000.000.000.00

B0.504.500.500.110.150.260.13

C1.004.001.000.450.490.940.47

D1.503.501.501.080.962.041.02

E2.003.002.001.241.122.361.18

F2.502.502.501.601.322.921.46

G3.002.003.001.801.803.601.80

H3.501.503.501.931.923.851.93

I4.001.004.002.182.114.292.14

J4.500.504.502.352.144.492.25

K5.000.005.002.562.184.742.37

Graph 6.1.1: The straight line of glucose standard curve

1.15 Preparation of synthetic and CMC mediaChemical composition% compositionPreparation in 1000 ml (g)

K2HPO40.080.80

KH2PO40.020.20

MgSO4.7H2O0.020.20

NaCl0.020.20

NaNO30.11.0

CaCO30.0010.01

Yeast extract0.050.5

CMC/Bagasse0.55.0/12.5

The composition above is then mixed with 1000ml distilled water. The pH was adjusted to pH 7 and sterilized at 121C for 15 minutes.

References

Samariha A, & A, K. (2011). NSSC for bagasse. BioResources, 6(3), 3313-3323. Boophthy, R. (2004). Use of post-harvest sugarcane residue in coastal reclamation: A feasibility study. SugarCane International, Jun/Feb, 9-13. Banerjee, R., & Pandey, A. (2002). Bio-industrial applications of sugarcane bagasse: a technological perspective. International sugar journal, 104(1238), 64, 66-67. Sanchez, O. J., & Cardona, C. A. (2008). Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresource technology, 99(13), 5270-5295. Sun, J., Sun, X., Zhao, H., & Sun, R. (2004). Isolation and characterization of cellulose from sugarcane bagasse. Polymer Degradation and Stability, 84(2), 331-339. Pandey, A., Soccol, C. R., Nigam, P., & Soccol, V. T. (2000). Biotechnological potential of agro-industrial residues. I: sugarcane bagasse. Bioresource technology, 74(1), 69-80. Peng, F., Ren, J.-L., Xu, F., Bian, J., Peng, P., & Sun, R.-C. (2009). Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. Journal of agricultural and food chemistry, 57(14), 6305-6317. Howard, R., Abotsi, E., Van Rensburg, E. J., & Howard, S. (2004). Lignocellulose biotechnology: issues of bioconversion and enzyme production. African Journal of Biotechnology, 2(12), 602-619. Malherbe, S., & Cloete, T. E. (2002). Lignocellulose biodegradation: Fundamentals and applications. Reviews in Environmental Science and Biotechnology, 1(2), 105-114. Kadla, J. F., & GILBERT, R. D. (2000). Cellulose structure: A review. Cellulose chemistry and technology, 34(3-4), 197-216. Saha, B. C. (2003). Hemicellulose bioconversion. Journal of Industrial Microbiology and Biotechnology, 30(5), 279-291. McMillan, J. D. (1994). Pretreatment of lignocellulosic biomass. Paper presented at the ACS symposium series (USA).Prez, J., Munoz-Dorado, J., de la Rubia, T., & Martinez, J. (2002). Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. International Microbiology, 5(2), 53-63. Sjstrm, E. (1993). Wood chemistry: fundamentals and applications: Gulf Professional Publishing.AlAni, F., & Smith, J. (1988). Effect of chemical pretreatments on the fermentation and ultimate digestibility of bagasse by Phanerochaete chrysosporium. Journal of the Science of Food and Agriculture, 42(1), 19-28. Doran, J. B., Aldrich, H., & Ingram, L. (1994). Saccharification and fermentation of sugar cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway. Biotechnology and bioengineering, 44(2), 240-247. Rodriguez-Vazquez, R., Villanueva-Ventura, G., & Rios-Leal, E. (1992). Sugarcane bagasse pith dry pretreatment for single cell protein production. Bioresource technology, 39(1), 17-22. Bravo, O., Ferrer, A., Aiello, C., Ledesma, A., & Dvila, M. (1994). Growth of Chaetomium cellulolyticum in solid-state fermentation of sugar cane bagasse treated with water and alkali at several liquid/solid ratios. Biotechnology letters, 16(8), 865-870. Du Toit, P., Olivier, S., & Van Biljon, P. (1984). Sugar cane bagasse as a possible source of fermentable carbohydrates. I. Characterization of bagasse with regard to monosaccharide, hemicellulose, and amino acid composition. Biotechnology and bioengineering, 26(9), 1071-1078. Wood, T. M., & Bhat, K. M. (1988). Methods for measuring cellulase activities. Methods in enzymology, 160, 87-112.

14