1. OBJECTIVES

• To enhance bioethanol production using OPFJ as substrate
• To optimized biotehanol production using various fermentation mode

2. INTRODUCTION AND RESEARCH BACKGROUND

Brazil and USA is the first producers with the world ethyl alcohol production about 51000 mills liters with an average of 73% of produces ethanol worldwide corresponds to fuel ethanol, 17% to beverage ethanol and 10% to industrial ethanol (Eufrozina NIGA, 2009). Bio-ethanol can be a product from an edible source which known as first generation bio-ethanol such as sugarcane and corns. Second generation of bio-ethanol is from lignocelluloses biomass is from non-edible source such as palm oil biomass. This make second generation of bio-ethanol is a better choice to replace fossil fuel without disturbing food sources. By converting the waste into valuable product we can reduce the environmental problem. Lignocelluloses material contain about 50% – 60% carbohydrate in the form of cellulose and hemicelluloses which may be fermented to ethanol and 20%-35% of lignin (Mats Galbe et al, 2007). Nowadays palm oil tree is one of the main source for the production of bio-ethanol but there are not much attention is been given to oil palm sap which are product that come from the tree component such as oil palm trunk and oil palm fronds.

Malaysia is the world’s largest exporter of palm oil product. Malaysia produces a large amount of agro-industrial residue with oil palm industry itself produced about 33 million tons of residues annually in the form of empty fruit bunch, fiber and shell (MPOB, 2009; Mohamed and Lee, 2006). Malaysia has produced about 51 million tons of oil palm fronds with 53% of the total palm biomass in year 2008 (Goh et al, 2010; MPOB, 2009). Bio-fuel that produces from palm oil tree is an environmental friendly therefore many interest are being shown to this sources. Mostly, the major parts of the solid biomass from the oil palm tree are being leave behind on the plantation is to be found as oil palm fronds.

It has been reported that about 46837K tons of oil palm fronds has been produced in Malaysia in the year 2007 as an agriculture wastes. Palm oil frond is one of the useful raw materials for the production of bio-ethanol which is environmental friendly way. Pre-treatment of the oil palm frond need to be done to achieve a good bio-ethanol production. However, ethanol production from lignocellulosic biomass is relatively expensive because of the latest technologies. The main contradict are low yield and cost of the hydrolysis process (Sun and Cheng, 2002).

Thus, oil palm frond juice is being introduced as another source for the production of bio-ethanol. Previous study has been shown that oil palm juice is suitable to used as fermentation feedstock because there was no inhibition on microbial growth or product formation, there were no impurities, it was easy to be operated, and it had no risk on health and safety (Zahari et al, 2012). Oil palm frond sap has been proved by Zahari et al. (2012) contains high sugar content which is 76.09 ± 2.85 g/l. There has been reported by Eze and Ogan (1988) that sucrose is the dominant sugar in the oil palm frond sap that consist of 10% w/v, as for glucose and fructose consist of <1.0% w/v (Kosugi et al, 2010. However in Malaysia, a study shows that glucose is the dominant sugar in the oil palm sap (Kosugi et al., 2010). Since there are many studies show that oil palm juice contains higher sugar content, a high level of the production of bio-ethanol from oil palm sap is further study.

A study by Nwachukwu (2008) shows that improving yeast resistance by protoplast fusion increased yields of ethanol by 16% v/v. Apart from that, types of fermentation also one of the factor that improve the production of bio-ethanol. Thus, this project aim is to increase the yield of the bio-ethanol production using oil palm frond juice by various type of fermentation process.

3. PROBLEM STATEMENT

Uses of natural sources like petroleum is been used over the decades and the demand of this sources are being increases from time to time. Over the century, there are an increasing of energy consumption with the increasing of world population, thus more growing countries become industrialized which there are possibility that the sources will be depleted one day. Apart from that, petroleum is harmful to human and environment and with the increasing of fossil fuel will lead to increasing of carbon dioxide that eventually leads to global warming issue. Thus, an alternative sources of the fuels is been quest to overcome the shortness of the fossil fuels. Bio-ethanol is one of the solutions to this problem. This is because bio-ethanol is a form of renewable energy source which are easy available, low cost and most efficient bio-fuel. Bio-ethanol offers a great advantages because it can be produced from various feedstock such as corn, sugarcane, red seaweed part sugar beet and many more (Mohd Dinie et al, 2013).Apart from that , bio-ethanol help in reducing air pollution and carbon dioxide accumulation.

Nowadays, high demands of fuel are been constantly increase over the world. Brazil is been known as the largest producer of the sugarcane and a most competitive producer of the bio-ethanol in the world. The arises of bio-ethanol from sugarcane is a prove that energy sources are being run out and constant increasing of fuel cars around the world. Fossil energy gives an impact to the atmosphere because the burning of the petroleum result in increasing of carbon dioxide emission to the environment which is the main causes of greenhouse effect. Production of bio-ethanol is increase significantly because many countries are looking for reducing oil imports, increasing rural economies and for better air quality (Eufrozina NIGA, 2009).

4.0 METHOD

4.1 Raw material preparation and Juice Clarification. (Potential Utilization of Sap from Oil Palm (Elaeis guineensis) for Lactic Acid Production by Lactobacillus casei by S. Chooklin et al, 2011)

1. Oil palm frond (OPF) will be harvest from oil palm tree from a local oil palm plantation.
2. The leaves from the frond will be cut off and discard.
3. The harvest OPF will be press using sugar-cane machine press as soon as possible after the frond was harvested.
4. OPF juice produced will be collect and filtered using coffee filter.
5. The filtered OPF juice will be centrifuged at 10,000 rpm for 15 minutes.
6. Supernatant will be stored at -20°C before use.

4.2 OPF Juice Sugar Composition (Ethanol Production Using Immobilized Saccharomyces cerevisiae in Lyophilized Cellulose Gel by Eleonora Winkelhausen et al, May 2010)

HPLC type:HPLC (Shimadzu Japan ), equipped with refractive index Detector

Type of column: APS-Hypersil column ( diameter of 250mm x 46mm)

Mobile phase:70% Acetonitrile and 30% de-ionized water

Column temperature:40°C with maximum operating temperature of 80°C

Flow rate: 0.6 ml/min

Sample volume: 20µl

Pressure: 10MPa

4.3 Pure Culture Establishment (Study on Bioethanol Production from Oil Palm Trunk (Opt) Sap by Using Saccharomyces Cerevisiae Kyokai No.7 by Nina Farhana 2010) (Isolation of Microorganism from Oil Palm Sap by Nurul Nadia Ummira, 2011)

1. 100mL of the Nutrient Broth Medium supplemented with g/L; Yeast Extract, 5; Peptone, 10 and 15% of glucose solution, 50 (Jamaludin, 2010) will be measured in a 250mL conical flask and will be autoclaved at 121 C in 15 minutes.
2. Saccharomyces cerevisiae will be subculture into the Nutrient Broth.
3. The mixture will be incubated for 24 hours at 150 rpm, 30^oC.
4. The suspension obtained will be subculture on the Nutrient Agar slant and will be incubated for another 2-3 days.
5. The Nutrient Agar slants will be stored in refrigerator at 4^oC.

4.4 Inoculums preparation (Study On Bio ethanol Production From Oil Palm Trunk (OPT) Sap by Using Saccharomyces Cerevisiae Kyokai No.7 by Nina Farhana 2010) (Optimization study of ethanol fermentation from oil palm trunk, rubber wood and mixed hardwood hydrolysates using Saccharomyces cerevisiae by K.L. Chin et al , 2010)

1. About 3 – 4 loop of pure culture will be transferred from agar slant into 100 mL of sterile Nutrient Broth in 250 ml conical flask plug with sterile cotton.
2. The flask will be incubated for 24 hours at 30°C and 150 rpm until reach standard initial concentration. The cell concentration will be standardized to 0.2 – 0.4 g/L (OD = 4.2) determined by turbidimetry at 600 nm.

4.5 Fermentation

a) Batch fermentation will be performed for the optimization of bio-ethanol production.

b) 20% (v/v) of inoculums size will be inoculated into the bioreactor with the condition of pH 4.5 and temperature of 30ºC , air flow rate of 1 vvm, agitation of 200 rpm with the initial 0₂ concentration of 30 %.

c) The fermentation process will be carried out for 42 hour and sample will be taken out every 3 hour and analyze for bio-ethanol production, sugar and cell biomass.

d) All experiment will be run in triplicate.

4.5.1 Batch fermentation.

4.5.1.1 Effect of sugar concentration (Effect of Cultural Conditions on Ethanol Production by Locally Isolated Saccharomyces Cerevisiae Bio-07 by Arifa Tahir et al, 2010)

1. OPF juice volume use will be varied in the range of 30% to 50%.

(30%, 40%, 50%)

4.5.1.2 Effect of agitation speed (Optimization of Fermentation Medium for the Production of Ethanol from Jaggery Using Box-Behnken Design by Mary Anupama.P et al 2010)

1. The fermentation will be carried out at different agitation rate in the range of 125 rpm to 175 rpm.

(125, 150, 175)

4.5.2 Fed Batch fermentation

4.5.2.1 Effect of feeding rate (Production of Ethanol by Fed- Batch Fermentation by Ngoh Gek Cheng et al, 2009)

The fed batch fermentation will be run according to the best optimized condition from the batch fermentation condition.

1. Substrate will be feed continuously into the bioreactor using peristaltic pump at glucose concentration (glucose concentration from OPFJ?) varying from 2 ml to 8 ml at feeding rate of 2 ml/h .

(2 ml , 4 ml, 8 ml)

4.5.2.2 Effect of feed time (Production of Ethanol by Fed- Batch Fermentation by Ngoh Gek Cheng et al, 2009)

a) Substrate will be feed from the interval of every one hour to three hour at feeding rate of 2 ml/h.

( 1 hour , 2 hour , 3 hour )

4.6 Harvesting (Study on Bioethanol Production from Oil Palm Trunk (Opt) Sap by Using Saccharomyces Cerevisiae Kyokai No.7 by Nina Farhana, 2010)

1. Each batch of fermentation will be terminated according to optimized time period.

b) Sample (15 ml) will be taken out and the samples will be kept in refrigerator at

4^oC before analyze for bio-ethanol production, sugar and cell biomass.

4.7 Yeast strain and it improvement (High-level Production of Ethanol during Fed Batch Ethanol Fermentation with a Controlled Aeration Rate and Non-Sterile Glucose Powder Feeding of Saccharomyces cerevisiae by Hyen-beom-seo et al, 2009)

a) Saccharomyces cerevisiae will be growth on Nutrient Broth (yeast extracts, 10g/l; peptone, 20g/l; glucose, 20g/l and agar, 20g/l) slant in a 30 ml universal bottle.

b) The slant culture will be exposed with UV light (6 watt, 254nm) for 15 second interval at a distance of approximately 7 cm from the slant.

c) A loopful of irradiated slant culture will be streak onto a Nutrient Broth agar plate and subsequently incubated for 3 days at 27ºC.

d) After that the colonies will be incubated in Nutrient Broth medium containing 100 g/l of ethanol at 27ºC while shaking at 100 rpm for 5 days to select resistance colonies.

4.8 Analysis method

4.8.1 Sugar content by HPLC (Oil Palm Fronds Juice as Future Fermentation substrate: A Feasibility Study by Che Mohd Hakiman Che Maail et al, 2014)

HPLC type:HPLC (Shimadzu Japan ), equipped with refractive index Detector

Type of column: APS-Hypersil column ( diameter of 250mm x 46mm)

Mobile phase:70% Acetonitrile and 30% de-ionized water

Column temperature:40°C with maximum operating temperature of 80°C

Flow rate: 0.6 ml/min

Sample volume: 20µl

Pressure: 10MPa

4.8.2 Cell dry determination (Study on Bioethanol Production from Oil Palm Trunk (Opt) Sap by Using Saccharomyces Cerevisiae Kyokai No.7 by Nina Farhana 2010)

1. Cell suspension will be vaccum-filtered using 0.45µm filter paper
2. Cell suspension for each samples will have to be rinsed 2x with distilled water.
3. Each sample will be dried at 70°C more than 24 hours until constant weight achieved.
4. Dried filter paper will be weighed on a analytical balance to measure the biomass.

4.8.3 Ethanol concentration using GC (Production of Ethanol by Fed –Batch Fermentation by Ngoh Gek Cheng et al, 2009)

Column : RT-Q-BOND (inner diameter of 0.32 mm)

Carrier gas: Helium gas

Detector: Flame ionization detector (FID)

Temperature : 200 C

Flow rate : 21.9 mL/min

Pressure : 71.1 kPa

Holding time : 5 minutes

% bioethanol yield is calculated using this formula:

5.9 OPTIMIZATION OF ETHANOL FERMENTATION

1. RSM analysis will be conducted using Design Expert Software.
2. The optimized parameters will be verified by running the new fermentation as suggested.

6.0 ANALYSIS

The significance of difference between each test variable will be determined using one way ANOVA analysis and Least Significance Test, computed using SPSS version 21.0 software. All tests will be done with a confidence interval of 95%.

7.0 REFERENCES

1. Ngoh Gek Cheng, M. H., Andri Chahyo Kumoro, & Tham, C. F. L. A. M. 2009. Production of Ethanol by Fed-Batch Fermentation. Pertanika J. Sci. & Technol., 17, 399 – 408.
2. Hyeon-Beom Seo, Seung Seop Kim,Hyoen0Yong Lee and Kyung Hwan Jung (2009).High level Production of Ethanol during Fed-Batch Ethanol Fermentation with a controlled Aeration Rate and Non-Sterile Glucose Powder Feeding of Saccharomyces cerevisie .Biotechnology and Bioprocess Engineering,14, 591-598.
3. Jamaludin, N. F. M. 2010. Study On Bioethanol Production From Oil Palm Trunk (Opt) Sap By Using Saccharomyces Cerevisiae Kyokai No.7(ATCC 26422). Bachelor of Chemical Engineering (Biotechnology), Universiti Malaysia Pahang.
4. Zahari, M. A., Zakaria, M. R., Ariffin, H., Mokhtar, M. N., Salihon, J., Shirai, Y. & Hassan, M. A. 2012. Renewable sugars from oil palm frond juice as an alternative novel fermentation feedstock for value-added products. Bioresour Technol, 110, 566-71.

5. Che Mohd Hakiman Che Maail, Hidayah Ariffin,Mohd Ali Hassan,Umi Kalsom Shah and Yoshihito Shirai (2014).Oil Palm Frond Juice as Future Fermentation Substrate : A Feasibility Study.Bioresource Technology,110,566-71.
6. Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar,Anal. Chem.,31,426, 1959.

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