Optimization and Kinetic Modeling of Ethanol Production from Oil Palm Frond Juice in Batch Fermentation

Available online at www.sciencedirect.com

ScienceDirect Energy Procedia 79 (2015) 111 – 118

2015 International Conference on Alternative Energy in Developing Countries and Emerging Economies

Optimization and Kinetic Modeling of Ethanol Production from Oil Palm Frond Juice in Batch Fermentation Tussanee Srimachaia, Kamchai Nuithitikula*, Sompong O-thongb,c, Prawit Kongjand,e, Kiattisak Panpongf,g a

School of Engineering and Resources, Walailak University, Nakhon si thammarat 80161, Thailand b Department of Biology, Faculty of Science, Thaksin University, Phatthalung 93110, Thailand Microbial Resource Management Research Unit, Faculty of Science, Thaksin University, Phatthalung 93110, Thailand d Chemistry Division and eBio-Mass Conversion to Energy and Chemicals (Bio-MEC) Research Unit, Department of Science, Faculty of Science and Technology, Prince of Songkla University (PSU), Muang, Pattani 94000, Thailand f Songkhla Rajabhat University (Satun Campus) and gDepartment of Engineering, Faculty of Industrial Technology, Songkhla Rajabhat University, Songkhla 90000, Thailand c

Abstract Ethanol was produced from oil palm frond juice (OPFJ) by using Saccharomyces cerevisiae yeast cells. Experiments were operated in a batch mode by varying the oil palm age between 3-4, 4-7, 7-10, 10-20 and 20-25 years. Experimental results showed that OPFJ at 3-4 years gave the highest concentrations of glucose and ethanol as 31.26 and 11.50 g/l. Ethanol yield was 0.39 g-ethanol/gglucose, which estimated as 76.47% of the theoretical yield (0.51 g-ethanol/g-glucose). Additionally, the Monod and modified Gompertz equations were used to describe the kinetics of substrate utilizationand product formation. The values of the kinetic parameters in Monod and modified Gompertz equations of OPFJ extracted from oil palm aged 3-4 years were evaluated: maximum specific growth rate (μmax) was 0.29 g/g-VSS.hr, saturation concentration (Ks) 47.05 g/l, maximum ethanol concentration (Pm) 11.50 g/l, maximum ethanol production rate (rp,m) 0.24 g./l.hr and lag phase 0.12 hr. Both models fitted well to the experimental data, which had the high regression coefficient values (R2>0.95). © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2015 The Authors. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Organizing Committee of 2015 AEDCEE. Peer-review under responsibility of the Organizing Committee of 2015 AEDCEE Keywords: Ethanol production; Oil palm frond juice; Kinetic modeling; Batch fementation

1. Introduction Presently, the primary energy obtained from petroleum resource is expensive and running out in the near future. The on-going increase of the human population results in increased demand for energy. Thus, it is inevitably to find alternative energy such as ethanol which is produced from agricultural and agro-industrial wastes. Ethanol is an interesting fuel which supports a sustainable economy by reducing the use of petroleum, reducing CO 2 accumulation and reducing particulate matter and NOx emission from combustion. Additionally, using ethanol-basedfuel leads to zero net CO2 output into the atmosphere since it is recycled through photosynthesis [1-3]. In 2005 and 2006, the worldwide production capacities of ethanol were about 45 and 49 billion liters, respectively and the total demand in 2015 is expected to be over 115 billion liters [4].

* Corresponding author. Tel.: +66 75672329; fax: +66 75672399. E-mail address: [email protected].

1876-6102 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Organizing Committee of 2015 AEDCEE doi:10.1016/j.egypro.2015.11.490

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Currently, ethanol produced by using starch and sugar as raw materials is dominant in the market. However, the use of starch- and sugar-based materials has led to a “food vs. fuel” conflict due to the increasing world population [5]. Therefore, using the lignocellulosic materials is promising. Lignocellulosic materials that can be used to produce ethanol include corn stalk, oil palm biomass, rice straw, bagasse etc. Typically, lignocellulosic material is a complex carbohydrate polymer containing cellulose (40-50%), hemicellulose (25-35%) and lignin (15-20%). The main problem of using lignocellulosic material feedstock is the poor hydrolysis and high cost of cellulolytic enzymes [6]. Additionally, the ethanol production of untreated lignocellulose has low yield due to the resistance of high crystalline cellulose to enzymatic hydrolysis [5]. For these reasons, the ethanol production from lignocellulosic feedstocks requires a pre-treatment step to open up the structure and reduce the crystallinity of lignocellulose [7]. It is accepted that the pre-treatment is a “key process” of the ethanol production from lignocellulose [8].There are several pretreatment processes: chemical (e.g. acids and alkalis) and physical (e.g. stream explosion, microwave) treatmentsas well as the combination of bothmethods [5]. Oil palm frond (OPF) is a sustainable agriculture waste because it is an enormous waste obtained from the harvest of fresh fruit bunch (FFB). OPF has no value.In 2010, about 7 million tons (wet weight) of OPF were generated in Thailand [9]. The zero cost of OPF could reduce the overall production cost of ethanol [10]. OPF contains large amount of carbohydrates in the form of simple sugars which can be suitably used as a raw material for ethanol production. However, using high temperature and pressure and cellulose enzymes for converting OPF to sugars makes the overall process costly. To solve this problem, sugar can be obtained from OPF simply by pressing the fresh OPF to extract the juice. Zahari et al. [11] reported that 25 kg of fresh OPF can be converted to 12.5 kg of OPFJ by using sugarcane pressing machines. OPFJ consists of glucose as a major sugar component about 23 g/L [12]. This research therefore aimed to study the production of ethanol from OPFJ. OPFJ were received from oil palm trees with different ages in order to investigate the effect of oil palm age on the yield of ethanol.Kinetic modeling is important in designing and controlling the bioprocess efficiently [13-14]. This researchthereforeexamined the optimization and kinetic modeling of ethanol production from OPJF in a batch fermentation process usingSaccharomyces cerevisiae yeast cells. Product formation and substrate utilization models were employed. Nomenclature P Pm

Ethanol concentration (g/l) Potential maximum ethanol concentration (g/l)

rp ,m

Maximum ethanol production rate (g/l-hr)

tL t Kd KS Se θH μ, μmax

Lag time fromthe beginning of fermentation to exponential ethanol production (hr) Fermentation time (hr) Death rate constant (per day) Half saturation concentration (g/l) Substrate concentration in the effluent (g/l) Retention time (hr) Specific growth rate (per hr) Maximum specific growth rate (per hr)

2. Materials and Methods 2.1. Oil palm frond juice (OPFJ) The fresh oil palm frond (OPF) was received locally fromThasala district, Nakhon Si Thammarat, Thailand. The fresh OPF without leaves was cut into 1.0 m length. The OPF was selected from oil palm trees having different ages: 3-4, 4-7, 7-10, 10-20 and 20-25 years old. The oil palm frond juice (OPFJ) was extracted by pressing OPF with a conventional sugarcane press machine. The OPFJ was then centrifuged at 15,000 rpm for 15 min to remove solid materials and filtered. The filtrate was stored at -20 0C and put aside at room temperature before using in the experiment. 2.2. Ethanol fermentation Ethanol fermentation from OPFJ was carried out in 250 ml flasks (working volume of 100 ml) without nutrient supplementation. All the flasks were sterilized by autoclaving. Then, 10% v/v of Saccharomyces cerevisiae was added

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to the fermentation flasks under aseptic technique. The mixtures were incubated at 37 0C with shaking at 150 rpm for 96 hours and samples were collected at 0, 24, 48, 72 and 96 hours to analyze the amount of reducing sugars and concentration of ethanol. 2.3.Kinetic model prediction The Modified Gompertz model as shown in equation (1) was able to describe the product (ethanol)formation as the fermentation proceeded [14]and therefore selected for this study. P

­° ½° ª rp ,m .exp(1) º Pm .exp ® exp « » .(t L  t )  1¾ Pm ¬ ¼ ¯° ¿°

(1)

The linear form of Monod model,equation (2), was used to model the utilization of the substrate (glucose). Plotting 1/μ against 1/Se gives a straight line from which μmaxand Ks can be determined from the intercept and slope. The substrate utilization can be predicted using equation (3) [15]:

1

P

KS 1 1  Pmax Se Pmax KS (Kd 

Se

1

TH

Pmax  K d 

(2)

)

(3)

1

TH

2.4. Calculation Sugar utilization, ethanol yield, ethanol productivity and fermentation efficiency were calculated using equations (4), (5), (6) and (7), respectively [16]:

Sugar utilization  %

Amount of original sugar – Amount of residual sugar x100 (4) Amount of original sugar

Ethanol yield  g  ethanol / g  glucose

Ethanol productivity  g / l  hr

Fermentation efficiency  %

Maximum ethanol concentration  g / l Utilized glucose  g / l

Maximum ethanol concentration  g / l Fermentation time  hr

Actual ethanol yield  g / l

Theoretical ethanol yield  g / l

x100

(5)

(6)

(7)

2.5. Analytical method The total sugars were analyzed by high performance liquid chromatography (HPLC) equipped with a refractive index detector at 500C. Samples (5.0 μl)were injected into a column (SH1011, 8.0x300 nm, Shodex) with 0.04 N H2SO4 and deionised water as the mobile phase (flow rate of 0.8 ml/min). The retention time of the samples was 20 min. The concentration of ethanol was analyzed by the dichromate reagent method [17]. The protein and total nitrogen were analyzed according to the standard method for examination of water and wastewater [18].

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3. Results and Discussion 3.1. Properties ofOPFJ The properties of OPFJ are shown in Table 1. The volumes of OPFJ extracted from oil palm trees aged 3-4, 4-7, 710, 10-20 and 20-25 years old with the sugarcane pressing machine were 550, 450, 400, 350 and 300 ml/kg of OPF, respectively. Glucose was found to be the main composition of sugars in all samples within the range of 6.67-31.26 g/l. The volume of OPFJ and concentration of glucose were found to decrease with the age of oil palm trees as shown in Fig1. When oil palm trees get older, glucose is more accumulated in the form of cellulose of oil palm frond solid (OPFS). The cellulose is a polysaccharide of glucose [19]. As a result, the concentration of glucose in OPFJ decreases. The concentration of glucose in OPFJ in this study is in agreement with the previous study of Yasin et al. [12] who reported that the concentration of glucose in oil palm juice was 23 g/l. The glucose concentration was important for metabolism of S. Cerevisiae to produce ethanol. The higher glucose content, the greater volume of ethanol produced. As shown in Table 1, the concentrations of fructose and sucrose of OPFJ were in the range of 1.02-4.69 and 0.09-2.81 g/l, respectively. This corresponds to the previous studies of Zahari et al. [11] and Yasin et al. [12] who stated that the fructose and sucrose contents in oil palm juice were 1.68 and 2.30 g/l, respectively. Both concentrations of fructose and sucrose were found to decrease with oil palm age. The concentrations of protein and nitrogen in OPFJ were in the range of 0.80-3.75 and 0.11-0.47 g/l, respectively. Similarly, these concentrations decreased whenthe age of oil palm increased. The nitrogen content of OPFJ was quite small compared to Yu et al. [20] who reported that filling 2.15 g/l of nitrogen into sweet sorghum stalk could increase the rate of ethanol production. The OPFJ had the pH between 4.23 and 4.56. This is suitable for ethanol production since the optimal pH for yeast growth in ethanol production was found between 4.5 and 5 [21]. The pH of OPFJ was independent of oil palm age. Since glucose is the most important substrate for the production of ethanol, the best age of oil palm trees should be 3-4 years. In conclusion, the age of oil palm significantly affected the concentrations of sugars in OPFJ. Table 1.Initial composition and properties of OPFJ Oil palm age (years)

Volume of OPFJ (ml/kg)

Glucose (g/l)

Fructose (g/l)

Sucrose (g/l)

Protein (g/l)

Total nitrogen (mg/l)

pH

3-4 4-7 7-10 10-20 20-25

550 450 400 350 300

31.26 24.43 20.43 10.87 6.67

4.69 3.66 3.06 1.63 1.02

2.81 2.20 1.84 0.98 0.09

3.75 2.93 2.45 1.30 0.80

0.47 0.35 0.31 0.16 0.11

4.43 4.23 4.41 4.32 4.56

Fig. 1. OPFJ volume and sugar concentration at different ages of oil palm trees

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3.2. Effect of oil palmage to ethanol production Fig 2 shows ethanol production from the fermentation process by S. cerevisiae cultivated in OPFJ. During the first 48 hr of fermentation, glucose in OPFJ from all different palm ages was rapidly consumed. The calculated ethanol yields during this period were 0.37-0.40 g-ethanol/g-glucose, which were equivalent to 72.78-78.43% of the theoretical ethanol yield (0.51g-ethanol/g-glucose). The ethanol yields in this research were similar to the study of Abdullah et al. [16] in which the ethanol yield from OPFJ was 0.38 g-ethanol/g-glucose. After 48 hr of fermentation, the glucose utilization and ethanol concentration slightly increased. The maximal ethanol concentrations at 96 hr of fermentation were 11.50, 8.80, 7.50, 3.79 and 2.34 g/l for oil palm ages of 3-4, 4-7, 7-10, 10-20 and 20-25 years, respectively. These were lower than that of Abdullah et al. [16]; the ethanol concentration produced from OPFJ was 18.67 g/l. The concentration of ethanol produced from S. cerevisiae was found to decrease with older oil palm trees. This is due to the decreased concentration of glucose. Cellulose is a linear polysaccharide polymer with many glucose monosaccharide units [22]. As the age of oil palm frond increases, the amount ofcelluloseincreases. The concentration of glucose in OPFJ will eventually decrease because glucose is used for the growth of the plants. To enhance the yield of ethanol, OPF must pass the pre-treatment process and enzyme hydrolysis to destroy the cellulose structure to glucose. The comparison in ethanol concentrations from different oil palm ages with theoretical ethanol concentrations was made as shown in Fig 3.

Fig.2. Glucose utilization and ethanol concentration during batch fermentation by S. cerevisiae in OPFJ

Fig.3. Comparison between experimental and theoretical concentrations of ethanol

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The significant fermentation parameters in ethanol production from OPFJ are summarized in Table 2. The comparison with other feedstocks using the sameS. cerevisiae was made. The results of this study showed that OPFJ has a potential as a non-food medium for ethanol fermentation. This is due to that the sugar utilization and ethanol yields were comparable to those of other feedstocks including sugar beet juice, sugarcanejuice and sweet sorghum juice [16, 23-25]. Table 2. Comparison of ethanol production from different juices by S. cerevisiae Media

Sugar utilization (%)

Max.ethanol concentration (g/l)

Ethanol productivity (g/l.hr)

Fermentation time (hr)

Ethanolyield (g-ethanol/gglucose)

Fermentation Efficiency (%)

Reference

OPF juice (3-4Y)

94.05

11.50

0.12

96

0.39

76.52

This study

OPF juice (4-7Y)

92.14

8.80

0.09

96

0.39

76.78

This study

OPF juice (7-10Y)

91.39

7.50

0.08

96

0.40

79.63

This study

OPF juice (10-20Y)

89.51

3.79

0.04

96

0.39

76.37

This study

OPF juice (20-25Y)

95.20

2.34

0.02

96

0.37

72.55

This study

OPF juice

86.39

18.67

0.25

72

0.38

74.51

[16]

Sugar beet juice

100

59.89

0.77

78

0.43

78.80

[23]

Sugarcane juice

98.00

67.00

0.93

72

0.40

78.43

[24]

Sweet sorghum juice

100

72.43

1.01

72

0.48

94.60

[25]

3.3. Prediction of kinetic parameters The kinetic parameters of ethanol formation model were estimated by applying the modified Gompertz model,equation (1), to the experimental data. The fitted curves are shown in Fig 4. The calculated correlation coefficients (R2) of all curves were over 0.99, suggesting that the modified Gompertz model was able to adequately describe the formation of ethanol by S. cerevisiae in OPFJ. The parameters of the modified Gompertz model were calculated and summarized in Table 3. In this work, Pm, rp,mand tL at different oil palm ages were found in the range of 2.34-11.50 g/l, 0.05-0.24 g/l.hr and 0.12-0.85 hr, respectively. The values of Pm and rp,m were found to decrease with the increased oil palm age whereas the trend of tL was in an opposite direction.

Fig.4.Fitting the modified Gompertz model to the experimental data of ethanol production from OPFJ

The kinetic parameters of glucose utilization were determined by applying Monod model, equations (2)-(3), to the experimental data as shown in Fig 5. The Monod model typically considers isothermal system and is based on a single growth rate [26]. The correlation coefficients (R2) values were higher than 0.95. This implies that the Monod model is

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a suitable model explaining the glucose consumption of S. Cerevisiae for the ethanol production from OPFJ. The kinetic parameters of the Monod model of this study were compared to the previous studies from other feedstocks as reported in Table 3. The values of μmax and Kspredicted by the Monod model were found in the ranges of 0.11-0.29 g/g-VSS.hr and 1.82-47.05 g/l, respectively. These ranges were in agreement with the previous studies [26-27]. When the oil palm trees become older, the values of μmax and Kswere found to decrease. Table 3.Comparison in the parameters of Modified Gompertz and Monod models with previous studies Media

Modified Gompertz model

Monod model

References

Pm(g/l)

rp,m(g/l.hr)

tL(hr)

μmax(g/g-VSS.hr)

Ks(g/l)

OPF juice (3-4Y)

11.50

0.24

0.12

0.29

47.05

This study

OPF juice (4-7Y)

8.80

0.18

0.58

0.26

45.37

This study

OPF juice (7-10Y)

7.50

0.16

0.65

0.24

38.02

This study

OPF juice (10-20Y)

3.79

0.08

0.77

0.15

10.21

This study

OPF juice (20-25Y)

2.34

0.05

0.85

0.11

1.82

This study

Sugar beet juice

73.31

4.39

1.04

*ND

*ND

[14]

Whey

*ND

*ND

*ND

0.32

10.50

[26]

Xylose

*ND

*ND

*ND

0.23

1.67

[27]

*ND = Not determined

Fig.5.Fitting the Monod model to the experimental data for glucose consumption by S. Cerevisiae in OPFJ

4. Conclusions This study showed that OPFJ is a potential fermentation feedstock for S. cerevisiae to produce ethanol. The age of oil palm significantly affected the concentration of sugar in OPFJ. When the age of oil palm increased, the concentration of sugar, particularly glucose, decreased significantly. The OPFJ obtained from oil palm aged 3-4 years old had the highest concentration of glucose which in turn gave the highest yield of ethanol (0.39 g-ethanol/gglucose). The production of ethanol and utilization of glucose could be well predicted with the Modified Gompertz and Monod models, respectively. Acknowledgments The authors would like tothankthe Office of Higher Education Commission (OHEC) for fundingthisresearch. This research project would not have been possible without extensive assistance of the staff from the Microbial Management Research Unit (MRM-TSU), Faculty of Science, Thaksin University, Thailand.

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