九州工業大学学術機関リポジトリ

Title Efficient Bioethanol Production From Oil Palm Frond Petiole

Author(s) Sharifah Soplah Binti Syed Abdullah

Issue Date 2015-03

URL http://hdl.handle.net/10228/5468

Rights

Kyushu Institute of Technology Academic Repository

EFFICIENT BIOETHANOL PRODUCTION FROM OIL PALM

FROND PETIOLE

SHARIFAH SOPLAH BINTI SYED ABDULLAH

March 2015

Department of Biological Functions and Engineering

Graduate School of Life Science and Systems Engineering

Kyushu Institute of Technology

EFFICIENT BIOETHANOL PRODUCTION FROM OIL PALM

FROND PETIOLE

by

SHARIFAH SOPLAH BINTI SYED ABDULLAH

March 2015

Department of Biological Functions and Engineering

Graduate School of Life Science and Systems Engineering

Kyushu Institute of Technology

ii
 ACKNOWLEDGEMENT

In the name of Allah, the Most Gracious and the Most Merciful. Alhamdulillah, all

praises to Allah for the strengths and His blessing in completing this thesis. Special

appreciation goes to my supervisor, Prof. Dr. Yoshihito Shirai for his supervision and

constant support. His invaluable help of constructive comments and suggestions

throughout the experimental and thesis works have contributed to the success of this

research. I would also like to extend this appreciation to Prof. Dr. Mohd Ali Hassan of

Universiti Putra Malaysia, Malaysia, for his meticulous supervision while advising my

study. My sincerest thanks to the examination committee members, Prof. Dr. Haruo

Nishida, Assoc. Prof. Dr. Yoshito Ando and Assoc. Prof. Dr. Toshinari Maeda for their

critical comments and significant contributions to this thesis. My gratitude also

extended to the Majlis Amanah Rakyat (MARA) and Universiti Kuala Lumpur for the

financial support and study leave throughout the years.

Sincere thanks to all my friends especially EB members, particularly Dr. Ezyana, Dr.

Muhaimin, Dr. Amir, Faiz, Che Hakiman, Sharmila, Huzairi and others for their

kindness and moral support during my study. Thanks for the friendship and memories.

Not forgotten to the staff of Faculty of Biotechnology and Biomolecular Sciences,

especially En. Rosli and Pn. Rosema, thank you very much.

No words can be expressed to appreciate my beloved husband Mr. Ahmad Kamal B.

Abdullah, my children Nur Syamim Fatihah, Nur Izzah Az Zahra and Ahmad Zharif

Aiman for their endless love, prayers and encouragement. Also not forgetting my late

iii
parents Umi and Abah, sisters and brothers, and last but not least my family in law for

their love and care. To those who indirectly contributed in this research, your kindness

means a lot to me. Thank you very much.

iv
 TABLE OF CONTENTS

CONTENT PAGE

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS v

LIST OF FIGURES x

LIST OF TABLES xiii

LIST OF ABBREVIATIONS xiv

PUBLICATIONS AND CONFERENCES xvi

ABSTRACT xviii

CHAPTER 1:

INTRODUCTION AND LITERATURE REVIEW 1

1.1 Introduction ....................................................................................................... 1

1.2 Objectives of the study...................................................................................... 2

1.3 Bioethanol as biofuel ........................................................................................ 3

1.4 Major bioethanol feedstock from sugar and starchy crops ............................... 6

1.5 Non-food sugar feedstocks for bioethanol production ...................................... 7

1.6 Liquid non-food sugar feedstock for bioethanol production ............................ 9

1.6.1 Sweet sorghum stalk juice .................................................................... 10

v
 1.6.2 Molasses ............................................................................................... 11

1.7 Potential of oil palm frond juice as non-food sugar feedstock for

bioethanol production ..................................................................................... 14

1.7.1 Physical characteristics of oil palm frond juice .................................... 17

1.7.2 Current utilisation of oil palm frond ..................................................... 18

1.8 Bioethanol processes ....................................................................................... 19

1.9 Treatment and storage of liquid feedstocks for bioethanol production .......... 22

CHAPTER 2:

OIL PALM FROND JUICE AS A NOVEL AND COMPLETE

NON-FOOD MEDIUM FOR BIOETHANOL FERMENTATION 26

2.1 Introduction ..................................................................................................... 26

2.2 Materials and methods .................................................................................... 27

2.2.1 Oil palm frond and sugarcane juices..................................................... 28

2.2.2 Bioethanol fermentation using OPF and sugarcane juices ................... 29

2.2.3 Effect of heat sterilisation ..................................................................... 30

2.2.4 Effect of OPF juice supplemented with yeast extract and

peptone on bioethanol production ........................................................ 30

2.2.5 Analytical methods ............................................................................... 31

2.2.6 Calculation ............................................................................................ 32

2.3 Results and discussion .................................................................................... 32

vi
 2.3.1 Nutrient composition of OPF and sugarcane juices ............................. 32

2.3.2 Bioethanol production from fresh OPF and sugarcane juices .............. 36

2.3.3 Sugars utilisation and microbial growth profile in OPF and

sugarcane juices .................................................................................... 39

2.3.4 Effect of OPF juice sterilisation on bioethanol production .................. 42

2.3.5 Effect of nitrogen source supplementation on bioethanol

fermentation .......................................................................................... 45

2.4 Conclusion ...................................................................................................... 49

CHAPTER 3:

EFFECTS OF OIL PALM FROND JUICE CONCENTRATION AND

MILD TEMPERATURE STORAGE ON GLUCOSE CONTENT FOR

BIOETHANOL PRODUCTION 50

3.1 Introduction ..................................................................................................... 50

3.2 Materials and methods .................................................................................... 51

3.2.1 Oil palm frond....................................................................................... 52

3.2.2 OPF juice concentration and storage .................................................... 53

3.2.3 Oil palm frond juice analyses ............................................................... 53

3.2.4 Determination of total sugars................................................................ 54

3.2.5 Bacterial counts .................................................................................... 54

3.2.6 Bioethanol fermentation ....................................................................... 55

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3.3 Results and discussion .................................................................................... 55

3.3.1 Effect of OPF juice concentration on glucose concentration,

density and water activity ..................................................................... 55

3.3.2 Effect of OPF juice concentration and storage temperature on

glucose degradation profile................................................................... 58

3.3.3 Bacterial counts and pH profile of OPF juice during storage ............... 65

3.3.4 Microbial community in oil palm plantations ...................................... 69

3.3.5 Potential of bioethanol production from the stored OPF juice ............. 70

3.4 Conclusion ...................................................................................................... 73

CHAPTER 4:

EFFICIENT FERMENTABLE SUGARS PRODUCTION AND

BIOETHANOL PRODUCTION FROM OIL PALM FROND BY

INTEGRATED TECHNOLOGY APPROACH TO AN EXISTING

PALM OIL MILL 74

4.1 Introduction ..................................................................................................... 74

4.2 Materials and methods .................................................................................... 75

4.2.1 Process description ............................................................................... 77

4.2.2 Fermentable sugar production cost ....................................................... 82

4.2.3 Bioethanol production cost ................................................................... 85

4.3 Results and discussion .................................................................................... 86

viii
 4.3.1 Energy cogeneration and utilisation at palm oil mill and

biorefinery plant .................................................................................... 86

4.3.2 Fermentable sugar production cost ....................................................... 91

4.3.3 Production cost of bioethanol production from OPF juice ................... 96

4.3.4 Future direction of bioethanol generation in Malaysia ....................... 101

4.4 Conclusion .................................................................................................... 106

CHAPTER 5:

CONCLUDING REMARKS AND SUGGESTIONS FOR FUTURE

RESEARCH 107

References 110

ix
 LIST OF FIGURES

FIGURE PAGE

1.1. Schematic pretreatment of lignocellulosic material. 9

1.2. The morphology of sweet sorghum tree. (a) Sweet sorghum tree,

(b) sweet sorghum grain and (c) sweet sorghum stalks are pressed

for juice 11

1.3. Processing flow diagram of sugar beet. 12

1.4. Direct production of white sugar from sugarcane juice or sugar 13

1.5. Percentage of solid oil palm biomass distribution. Oil palm frond (OPF),

mesocarp fibre (MF), oil palm trunk (OPT), empty fruit bunch (EFB)

and palm kernel (PKS). 14

1.6. Solid biomass distribution at oil palm plantation and palm oil mill. 16

1.7. Process flow of OPF juice production, (a) oil palm tree,

(b) petiole part of OPF and (c) OPF juice. 18

1.8. Flowchart with the main raw materials and processes used for ethanol

production. 21

1.9. Block flow diagram of the conventional sugarcane juice preparation

and treatment for bioethanol production. 24

2.1. Diagram of overall experimental outline 28

2.2. Process flow diagram of bioethanol production from OPF juice 29

2.3. Bioethanol production during batch fermentation by S. cerevisiae

(Bakers yeast) in fresh OPF juice, fresh sugarcane juice and control

medium. 37

x
2.4. Sugars utilisation by Saccharomyces cerevisiae during fermentation

in synthetic medium(a), OPF juice (b) and sugarcane juice (c). 41

2.5. Microbial growth profile during bioethanol production by

S.cerevisiae in OPF and sugarcane juices. 42

2.6. Bioethanol production, sugars consumption and pH profile in

(a) heat sterilized OPF juice and (b) Fresh OPF juice 44

2.7. Bioethanol production and sugars consumption in (a) Yeast extract and

peptone supplemented OPF juice, (b) Non-supplemented OPF juice

and (c) control medium. 47

3.1. Diagram of overall experimental outline for the effect of storage 52

3.2. Effect of OPF juice concentration (percentage of water removal) on

glucose concentration, juice density and water activity. 56

3.3. Glucose degradation profile in OPF juice stored at different

temperatures (a) 30°C, (b) 40°C, (c) 50°C and (d) 60°C 59

3.4. HPLC chromatograms showing the change of sugar profile over time

at 50°C in (a) sucrose solution and (b) 50% concentrated juice 61

3.5. Hydrolysis of sucrose to glucose and fructose during 20 days of storage

at 50°C. 64

3.6. Bacterial growth in OPF juice stored at different temperatures

(a) 30°C, (b) 40°C, (c) 50°C and (d) 60°C. 66

4.1. Schematic diagram of the integrated OPF fermentable sugars and

biorefinery plant for the production of bioethanol to existing

palm oil mill (POM). Average distance from each POM to

biorefinery plant is 80 km radius. 77

xi
4.2. Schematic flow diagram of biorefinery concept for the production of

bioethanol and from fresh OPF. 78

4.3. Schematic diagram of integrated technology concept of renewable

sugars recovery from OPF at one of the POMs. 90

4.4. Overall mass balance for the production of fermentable sugars from

oil palm frond (OPF) from 6 palm oil mills and subsequently

bioethanol production at a centralised biorefinery plant. 94

xii
 LIST OF TABLES

TABLE PAGE

1.1. List of countries with the blending mandate of bioethanol to gasoline. 4

1.2. Comparison of bioethanol and gasoline 5

1.3. World's total production of fuel ethanol (billion litres) from year

2004 to 2013. 6

1.4. Lignocellulosic biomass sources with respective categories. 8

2.1. Nutrient composition of fresh OPF juice and sugarcane juice 35

2.2. Comparison of bioethanol production from different juices. 38

2.3. Comparison of bioethanol production from various renewable carbon

sources. 48

3.1. pH profile of various concentrated OPF juice stored at different

temperatures for 20 days 67

3.2. Comparison of parameters by freshly prepared 50% concentrated OPF

juice with stored juice at 50°C. 72

4.1. Estimated energy and utility requirement for bioethanol production

from OPF 88

4.2. Estimated total OPF processed and sugar produced at 6 palm oil mills

per year. 92

4.3. Cost estimation for renewable sugars production from 345,600 t/y

of oil palm frond (OPF) processed. 96

4.4. Production cost of bioethanol from OPF at centralized biorefinery plant. 98

4.5. Production cost of bioethanol from various feedstocks 100

4.6. Potential value of sugar and ethanol from OPF and EFB 103

xiii
 LIST OF ABBREVIATIONS

% Percent

°C degree celcius

aw water activity

C/Co final glucose concentration per initial glucose concentration

CFU colony forming unit

CPO crude palm oil

d day

EFB empty fruit bunch

FFB fresh fruit bunch

FGB first generation bioethanol

g gram

g/g gram per gram

g/l gram per litre

g/l.h gram per litre per hour

GWh Gigawatt hour

h hour

kg kilogram

kg/m3 kilogram per cubic metre

kJ/kg kilo joule per kilogram

KWh kilowatt hour

l litre

mbar milibar

MF mesocarp fibre

MIA Malaysian Innovation Agency

min minute

xiv
MJ mega joule

ml mililitre

mm milimetre

MPIC Ministry of Plantation Industries and Commodities

MPOB Malaysia Palm Oil Board

MPOC Malaysia Palm Oil Council

OPF oil palm frond

OPT oil palm trunk

PKS palm kernel shell

POM palm oil mill

ppm part per million

rpm rotational per minute

SGB second generation bioethanol

t/y tonne per year

USDA United States Department of Agriculture

v/v volume per volume

w/v weight per volume

w/w weight per weight

Y P/S yield of product per substrate

YPD yeast potato dextrose

xv
 PUBLICATIONS AND CONFERENCES

1. Abdullah, S.S.S., Shirai, Y., Bahrin, E.K., Hassan, M.A., 2015. Fresh oil palm

frond juice as a renewable, non-food, non-cellulosic and complete medium for

direct bioethanol production. Ind. Crops Prod., 63, pp. 357-361.

doi:10.1016/j.indcrop.2014.10.006 (IF: 3.208, Q2)

2. Zahari, M.A.K.M., Abdullah, S.S.S., Roslan, A.M., Ariffin, H., Shirai, Y.,
Hassan, M.A., 2014. Efficient utilization of oil palm frond for bio-based
products and biorefinery. J. Clean. Prod. 65, 252–260. (IF: 3.398, Q1)

3. Abdullah, S.S.S., Hassan, M.A., Shirai, Y. Oil palm frond juice as a novel and
complete non food medium for ethanol production, in: Bioenergy Korea
Conference 2013 International Symposium. Chonnam National University,
Gwanju, Korea, Jeju Island, South Korea, p. 411. 12 -14 November 2013.

4. Abdullah, S.S.S., Shirai, Y. and Hassan, M.A. Alternative storage method of

oil palm frond juice for ethanol production. Symposium of Applied
Engineering and Sciences (SAES 2013). Universiti Putra Malaysia. 30th
September – 1st October 2013.

5. Abdullah, S.S.S., Shirai, Y. and Hassan, M.A. Efficient bioethanol

production from oil palm frond juice by integrated technology approach to an
existing palm oil mill. Symposium of Applied Engineering and Sciences

xvi
 (SAES 2014). Kyushu Institute of Technology, Japan. 20th – 21st December
2014.

6. Abdullah, S.S.S., Shirai, Y., Bahrin, E.K., Hassan, M.A. Effects of oil palm
frond juice concentration and mild temperature storage on glucose content for
bioethanol production.(manuscript preparation for submission to Journal of
Bioresource Technology, IF: 5.039, Q1)

7. Abdullah, S.S.S., Shirai, Y.,Mohd Ali, A.A., Hassan, M.A, Ahmad. Efficient

fermentable sugars production and bioethanol production from oil palm frond

by integrated technology approach to an existing palm oil mill. (manuscript

preparation for submission to Journal of Bioresource Technology, IF: 5.039,

Q1)

xvii
 ABSTRACT

The growing interest in bioenergy and particularly in second generation bioethanol

(SGB) is a great challenge as the development of lignocellulose-related technologies

are not very well established in the world. Another major constraint is the relatively

higher cost of SGB, both in terms of investment costs and final energy costs. This

causes the commercialization of research findings on SGB faces stiff competition from

fossil fuels. Hence, this study was aimed to produce SGB but using a straight forward

technology of first generation bioethanol from sugarcane juice. A newly identified

lignocellulosic material having such characteristics is the fresh oil palm frond (OPF).

OPF is the largest biomass source in the palm oil industry contributing 61% of total

biomass. Fresh OPF juice can be readily obtained by just pressing the fresh OPF

petiole, similar to sugarcane juice. In the first chapter, a literature studies was carried

out to understand the gap in the research for liquid non-food feedstocks as fermentation

medium for bioethanol production, particularly in the treatment and storage of the

feedstock.

In the second chapter, the potential of fresh OPF juice as a complete non-food medium

for direct bioethanol production was evaluated. OPF juice contained sugars and other

nutrients such as nitrogen, magnesium, calcium, zinc, phosphorus and sulphur, making

it a potential medium for bioethanol fermentation. A promising yield of 0.38 g

bioethanol per g sugars consumed was obtained after 24 hours of fermentation of fresh

xviii
OPF juice without nutrient supplementation and without pH correction, which is

comparable to synthetic medium as well as the bioethanol yield from sugarcane juice

in the Brazilian bioethanol industry (0.40 g/g). Therefore, this study provides an

opportunity for the use of fresh OPF juice as a new renewable, non-food and non-

cellulosic feedstock for the bioethanol industry.

The major challenge of using liquid feedstock as a fermentation medium is rapid

degradation of sugars during storage. Therefore in the third chapter, the effect of OPF

juice concentration and mild temperature storage on glucose content were discussed.

The OPF juice was concentrated by evaporation of 30-70% (v/v) of water and stored

at different temperatures (30-60°C) for 20 days. Regardless of OPF juice

concentration, glucose content was declined rapidly (C/Cₒ = 80%) at 30 and 40°C,

while it remained stable at 50 and 60°C. Despite the high concentration of OPF juice

(70%) did not significantly reduced the aw (0.93), the microbial spoilage in the OPF

juice was inhibited when stored at 50°C and 60°C. Consequently, considering the

minimum size of storage container, 50% concentrated OPF juice and 50°C storage was

the alternative treatment condition of OPF juice prior to fermentation.

In order to determine the feasibility of bioethanol production from OPF at industrial

scale, the fourth chapter discuss the potential of developing a new approach of

integrating a biorefinery plant for bioethanol production to an existing palm oil mill

(POM). The concept proposed the production of fermentable sugars from OPF at six

neighbouring POMs before being transported to the nearest biorefinery plant for

bioethanol production which is located at one of the POMs. The production cost of

fermentable sugars was estimated at $72/t OPF, resulting 3 times of profit based on

xix
current market price. The biorefinery plant has the capacity of producing 73.7 million

litres of bioethanol per year from 345,600 tonnes fresh OPF petioles based on 51%

yield. Current POM was estimated to generate an excess steam amounting to 177,000

tonnes and 5.9 GWh of electricity per year which is sufficient to produce fermentable

sugars from OPF. However, minimum additional steam (9,000 t/y) required to produce

bioethanol can be obtained by exploiting the OPF fibre residue as biofuel for boiler.

The low production cost of bioethanol from OPF by integrated approach at $ 0.46 per

litre is similar to production cost of corn bioethanol and cheaper than the current SGB

cost. This finding suggests that an integrated approach is the most economically

feasible option to commercialize bioethanol production in the near future, provided

that the government make a move towards the commercialization by introducing a

policy on SGB.

xx
 CHAPTER 1:

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Bioethanol is widely recognised these days as a very promising alternative source of

energy due to many positive attributes. Bioethanol is usually used as transportation

fuel by blending with gasoline up to 25% to increase octane and fuel oxygen content,

which further helping to improve overall fuel combustion (McMillan, 1997). In

addition, the complete combustion of bioethanol produces only carbon dioxide and

water which later absorbed by the plant based renewable feedstock to complete the life

cycle. The development of environmental friendly bioethanol will go a long way in

protecting the next generation from the negative consequences of global warming (Tye

et al., 2011).

The first generation bioethanol (FGB) is produced from edible source mainly

sugarcane and corn. The technology of bioethanol production from sugarcane juice

has been commercialised for over three decades resulting the lowest production cost

worldwide compared to other sugar or starch-derived bioethanol (Wang et al., 2014).

However, FGB was criticized for their negative impact on world food security. In this

respect, second generation bioethanol (SGB) offers great promise to replace fossil

fuels. The SGB is derived from non-edible sources such as lignocellulosic biomass

which mainly comes from agricultural wastes. Besides eliminating the feud of food

1
and fuel supply, it can reduce the cost of sugar feedstock. Nevertheless, high cost of

lignocellulosic pretreatment technology and cellulolytic enzymes has resulting the

delay in the commercialisation of SGB.

1.2 Objectives of the study

Therefore, in this study we use non-edible source of feedstock and interestingly the

processing technology is simple and similar to FGB production. As the biggest

commodity planted in Malaysia, oil palm also generate huge amount of biomass of

which oil palm frond (OPF) contributes the largest portion. Although similar amount

of pruned OPF and harvested fresh fruit bunch (FFB) are obtained every year with

104.1 million tonnes (wet weight) but OPF still received less attention compared to

other biomass (MPOB, 2014). The main issues arisen from the usage of OPF as sugar

feedstock are the establishment of a collection system for OPF, which presently remain

in the plantations. Furthermore, the juice extracted from the fresh OPF petiole is rich

in sugars and nutritional content (Abdullah et al., 2013; Zahari et al., 2012), which

results in rapid degradation of sugars during storage.

Hence, this research was aimed to :

1. exploit the OPF juice as a renewable, non-food, non-cellulosic and complete

medium for direct bioethanol production.

2. investigate the effects of OPF juice concentration and mild temperature storage

on glucose content in OPF juice.

3. evaluate the feasibility of bioethanol commercialization from OPF by

introducing integrated technology approach of biorefinery plant for bioethanol

production to an existing palm oil mill (POM).

2
1.3 Bioethanol as biofuel

Biofuels are solid, liquid or gaseous fuels made from plant matter and residues, such

as agricultural crops, municipal wastes and agricultural and forestry by-products.

Liquid biofuels can be used as an alternative fuel for transport, as can other alternatives

such as liquid natural gas (LNG), compressed natural gas (CNG), liquefied petroleum

gas (LPG) and hydrogen (Balat and Balat, 2009). Solid fuels are generally in solid

form partially used for heating, cooking or electricity production such as wood chips,

biochar, wood pellets and briquettes.

To be a viable alternative biofuel, bioethanol must present a high net energy gain, have

ecological benefits, be economically competitive and able to be produced in large

scales without affecting the food provision (Šantek et al., 2010). Currently bioethanol

is the main biofuel used in the world and its use is increasingly widespread. The

worldwide prospects are the expansion of the production and consumption of ethanol

(Gupta and Verma, 2015; Mussatto et al., 2010). Bioethanol global production

worldwide keep on increasing from 31 billion litres in 2004 to 87.2 billion litres in

2013 (Gupta and Verma, 2015). With all of the new government programs in America,

Asia, and Europe in place, total global fuel bioethanol demand could grow to exceed

125 billion litres by 2020 (Balat and Balat, 2009). The ethanol blending mandate

implemented in various countries are summarised in Table 1.1.

3
Table 1.1. List of countries with the blending mandate of bioethanol to gasoline.

Country Bioethanol as biofuel References

Brazil 100% anhydrous ethanol and Solomon et al. (2007)
22 – 26% mixture of ethanol to Prasad et al. (2007)
gasoline

United States Mixture of 85% or 10% ethanol to Mussatto et al.

gasoline (2010)

India Mandatory blending of 5% ethanol to Sorda et al. (2010)

gasoline and planning to increase to
10%

Sweeden 5% blending of ethanol to gasoline Soccol et al. (2010)

Canada Blending of up to 10% ethanol to Soccol et al. (2010)

gasoline

China Blending of up to 10% ethanol to Soccol et al. (2010)

gasoline

Thailand E10 (10%), E20 (20%) and E85 (85%) Silalertruksa and
ethanol blend to gasoline Gheewala (2009)

Germany E10 (10%) ethanol mixed to gasoline Spencer (2010)

Japan 3% of ethanol introduced to gasoline Orellana and Neto

(2006)

The Malaysian market for bioethanol is potentially much larger than the market for

biodiesel, because a much larger proportion of the vehicle fleet runs on gasoline (Tye

et al., 2011). Moreover, Wiloso et al. (2012) have found from life cycle assessment

(LCA) that second generation bioethanol performs better than fossil fuel at least for

the two most studied impact categories, net energy output and global warming. Table

1.2 summarized the advantages and disadvantages of both bioethanol and gasoline as

transport fuel.

4
Table 1.2. Comparison of bioethanol and gasoline
Bioethanol Gasoline/petrol
Advantages: Advantages:
 Renewable biofuel as it is derived  It is less corrosive on engines,
from renewable sources of though more recently non-
feedstock. corrosive materials to alcohol have
 Help in reducing CO2 build up in been designed.
two important ways: by displacing  It provides more energy per unit of
the use of fossil fuels, and by volume, thus allowing for smaller
recycling the CO2 that is released fuel tanks.
when it is combusted as fuel.  It has a lower ignition temperature,
 The combustion of ethanol is cleaner and therefore leads to easier starts
(because it contains oxygen). in the winter.
Consequently, the emission of toxic
substances is lower.
 Bioethanol produces less CO, and
therefore lowers greenhouse gas
emissions.
 Contain very low sulfur and nitrogen
level.
 Bioethanol has a higher octane
number, broader flammability
limits, higher flame speeds.
 Can be used as petrol
additive/substitute.

Disadvantages: Disadvantages:
 Bioethanol has lower energy density  Non renewable fuel - petroleum
than gasoline (bioethanol has 66% of derived.
the energy that gasoline has).  Petroleum has a lower octane
 Its corrosiveness, low flame number
luminosity and lower vapour  It produces more dangerous and
pressuremaking cold starts difficult. threatening pollutants, thus has
 Bioethanol can miscible with water higher toxicity.
making it toxicity to ecosystems.  It is more likely to explode and
 Bioethanol produces more burn accidentally.
aldehydes and may lead to increases  It is more threatening to the
in nitrogen monoxide (NO). environment if spilled or leaked.
 It leaves a residue gum on
surfaces where it is stored, and
the fuel leaves carbon deposits in
combustion chambers.

5
  It requires extensive pipeline
networks, and incredibly risky,
expensive exploration and
development.

Source: Demirbas (2008); Hira and de Oliveira (2009)

1.4 Major bioethanol feedstock from sugar and starchy crops

Bioethanol production from sugar crops such as sugarcane and sugar beet account for

about 40% of the total bioethanol produced and nearly 60% corresponding to starch

crops (Mussatto et al., 2010). Data in Table 1.3 shows the distribution of bioethanol

worldwide and the feedstocks, respectively.

Table 1.3. World's total production of fuel ethanol (billion litres) from year 2004 to
2013.

Source: Adapted from Gupta and Verma (2015)

6
US is leading the bioethanol production worldwide accounted 58% of total production

using corn as the main feedstock. Brazil is the world second largest bioethanol

producer from sugarcane accounted for 29%. China is the third largest bioethanol

producer in the world using sugarcane, cassava and yams, while the European Union

by wheat and sugar beet. It can be seen that most of the countries are relying on sugar

and starchy crops as bioethanol feedstocks, where these crops impose problem of food

insecurity. Moreover, the drawback in producing bioethanol from sugar or starch is

that the feedstock tends to be expensive and demanded by other applications as well.

1.5 Non-food sugar feedstocks for bioethanol production

The ethical concerns about the use of food as bioethanol feedstocks have encouraged

research efforts to be more focused on the potential of inedible feedstock alternatives.

The best candidate is lignocellulosic biomass materials constitute a substantial

renewable substrate for bioethanol production that do not compete with food

production and animal feed. These cellulosic materials also contribute to

environmental sustainability (Demirbas, 2008). Furthermore, lignocellulosic biomass

can be supplied on a large-scale basis from different low-cost raw materials which can

be grouped into four categories. Wood residue is by far the largest current source of

biomass for energy production, followed by municipal solid waste, agriculture

residues and dedicated energy crops (Lin and Tanaka, 2006). Examples of the

respective lignocellulosic categories are summarized in Table 1.4.

However, lignocellulosic based feedstock is a recalcitrant material that requires an

intensive labour and high capital cost for processing as the main challenge is the

7
pretreatment of the lignocellulosics. The lignocellulosic complex is made up of a

matrix of cellulose and lignin bound by hemicellulose chains in which during the

pretreatment, this matrix should be broken in order to reduce the crystallinity degree

of the cellulose (Fig. 1.1). The aims of pretreatment is to remove lignin and

hemicellulose, to reduce crystalline cellulose and to increase in the porosity of the

material (Sánchez and Cardona, 2008). All these obstacles are yet to be resolved

resulting economic unfeasibility of the bioethanol production from lignocellulosic

biomass (Limayem and Ricke, 2012).

Table 1.4. Lignocellulosic biomass sources with respective categories.

Types of lignocellulosic feedstock
Municipal and industrial Agricultural
Wood residues Energy crops
solid waste residues
Hardwood Garbage household Corn stover Switchgrass
Softwood Processing papers Corn stalk
Sawdust Food-processing by- Wheat straw
Woodchips products Rice straw
Black liquors and pulps Sugarcane bagasse

8
Figure 1.1. Schematic pretreatment of lignocellulosic material.

Source: Haghighi Mood et al., (2013)

1.6 Liquid non-food sugar feedstock for bioethanol production

An alternative route to derive bioethanol from non-food sugar feedstock is by

eliminating the recalcitrant pretreatment step of the lignocellulosic complex by

squeezing the sugar juice from the stem. Similar to sugar cane, the juice can then serve

as feedstock for production of first generation bioethanol. There are some examples of

lignocellulosic materials which have this characteristics such as sweet sorghum stalk

juice, cassava waste, molasses and the newly identified sugar feedstock in this study

namely OPF juice.

9
1.6.1 Sweet sorghum stalk juice

Sorghum tree is a hardy grass looks like corn stalk grows up to 4 meters of height.

Stalk or stem of the tree is pressed for juice, while the seeds are for food (Fig. 1.2).

Sweet sorghum is an attractive feedstock for bioethanol production because of its high

fermentable sugars content and very high yield of green biomass (20 - 30 dry tons/ha),

low requirement for fertilizer, high efficiency in water usage (1/3 of sugarcane and 1/2

of corn), short growth period (120 - 150 days) and it is well adapted to diverse climate

and soil conditions. Studies on many aspects of bioethanol production from sweet

sorghum have been conducted during the past two decades (Wu et al., 2010). The juice

extracted from the fresh stem is usually contain approximately 16 - 18% fermentable

sugar composed of sucrose, glucose, and fructose and therefore, can be readily

fermented to alcohol (Jin et al., 2012). Technical challenges of using sweet sorghum

for biofuels are a short harvest period for highest sugar content and fast sugar

degradation during storage. At room temperature about 12-30% fermentable sugar can

be lost in 3 days and 40-50% in 1 week (Wu et al., 2010).

10
 (b)

(c)

(a)

Figure 1.2. The morphology of sweet sorghum tree. (a) Sweet sorghum tree, (b)
sweet sorghum grain and (c) sweet sorghum stalks are pressed for juice
Sources:
http://arkansasagnews.uark.edu/Sweet_Sorghum_Vert2.jpg
http://vivianreiss.com/wp-content/uploads/2010/10/sorghum-050-1024x768.jpg
http://i.ebayimg.com/00/s/MTIwMFgxNjAw/z/12AAAOxyOlhSze9A/$_35.JPG

1.6.2 Molasses

Molasses is the dark, sweet, syrupy byproducts of sugar manufacturing process usually

from sugar beet or sugarcane. Sugar beet crops are grown in most of the EU-25

countries, and yield substantially more bioethanol per hectare than wheat (EUBIA,

2012). The advantages with sugar beet are a lower cycle of crop production, higher

yield, and high tolerance of a wide range of climatic variations, low water and fertilizer

11
requirement (Balat et al., 2008). Sugar beet and intermediates from beet processing are

very good raw materials for bioethanol production due to their content of fermentable

sugars, which can be directly used for fermentation without any modification (Dodić

et al., 2012). The general process flow of sugar beet is shown in Fig. 1.3. Beet molasses

contain large portion of sucrose as compared to glucose and fructose. The high content

of fermentable sugars up to 54% sugars (Nigam, 1999) provides high ethanol

productivity (Hatano et al., 2009). In European countries, beet molasses are the most

utilized sucrose-containing feedstock (Cardona and Sánchez, 2007). Sugar beet pulp

and molasses, the two main by-products of the sugar industry, are produced in large

amounts annually. Both contain considerable amounts of carbohydrate. Beet pulp

contains cellulose, hemicellulose and pectin as its main constituents which can be

enzymatically degraded to obtain fermentable sugars.

Figure 1.3. Processing flow diagram of sugar beet.

Source:
http://www.nzdl.org/gsdl/collect/envl/archives/HASH01ee/651bfb78.dir/P30.gif

12
In Thailand, from the total national molasses production of about 3 million tonnes a

year, the surplus 30–35% is potentially available for the production of 0.8 million litres

bioethanol a day (Sriroth et al., 2003). Indian distilleries almost exclusively use

sugarcane molasses for ethanol production (Satyawali and Balakrishnan, 2008).

Molasses contains around 50% of sugar content that is fermented by yeast during the

ethanol conversion process.

Figure 1.4. Direct production of white sugar from sugarcane juice or sugar

Source: Rein et al. (2007)

Fig. 1.4 shows the process flow diagram of sugar production from sugarcane, where

molasses is high value byproduct. To produce a litre of ethanol, around 4 kg of

molasses is required. However, this could vary based on the sugar content in molasses

13
and conversion efficiency of the ethanol plant. About 37% of the molasses available

in Thailand is consumed for ethanol production (Silalertruksa and Gheewala, 2009).

1.7 Potential of oil palm frond juice as non-food sugar feedstock for
bioethanol production

As of June 2013, it is estimated that 5.1 million hectares of agricultural land was

planted with oil palm trees all over Malaysia (MPIC, 2013) with planting density

works out to 148 palms per hectare (Basiron, 2007). The oil palm tree has an average

productive life-span of about 25 to 30 years and produces fruit bunches from three

years of age after field planting. In each productive year, an oil palm tree may produce

between 8 to 12 bunches of fruit, with two fronds are pruned for every bunch of fresh

fruit harvested (Sime Darby, 2014). It is estimated that the same ratio of fresh oil palm

frond (OPF) petiole to fresh fruit bunch (FFB) produced yearly (MPOC, 2010). OPF

is the most abundant solid biomass of palm oil industry accounted for 61% as shown

in Fig. 1.5.

PKS
OPT EFB 5%
17% 8%
MF
9%

OPF
61%

Figure 1.5. Percentage of solid oil palm biomass distribution. Oil palm frond (OPF),
mesocarp fibre (MF), oil palm trunk (OPT), empty fruit bunch (EFB) and palm kernel
(PKS) (Zwart, 2013).
14
The volume of solid biomass from palm oil industry is projected to increase to 85–110

million dry tonnes by 2020 (MIA, 2013), in which 75% found in the plantations as

fronds and trunks, whilst only 25% is generated in the mills comprise of empty fruit

bunch (EFB), mesocarp fibre (MF) and palm kernel shell (PKS) during the extraction

of palm oil (Fig. 1.6).

15
 At oil palm plantation At palm oil mill

Oil palm frondb Palm kernel shelle

Fresh fruit bunchc

Mesocarp fiber

Oil Palm treesa

Oil palm trunkd

Empty fruit bunchf
Figure 1.6. Solid biomass distribution at oil palm plantation and palm oil mill.

16
a
http://archives.thestar.com.my/archives/2007/4/17/business/b_pg05palm.jpg

b
http://www.mpoc.org.my/images%5Cphoto%5Cshow_oil-palm-field-recycling-pruned-fronds.jpg

c
http://archives.thestar.com.my/archives/2012/9/6/business/p8-oilpalmfr.JPG

d
http://www.palmwood.com.my/images/wood-oil-palm-tree1.jpg

e
http://web.tradekorea.com/upload_file2/product/369/P00232369/cbe9caa5_9bd1074f_2f01_4d5b_a4
d7_67b94cbdc535.jpg

f
http://inovasibiomasa.blogspot.com/2013/06/pembriketan-tankos-sawit-untuk.html

1.7.1 Physical characteristics of oil palm frond juice

The oil palm frond is approximately 2-3 metres long and weighs about 10 kg (wet

weight) (MIA, 2013). It consists of the petiole and many long leaflets on either side of

the stem. The OPF petiole which is about 1 meter length from the oil palm trunk is cut

and transported to the palm oil mill for sugar juice extraction (Fig. 1.7). About half of

the OPF petiole capacity will be converted to OPF juice by simple pressing which is

considered as big potential for fermentation medium. The juice was found rich in

sugars (78.42 g/l)mainly glucose (73%) and some portion of sucrose (25%) and

fructose (2%) (Zahari et al., 2012). Furthermore, the OPF juice is rich in minerals and

nutrients which are essential for bacterial growth during fermentation(Abdullah et al.,

2015). Due to this characteristics, OPF juice has been identified as a fermentation

feedstock for poly(3-hydroxybutyrate) and bioethanol production (Zahari et al., 2014).

The remaining OPF part is left at the oil palm plantation was known to contain most

of the nutrients suitable as natural fertilizer as soil cover (MIA, 2013).

17
 Oil palm frond
(a) (b) (c)

Figure 1.7.Process flow of OPF juice production, (a) oil palm tree, (b) petiole part
of OPF and (c) OPF juice.

1.7.2 Current utilisation of oil palm frond

There is only one common practice to manage pruned OPF in the plantation area by

the oil palm settlers. The oil palm fronds are left rotting between the rows of palm

trees, mainly for soil conservation, erosion control and ultimately the long-term benefit

of nutrient recycling (Abu Hassan et al., 1994). In addition, OPF and trunks are used

for the manufacturing of pulp, paper and fibreboard in the wood based industry (Chew

and Bhatia, 2008). On the other hand, application of whole OPF as feed for ruminant

livestock has been the subject of research since 1991 (Islam, 1999; Khamseekhiew et

al., 2002; Zahari et al., 2002). However, the application of OPF as ruminant feed is not

very convenient due to its low feed quality resulting from high fibre content, low

nitrogen content and low digestibility which finally leads to low feeding intake of the

ruminant livestock. Nevertheless, due to its huge quantity and availability throughout

the year, OPF has been widely used as a roughage source and as a component in

ruminant feed with an ideal percentage for livestock. The optimum level of OPF

inclusion for ruminant feeding is only 30% (Kum and Zahari, 2011). Abu Hassan

(1996) reported that animal performance is affected when the content of OPF is more

than 60%. Other findings have shown the potential of sugar recovery from the

18
lignocellulosic fibre of OPF for second generation bioethanol (Goh et al., 2010a;

2010b).

1.8 Bioethanol processes

Basically, bioethanol production is usually performed in three steps: (1) obtainment of

a solution of fermentable sugars, (2) fermentation of sugars into bioethanol and (3)

bioethanol separation and purification, usually by distillation–rectification–

dehydration (Demirbas, 2005). The step before fermentation, to obtain fermentable

sugars, is the main difference between the bioethanol production processes from

simple sugar, starch or lignocellulosic material (Fig. 1.8). Sugar crops such as

sugarcane juice, sugar beet juice and sweet sorghum juice need only a milling process

for the extraction of sugars to fermentation, becoming a relatively simple process of

sugar transformation into ethanol. In this process, bioethanol can be fermented directly

from the juices without needing the step of hydrolysis. Generally, the process of

bioethanol production from sugarcane consists of preparing, milling of cane,

fermentation process and distilling–rectifying–dehydrating. Currently bioethanol

fermentation is carried out mainly by fed- batch processes with cell recycle, and a

small part is produced through multi-stage continuous fermentation with cell recycle

(Sánchez and Cardona, 2008).

In processes that use starch from grains like corn, saccharification is necessary before

fermentation (Fig 1.8). In this step, starch is gelatinized by cooking and submitted to

enzymatic hydrolysis to form glucose monomers, which can be fermented by

19
microorganisms. The processes of bioethanol production using starchy crops are

considered well established.

On the other hand, the four basic process steps in producing bioethanol from

lignocellulosic biomass are: (1) pretreatment to render cellulose and hemicellulose

more accessible to the subsequent steps. Pretreatment generally involves a mechanical

step to reduce the particle size, chemical pretreatment (diluted acid, alkaline, and

solvent extraction), and physical pretreatment (steam explosion) to make the biomass

more digestible; (2) acid or enzymatic hydrolysis to break down polysaccharides to

simple sugars; (3) fermentation of the sugars (hexoses and pentoses) to bioethanol

using microorganisms; (4) separating and concentrating the ethanol produced by

distillation–rectification–dehydration (Sánchez and Cardona, 2008).

Note from Fig. 1.8 the calculated bioethanol yield from corn is greater than that from

sugar cane and OPF juice because of the higher amount of fermentable sugars

(glucose) that may be released from the original starchy material. However, the annual

bioethanol yield from each hectare of cultivated corn is lower than that for sugar cane

(Sánchez and Cardona, 2008). On the other hand, the bioethanol yield derived from

lignocellulosics are higher from sugarcane and varies from 140 - 330 L

bioethanol/tonne biomass depend on type of lignocellulosics material. Promising

bioethanol yield was expected from OPF fibre at 190 L/tonne OPF due to the high

hollocellulose content (80%, w/w) and subjected to 95% of sugar conversion by wet

disc milling and cellulase treatment (Zahari et al., 2015). Obviously, major sugars in

OPF was obtained from the pressed OPF fibre, if combined with OPF juice will lead

to higher bioethanol yield and productivity.

20
 Ethanol yield Output/input
(L/tonne) energy ratio

70a 8.0b
26c NA
(OPF juice) (OPF juice)

370a 1.34-1.53b

140-330a 6.0b
190c NA
(OPF fibre) (OPF fibre)

Figure 1.8. Flowchart with the main raw materials and processes used for ethanol productiond.
a b
Sources: Sánchez and Cardona (2008); Berg (2004) ; c Zahari et al. (2014b); d Mussatto et al. (2010)

21
In assessing bioethanol’s energy performance, net energy value is basic key indicator

to identify whether bioethanol production and consumption results in a gain or loss of

energy. It weighs the energy content of bioethanol against the energy inputs in the fuel

production cycle (Nguyen et al., 2008). The energy ratio greater than 1 indicated that

the bioethanol feedstock is an energy efficient resource. Among the three types of

feedstock, sugarcane bioethanol gave the highest energy ratio (8.0) followed by

lignocellulosic materials (6.0) and corn (1.34-1.53). There is no report available on the

energy ratio of bioethanol from OPF. However, it is expected that the energy ratio

value is comparable to sugarcane bioethanol as there is no cost incurred on the

production of OPF.

1.9 Treatment and storage of liquid feedstocks for bioethanol production

One of the main issues in dealing with liquid fermentation feedstock is the storage as

the medium is easily deteriorated by the microbial growth. Identified processing areas

that need to be addressed include, but are not limited to: (1) stabilisation of the raw

juice; (2) clarification of the raw juice to make it suitable for concentration; and (3)

concentration of the juice into stable syrup for efficient transport, storage, and year-

round supply (Andrzejewski et al., 2013). The preheating sweet sorghum stalk juice

with both milk of lime and polyanionic flocculant was clarified the juice to a very

acceptable level in short time. Most importantly, no fermentable sugars (sucrose,

glucose and fructose) losses were observed during clarification with temperature in the

range of 80-85°C and only a slight decrease with mildly acidic pH (~6.3-6.5). Another

research done by Kumar et al. (2013) identified a suitable pasteurization temperature

that was capable of preserving the fermentable sugars in sweet sorghum stalk juice and

22
maintained the sugar profiles reasonably well at near room temperature, i.e.

pasteurization at 90°C followed by storage at 35°C. The storage shelf life of the juice

was extended up to 21 days and also enabled efficient bioconversion of the juice to

ethanol.

On the other hand, same consideration also being paid on sugar beet as fermentation

feedstock since it is harvested for a short period each year. Therefore, the storage and

preservation of fermentable sugars from sugar beets would be required for yearlong

operation of beet ethanol plants. Conventional storage of sugar beets for table sugar

production consists of piling the crop in open storage fields and freezing the beets by

forced ventilation, taking advantage of the cold winter air. Another option is to

concentrate beet juiceby multiple effect evaporators to a high sugar concentration to

reduce storage volume and inhibit microbial growth. Therefore, during the rest of the

year, the concentrated raw juice needs to be diluted before use (Vučurović et al., 2012).

However, the purification of raw beet juice is yet another energy-intensive processing

step and is not essential if the juice is to be used for ethanol production. Hence,Vargas-

Ramirez et al. (2013) have found that combinations of pH ≤ 3.5 or pH ≥ 9.5 with

refractometric dissolved solids (concentration) ≥64.5% were effective in preserving

up to 99% of fermentable sugars in stored raw thick juice.

Final example is the first generation bioethanol feedstock namely sugarcane. The

general processes involved in sugarcane juice production and treatment is summarized

in Fig. 1.9. Before entering the extraction system, a cleaning systemremoves excessive

amounts of soil, rocks and trash coming with the sugarcane. A juice extraction system

23
separates bagasse and juice by compressing the cane. Bagasse is used as fuel for the

cogeneration system, and the raw juice is sent to the processing system (Palacios-

Bereche et al., 2014).

Sugarcane

Cleaning Sand, dirt, metals

Extraction of sugars Sugarcane baggase

Juice treatment Mud

Clarified juice

Juice concentration

Juice sterilization

Fermentation

Figure 1.9. Block flow diagram of the conventional sugarcane juice preparation and
treatment for bioethanol production.

Sugarcane juice contains impurities, such as minerals,salts, acids, dirt and fibre,

besides water and sugars. In order to be efficiently used as a raw material for ethanol

production through fermentation, those impurities must be removed by submitting the

juice to physical and chemical treatments. Screens and hydrocyclones are used in the

24
physical treatment to remove fibre and dirt particle, whereas in a subsequent chemical

treatment, phosphoric acid is added to sugarcane juice, to increase juice phosphates

content and enhance impurities removal during settlement, followed by the first

heating operation in which juice temperature increases from 30 to 70°C. Pre-heated

juice receives lime and is mixed with a recycle stream containing the filtrate obtained

at the cake filter, being then heated up again to 105°C. Hot juice is then flashed to

remove air bubbles, and a flocculant polymer is added to the de-aired juice, which is

fed to the settler. In the settler impurities are removed from the juice and two streams

are obtained: mud, which contains the impurities, and clarified juice. Clarified juice

contains around 15wt.% diluted solids, so it must be concentrated before fermentation

in order to achieve an adequate ethanol content that allows reduction of energy

consumption during product purification steps. Concentration is carried out in a five-

stage multiple effect evaporator up to 65 wt.% sucrose. Juice is sterilized prior feeding

the fermentation reactor,in order to avoid contamination, which would decrease

fermentation yields. During sterilization juice is heated up to 130°C during about 30

min and then rapidly cooled down to fermentation temperature.

25
 CHAPTER 2:

OIL PALM FROND JUICE AS A NOVEL AND COMPLETE

NON-FOOD MEDIUM FOR BIOETHANOL FERMENTATION

2.1 Introduction

The alarming ethical concerns on food security of sugarcane and starchy crops as sugar

feedstock has urged the researchers to find alternative feedstock for bioethanol

production. The widely recognised renewable feedstocks available are the sugars

derived from lignocellulosic materials. However, ineffective pretreatment and high

cost of hydrolytic enzymes are the main issues that hindering the commercialisation

of bioethanol from this feedstock. Therefore, efforts for bioethanol production have

been focused on the potential of non-edible feedstocks enriched with sugars (Limayem

and Ricke, 2012) such as sweet sorghum stalk juice.

The palm oil industry generates abundant biomass throughout the year of which OPF

contributes the largest portion among the oil palm solid biomass (EFB, OPT, PKS and

MF). OPF currently receive less attention as the plantation owners believed that whole

OPF is important for nutrient recycling and soil conservation to the oil palm tree, hence

the current practise is to leave the OPF at the plantation. However, Zahari et al. (2012)

reported that only petiole or basal part of the OPF contains large amount of sugars and

can be exploited to produce value added products. Hence, apart from the

lignocellulosic route, a direct route to derive bioethanol from OPF is by fermenting the

squeezed sugar juice from OPF petiole, as a new, green and renewable energy

26
feedstock (MIA, 2013). The OPF juice could provide good nutritional content for

bacterial growth during fermentation due to the presence of high amount of sugars

(glucose, sucrose and fructose), minerals and nutrients. We previously reported that

sterile OPF juice with supplementation of other nutrients has potential to be used as

substrate for poly(3-hydroxybutyrate) (Zahari et al., 2014; Zahari et al., 2012).

To date, there is no other reports on the usage of OPF juice as fermentation feedstock

for bioethanol production. Therefore, the aim of this study was to assess the potential

of fresh OPF juice as a complete fermentation feedstock for bioethanol production

without sterilisation, without pH adjustment and without additional nutrients.

Comparison of bioethanol production was made with fresh sugarcane juice. This work

is expected to provide useful information to assist interested parties in evaluating the

potential development of first generation bioethanol facility from OPF juice.

2.2 Materials and methods

The overall experimental outline in this chapter is summarised in Fig. 2.1 whereby the

potential of bioethanol production from OPF juice was compared to the sugarcane

juice. The effect of heat sterilisation and nitrogen source supplementation was carried

out in the later steps.

27
 OPF petiole

pressing

OPF juice Nutrient

composition

comparison sugarcane juice

Fermentation

Bioethanol

Effect of heat sterilized OPF juice

Effect of yeast extract and peptone

supplementation

Figure 2.1. Diagram of overall experimental outline

2.2.1 Oil palm frond and sugarcane juices

Fresh OPF was obtained from oil palm trees planted at Taman Pertanian Universiti,

Universiti Putra Malaysia, Serdang, Selangor, Malaysia. The petiole or basal part was

collected, while the leaves part was left at the oil palm plantation as natural fertiliser

and soil cover. The petiole was pressed and squeezed by three roller hydraulic press

machine (No.1 Mini mill 7.5kWx 4P x 1/195, 415v x 50 Hz, Matsuo Co.Ltd.,

Kagoshima, Japan) to obtain the juice (Fig. 2.2). The juice was centrifuged at 15,000g,

4°C for 15 min to remove solid materials (Zahari et al., 2012). The OPF juice was

stored in -20°C freezer for further experiments. On the other hand, fresh sugarcane

28
juice was obtained from a stall in Seremban, Negeri Sembilan, Malaysia. The juice

was centrifuged and stored in a similar manner.

Saccharomyces cerevisiae

Oil palm frond

(OPF) petioles

Bioethanol

Oil palm tree Pressing Fresh OPF juice Fermentation

OPF fiber

Figure 2.2. Process flow diagram of bioethanol production from OPF juice

2.2.2 Bioethanol fermentation using OPF and sugarcane juices

The commercial bakers yeast, Saccharomyces cerevisiae (Mauripan Baking Industry,

Malaysia) was inoculated on yeast potato dextrose (YPD) agar, which consisted of

glucose (20 g/l), peptone (20 g/l), yeast extract (10 g/l) and technical agar (10 g/l). This

culture was incubated at 30°C for 24 h. A loopful of yeast was pre-cultured in medium

containing 5 g yeast extract and 20 g glucose per litre. The inoculum was cultivated at

30°C for 12 h before centrifuging at 8000 rpm for 5 min to obtain the cell pellet and

introduced into the production medium.

The production media comprised fresh OPF juice and sugarcane juice. Both media

were fermented separately to observe the ability to produce bioethanol in batch system,

without nutrient supplementation and sterilisation. The initial pH for OPF and

29
sugarcane juices were 4.84 and 5.03, respectively. The pH of the juices were not

corrected for fermentation. A control medium was prepared to mimic OPF juice by

using a mixture of commercial sugars comprising of glucose (48 g/l), sucrose (12 g/l),

fructose (8 g/l), with other nitrogenous and mineral components such as peptone (20

g/l), yeast extract (10 g/l), KH2PO4 (1.0 g/l), Mg SO4.7H2O (0.1 g/l), CaCl2.2H2O (0.1

g/l) and (NH4)2SO4 (1.5 g/l). The fermentation was conducted in 250 ml flasks at 30°C,

150 rpm for 48 h. Samples were withdrawn and centrifuged at 10,000 rpm for 5 min.

The obtained cell free supernatant was used for the determination of bioethanol

produced and sugars consumed by the yeast. The growth of cells in the production

medium was determined by total plate count method on YPD agar and nutrient agar.

2.2.3 Effect of heat sterilisation

The inoculum was prepared similarly as section 2.2.2. The production media

comprised of fresh OPF juice and heat sterilised OPF juice at 115°C for 5 minutes.

Both media were fermented to observe the ability to produce bioethanol in batch

system, without nutrient supplementation and pH correction. The fermentation was

conducted in 250 ml flasksat 30°C, 150 rpm for 48 h. Samples were withdrawn and

centrifuged at 10,000 rpm for 5 min. The cell free supernatant obtained was used for

the determination of bioethanol produced and sugarsconsumed by the yeast.

2.2.4 Effect of OPF juice supplemented with yeast extract and peptone on

bioethanol production

Effect of nitrogen supplementation was studied by using two different production

media: i) OPF juice supplemented with 4 g/l of peptone and yeast extract, respectively;

30
and ii) OPF juice without nitrogen supplementation. Control medium was prepared to

mimic supplemented OPF juice by using mixture of commercial sugars: glucose,

sucrose and fructose, at the same concentration as in OPF juice. The initial pH and

sugar content in all OPF juice medium were not adjusted. All the Erlenmeyer flasks

were cultivated on a rotary shaker (150 rpm) at 30°C for 48 h. Samples were withdrawn

at time intervals for analyses of ethanol, residual sugars and pH.

2.2.5 Analytical methods

The analysis for elemental constituents in the juices (carbon, nitrogen, sulphur) was

determined using CNHS analyser (LECO, CNHS932, USA) whereas macro and

micronutrients were determined using Inductively Coupled Plasma (ICP) (Perkin

Elmer, 7300 DV, USA) (Omar et al., 2011). Free amino acids and total amino acids

were analysed according to the method described in Official Journal of the European

Communities 19.9.98, L257/16. Sucrose, glucose, fructose and bioethanol in the OPF

and sugarcane juices were determined by high performance liquid chromatography

(HPLC) (Shimadzu LC-20A series, Japan) using the Shodex sugar Na+ (KS-802)

column (8.0x300 mm) with a refractive index detector operated at 80°C. The mobile

phase was 100% water at a flow rate of 0.6 ml/min. The components were identified

by comparing their retention times with those standards under analytical conditions

and quantified by external standard method. A pH meter was used to determine the pH

of both juices.

31
2.2.6 Calculation

Sugar utilisation (Eq. 2.1), bioethanol yield (Y) (Eq. 2.2), volumetric productivity of

bioethanol (Eq. 2.3) and fermentation efficiency (FE) (Eq. 2.4) were calculated using

the following equations as described by Laopaiboon et al. (2009):

Grams of original sugar - Grams of residual sugar

Sugar utilisation (%) = × 100
Grams of original sugar
Equation 2.1

Bioethanol concentration (g/l)

Bioethanol yield (Y, g/g) =
Total utilised sugar (glucose, sucrose and fructose) (g/l)
Equation 2.2

Maximum bioethanol concentration (g/l)

Volumetric productivity (g/l.h) =
Fermentation time (h)
Equation 2.3

Actual yield
Fermentation Efficiency (FE, %) = × 100
Theoretical yield
Equation 2.4

2.3 Results and discussion

2.3.1 Nutrient composition of OPF and sugarcane juices

Table 2.1 shows the chemical compositions of OPF and sugarcane juices. The free

sugars content in both OPF and sugarcane juice were mainly comprise of sucrose,

32
glucose and fructose.The total free sugars in OPF juice obtained from this study was

55 g/l, which is lower than the previous results reported by Roslan et al. (2014) and

Zahari et al. (2012) at 78 g/l and 63 g/l, respectively. However, the percentage of

glucose in the juice is not significantly different at 76% from the total free sugars. The

total sugars concentration (sucrose, glucose and fructose) in fresh sugarcane juice was

three times higher than fresh OPF juice. Furthermore, the amount of sucrose and

fructose present in sugarcane juice were 8 folds higher than OPF juice which

comprised of 75 g/l sucrose and 26 g/l fructose, respectively. Kim and Day (2010)

reported that 96 g/l sucrose could be obtained from sugarcane juice and another 1 g/l

was glucose and fructose, respectively. On the other hand, glucose content in the OPF

juice was 20% higher than sugarcane juice. The important criteria of fermentation

medium is the absence of inhibitor. Both OPF and sugarcane juices did not contain the

inhibitors.

The chemical composition in the OPF juice may be influenced by many factors. For

the case of sugarcane, available moisture, soil, fertilisers, supplementary

irrigation,diseases, and temperature are the factors that can affect the agronomic

properties of cane yield, sucrose content and content of fibrous components (Benjamin

et al., 2014). Moreover, studies on other types of energy feedstocks such as sweet

sorghum (Zhao et al., 2009), switchgrass (Kim et al., 2011) and winter triticale

(Kučerová, 2007) have shown that the chemical composition of the biomass can vary

depending on genotype, location, year, age of crop, harvest batch, environmental and

cultivation parameters. In case of oil palm industry, there is no report on the effect of

the above mentioned factors on the oil palm frond properties. However, oil palm

growth and yield depend to a large extent on the physical and climatic characteristic

33
of the environment in which the palm is established. Oil palm fruit production is

generally determined by frond production, sex ratio, the extent of floral abortion, the

degree of survival of flora after anthesis and bunch weight. The oil palm yield is

reduced when trees are exposed to stressful conditions which is the low moisture level

(Rizal and Tsan, 2008). There might be a correlation between plant age and maturity

of FFB and chemical composition of OPF petiole which need further investigation.

34
 Table 2.1.Nutrient composition of fresh OPF juice and sugarcane juice
Type of fermentation media
OPF juice
Components Unit Sugarcane
Zahari et Roslan et
This studya juice
al., (2012) al., (2014)
Sucrose 9.85±1.11 19.94 16.77 75.26±3.34
Glucose g/l 41.78±2.64 57.21 44.34 34.24±4.89
Fructose 3.27±0.24 1.26 1.37 25.98±3.66
TN 0.0097 0.8 0.0172
%,
TC 0.0230 39 NA 0.0680
w/wb
S 1.1 0.4 ND
K 1920.00 23000 93.05
Mg 290.35 5000 166.15
P 11.90 200 38.75
Ca 1217.00 29000 732.00
Cu 0.30 2 1.60
ppmb
Zn 0.50 9 NA 4.00
B 3.80 2 4.20
Si 18.50 NA 14.15
Fe 73.25 66 18.60
Mn 22.60 2 3.10
Total amino g/kg 2.86 0.174 NA
acids
a
Data obtained are average of triplicate samples
b
Data obtained are average of duplicate samples

TN - Total nitrogen

TC - Total carbon

ND - Not detected

NA - Data not available

35
2.3.2 Bioethanol production from fresh OPF and sugarcane juices

Fig. 2.3. shows the profile of bioethanol production by S. cerevisiae cultivated in fresh

OPF juice, sugarcane juice and synthetic medium as control. During the first 24 h of

fermentation, OPF juice was rapidly consumed compared to sugarcane juice, giving

0.38 g/gbioethanol yield per sugars consumed. In addition, a similar trend of

bioethanol production was observed in control medium, where the maximum yield of

bioethanol was reached at 24 h of fermentation. Although, the fermentation of

sugarcane juice was completed at 48 h based on sugar consumption, but the juice

demonstrated the same yield of bioethanol (0.38 g/g). These values are comparable to

the yield of bioethanol per sugars consumed in control medium (0.40 g/g) and as

reported for sugarcane juice (Ramos et al., 2013). The bioethanol volumetric

productivity in OPF juice of 0.78 g/l/h was slightly lower compared to sugarcane juice

and control medium. A possible reason for this phenomenon is due to lower nitrogen,

zinc and phosphorus contents in OPF juice as compared to sugarcane juice (Table 2.1).

The available nitrogen was consumed by the yeast for metabolism and growth in the

fermentation media. Nitrogen source plays a vital role in accelerating the rate of sugars

utilisation and ethanol productivity (Laopaiboon et al., 2009; Zahari et al., 2014). Yu

et al. (2009) increased the rate of ethanol production by adding 0.77 g phosphorus and

2.15 g nitrogen into 1 L of sweet sorghum stalk juice medium. Furthermore, the

presence of some macroelements (K, Mg and S) and microelements (Fe, Cu, Mn, Ca

and Si) in both juices also have contributed to the bioethanol fermentation (Tamunaidu

et al., 2013).

36
 60
control medium
50
Ethanol concentration (g/l) Fresh sugarcane juice
40

30

20

10

0
0 10 20 30 40 50
Fermentation time (h)

Figure 2.3. Bioethanol production during batch fermentation by S. cerevisiae

(Bakers yeast) in fresh OPF juice, fresh sugarcane juice and control medium.

Table 2.2 summarised the significant fermentation parameters in bioethanol

production by fresh OPF and sugarcane juice in comparison to synthetic medium and

other fermentation feedstocks. The results of this study exhibited that OPF juice has

potential as a complete, non-food medium for bioethanol fermentation due to

comparable bioethanol yield and fermentation efficiency to other fermentation

feedstock such as sugarcane juice, sugarbeet juice and sweet sorghum stalk juice

(Massoud and El-Razek, 2011; Pavlecic et al., 2010; Ramos et al., 2013; Wu et al.,

2010). However, the OPF juice could be concentrated in order to get higher yield and

productivity of bioethanol.

37
Table 2.2. Comparison of bioethanol production from different juices.
Maximum Bioethanol
Bioethanol yield
Fermentation bioethanol Fermentation volumetric
Media Yeast strain per consumed References
mode concentration, time (h) productivity,
sugars, Y(g/g)
P (g/l) (g/l.h)
Control S. cerevisiae
mediuma Batch (Bakers yeast) 27.57±0.88 24 1.15±0.04 0.40±0.04 This study

Oil palm frond S. cerevisiae

juicea, Batch (Bakers yeast) 18.67±1.60 24 0.78±0.07 0.38±0.03 This study

S. cerevisiae
Sugarcane
Batch (Bakers yeast) 54.22±4.67 48 1.13±0.10 0.38±0.01 This study
juicea,
S. cerevisiae
Sugarcane
(indigenous
juiceb Batch 67.00 24 2.79 0.40 Ramos et al., 2013
fruit wine)

Sweet sorghum S. cerevisiae

Massoud and El-
juicec Batch (dry yeast) 10.70 72 0.15 0.20
Razek, 2011
Sugar beet Batch S. cerevisiae
59.89 78 0.77 0.43 Pavlecic et al., 2010
juicec
S. cerevisiae
Sweet sorghum
Batch (dry yeast 72.43 72 1.01 0.48 Wu et al., 2010
juiced
Ethanol Red)
a
Data are average of triplicate experiments
b
Sterilised and without nutrient supplementation
c
Sterilised with nutrient supplementation
d
Fresh juice with nutrient supplementation and pH correction

38
2.3.3 Sugars utilisation and microbial growth profile in OPF and sugarcane

juices

The trend of sugars utilisation by S. cerevisiae is presented in Fig. 2.4, whereby the

yeast was capable of utilising all sugars (sucrose, glucose and fructose) in control

medium. A complete composition of nitrogenous and mineral components in the

synthetic medium has resulted in complete utilisation of sugars by the yeast at 24 h of

fermentation.On the other hand, glucose was completely utilised at 36 h of

fermentation in OPF juice (Fig. 2.4b) and a longer period (48 h) was taken by the yeast

to totally consume the glucose in sugarcane juice (Fig. 2.4c). It is believed that the

high concentration of sucrose present in sugarcane juice required longer time to be

converted to glucose and fructose by a perisplamic invertase at the yeast cell envelope

during the fermentation process (Walker, 1998). The enzymatic conversion of sucrose

and utilisation of glucose and fructose in both juices continued until 48 h of

fermentation. Similar observation was also reported by Atiyeh and Duvnjak (2001),

where ethanol and fructose were produced from sucrose media by mutant S. cerevisiae

ATCC 36858. The rates of sucrose and fructose utilisation were slower than glucose

and a small amount of the sugars were still present in both juices at the end of

fermentation (Fig. 2.4b, c). Generally in yeast nutrition, cells transport glucose

preferentially and thus resulted in the different residual sugars throughout the

fermentative process. Approximately 0.5% and 6.4% of total sugars were detected in

sugarcane juice and OPF juice, respectively, at the end of the fermentation time.

Laopaiboon et al. (2009) reported the amount of residual sugars remaining in the

fermentation broth was dependent on supplemented nitrogen sources. The percentage

of total sugar utilisation in both juices were presumed as good for bioethanol

production as there was no additional nitrogen source supplemented in both media.

39
Since both OPF and sugarcane juices were fermented directly without sterilisation, the

S. cerevisiae and bacterial growth profile during the fermentation period were recorded

and presented in Fig. 2.5. The initial bacterial cells number present in OPF juice and

sugarcane juice were 5.1 and 4.7 log CFU/ml, respectively. On the other hand

approximately 6 log CFU/ml of S. cerevisiae was introduced to both juices at the

beginning of fermentation process. As the fermentation process was continued, the

bacterial numbers in both OPF and sugarcane juices increased until 12 h of

fermentation to about 6.3 log CFU/ml, but reduced thereafter until completely absent

at 24 h of fermentation. On the other hand, the yeast cells of S. cerevisiae increased

where the log phase is started from 12 to 36 h (~8 log CFU/ml) and reach stationary

phase thereafter. The cell growth and product formation kinetics of the S. cerevisiae

were found to be growth associated, similar with the previous report (Bakar et al.,

1992). The bioethanol produced in both OPF and sugarcane juice increased and has

reached maximum bioethanol concentration at 36 h of fermentation (Fig. 2.3). This

result explained the reduction and finally absence of bacterial or contaminant cells in

both OPF and sugarcane juices from 12 h onwards. The increment of bioethanol

concentration in both juices has led to inhibition of bacterial growth and subsequently

reduced the risk of contamination during the fermentation process (Soccol et al., 2010).

40
 60
(a)

Suagrs concentration (gl-1)

50

40

30

20

10

0
0 12 24 36 48
Fermentation time (h)

(b) 50
Sugars concentration (gl-1)

40

30

20

10

0
0 12 24 36 48
Fermentation time (h)

(c) 80
Sugars concentration (gl-1)

70
60
50
40
30
20
10
0
0 12 24 36 48
Fermentation time (h)

sucrose glucose fructose

Figure 2.4. Sugars utilisation by Saccharomyces cerevisiae during fermentation in

synthetic medium(a), OPF juice (b) and sugarcane juice (c).

41
 9.00
8.00
7.00
6.00
Log (CFU/ml)

5.00 Bacteria in OPF juice

4.00 Bacteria in sugarcane juice

Yeast in OPF juice
3.00
Yeast in sugarcane juice
2.00
1.00
0.00
0 10 20 30 40 50
Fermentation time (h)

Figure 2.5. Microbial growth profile during bioethanol production by S.cerevisiae in

OPF and sugarcane juices.

2.3.4 Effect of OPF juice sterilisation on bioethanol production

Fig. 2.6 shows the bioethanol production by S. cerevisiae when cultivated in fresh and

heat sterilised OPF juice medium. The initial sugars concentration in the juice was 56

and 48 g/l for fresh and heat sterilised OPF juice, respectively mainly comprised of 78

- 80% of glucose. The minor amount was sucrose and fructose which account for 14%

and 6%, respectively. The trend of sugars consumption was similar in both media

where the sugars were maximally consumed at 24 h of fermentation. Low amounts of

sucrose and fructose were detected at the end of fermentation and this indicated that

the yeast did not completely utilise the sugars. In S. cerevisiae, glucose and fructose

are the preferred carbon source and are transported into the cell by facilitated diffusion

(Mendes-Ferreira et al., 2011), thus resulted in the different residual sugars

42
consumption throughout the fermentative process. However, the amount of residual

sugars remaining was also dependent on supplemented nitrogen sources (Laopaiboon

et al., 2009). Although approximately 0.1% of glucose was retained in both OPF media

at the end of fermentation, it is presumed that there was good glucose consumption for

ethanol production as there was no additional nitrogen source supplemented in both

OPF juice media. On the other hand, only 30 - 40% of fructose was consumed by the

yeast, suggesting that the sugar was fermented under nitrogen starvation or high

concentration of ethanol. The difference in glucose and fructose consumption rate

maybe due to the differential transport mechanism across the plasma membrane or

differences in the hexose phosphorylation occurring inside the cell (Tronchoni et al.,

2009).

The pH of the juice was not corrected where the initial pH was approximately 4.9 to

5.0 which was in the optimum range for yeast growth and ethanol production

(Laopaiboon et al., 2009). However, profiles of pH during ethanol fermentation were

monitored. The pH of both media slightly decreased to 4.1 after 16 h and remained

relatively constant afterwards. This might be due to carbon dioxide production by S.

cerevisiae during fermentation which was able to dissolve in fermentation broth,

converted to carbonic acid, thus changing to carbonate ion and proton. Hence, pH of

the fermentation broth was constant (Shen et al., 2004).

43
 (a)
60 6

ethanol concentration, g/l

Sugar concentration, g/l
50 5

40 4

pH
30 3

20 2

10 1

0 0
0 8 16 24 31 48
Fermentation period, h

(b)
60 6
ethanol concentration, g/l
Sugar concentration, g/l

50 5

40 4

pH
30 3

20 2

10 1

0 0
0 8 16 24 31 48

Fermentation period, h

Glucose sucrose fructose ethanol pH

Figure 2.6. Bioethanol production, sugars consumption and pH profile in (a) heat
sterilized OPF juice and (b) Fresh OPF juice

The fermentation was completed when maximum ethanol concentration was produced

at 31 h in both media. There was no difference of bioethanol yield between fresh and

heat sterilised OPF juice at 0.39 g bioethanol/g sugars consumed, similar with previous

result. However, the productivity of bioethanol using fresh OPF juice (QP = 0.90 g/l.h)

was higher compared to sterilized OPF juice (QP = 0.66 g/ l.h). The low bioethanol

44
productivity obtained from heat sterilized OPF juice was resulted from the low

nitrogen content in fresh OPF juice as compared to fresh OPF juice. The available

nitrogen was consumed by the yeast for metabolism and growth in the fermentation

media. These results are comparable to the trend of ethanol production from non-

supplemented nipa sap with average ethanol yield of 0.45 g/g (Tamunaidu et al., 2013).

Similar observation was reported by Kosugi et al. (2010) where theoretical ethanol

yield was obtained from fermentation of oil palm trunk sap without additional nutrient.

The results obtained from this findings suggested that heat sterilisation on fresh OPF

juice does not affected the bioethanol yield. However, supplementation of OPF juice

with nitrogen source would enhance both parameters in bioethanol production.

2.3.5 Effect of nitrogen source supplementation on bioethanol fermentation

In order to improve the yield and productivity of bioethanol production, effect of yeast

extract and peptone supplementation were examined in this section. The results are

shown in Fig. 2.7. Overall, sugars in OPF juicewere completely consumed by the yeast

at the end of fermentation period (Fig. 2.7a,b), including sucrose. Since sucrose is non-

reducing sugar unlike glucose and fructose, the observed condition can be explained

by thepresence of invertase that breaks down sucrose during the fermentation. This is

supported by previous studies which reported that S.cerevisiae has the ability to

produce invertase enzyme (Badotti et al., 2008; Dodić et al., 2009; Wu et al., 2010).

The presence of nitrogen source in OPF juice brought positiveeffect on ethanol

production, whereby higher ethanol concentration was recorded compared to that in

non-supplemented OPF juice. Maximum ethanol productionwas obtained at 24 h and

45
36 h of fermentation in nitrogen supplemented and non-supplemented OPF juices,

respectively. Slower ethanol production innon-supplemented OPF juice is attributed

to the little amount of nitrogen source. It has been reported that deficient of nitrogen

source during fermentation resulted in slow rate of sugars utilisation and ethanol

productivity (Laopaiboon et al., 2009). This explains the low ethanol production in

non-supplemented OPF juice. Overall, OPF juice supplemented with peptone and

yeast extractyielded 0.49 g ethanol/g sugars. This value is comparable to that of

ethanol produced from oil palm trunk sap (Kosugi et al., 2010) and other renewable

resources as reported previously (Table 2.3). The productivity of bioethanol using

nitrogen supplemented OPF juice was also enhanced to 1.04 g/l/h with 33%

improvement compared to non-supplemented OPF juice. The promising yield and

productivity of bioethanol obtained in this study suggests that OPF juice is suitable as

fermentation feedstock for ethanol production.

46
(a) 60

ethanol concentration, g/l

Sugar concentration, g/l
40

20

0
0 4 8 12 24 36 48
Time (h)
(b)
60
ethanol concentration, g/l
Sugar concentration, g/l

40

20

0
0 4 8 12 24 36 48
Time (h)

(c)
60
ethanol concentration, g/l
Sugar concentration, g/l

40

20

0
0 4 8 12 24 36 48
Time (h)

glucose sucrose fructose ethanol

Figure 2.7. Bioethanol production and sugars consumption in (a) Yeast extract and
peptone supplemented OPF juice, (b) Non-supplemented OPF juice and (c) control
medium.

47
Table 2.3. Comparison of bioethanol production from various renewable carbon sources.

Sugar concentration Ethanol yield,

Strain Substrate Yp/s (g/g) References
(g/l)
Saccharomyces cerevisiae Oil palm trunk sap 55 0.48 Kosugi et al. (2010)
Kyokai no.7

Saccharomyces cerevisiae Glucose from residual 80 0.48 Awg-Adeni et al. (2013)

(commercial Bakers yeast, starch of sago hampas
Mauripan)

Saccharomyces cerevisiae Sugar beet molasses 100 0.41 Razmovski and Vučurović
(strain DTN) (2012)

Saccharomyces cerevisiae Sugar beet thick juice 100 0.43 Razmovski and Vučurović
(strain DTN) (2012)

Saccharomyces cerevisiae Sweet sorghum stalk 110 0.39 Guigou et al. (2011)
(dry baking yeast, juice (M81)
Fleischmann, Montevideo,
Uruguay)

Saccharomyces cerevisiae OPF juice 53 0.49 This study

(commercial Bakers yeast,
Mauripan)

48
2.4 Conclusion

A large amount of unexploited OPF could serve as a new renewable feedstock for

bioethanol production. The high bioethanol yield obtained from OPF juice is

equivalent to sugarcane juice. Therefore, fresh OPF juice demonstrated as a complete

non-food fermentation medium for bioethanol production without nutrient

supplementation and pH correction.In addition, heat sterilization was not significantly

affected the bioethanol yield from OPF juice. However, supplementation of OPF juice

with nitrogen sources was able to improve the yield of bioethanol. The unnecessary

pretreatment and enzymatic saccharification of the OPF juice has promoted the juice

as an attractive bioethanol feedstock. These results could be a fundamental reference

for future pilot scale of first generation bioethanol production from OPF juice.

49
 CHAPTER 3:

EFFECTS OF OIL PALM FROND JUICE CONCENTRATION

AND MILD TEMPERATURE STORAGE ON GLUCOSE

CONTENT FOR BIOETHANOL PRODUCTION

3.1 Introduction

The juice extracted from fresh oil palm frond (OPF) petiole was recently identified as

promising fermentation medium for bioethanol and PHB production (Zahari et al.,

2014). The juice provides good nutritional contents for yeast and bacterial growth

during fermentation due to the presence of high amount of sugars such as glucose,

sucrose and fructose; minerals and nutrients (Zahari et al., 2012; Abdullah et al., 2014).

However, the main technical challenge of using OPF juice as fermentation medium is

the rapid degradation of sugar juice during storage. There are some methods which

normally practiced to overcome this issue for storing liquid sugar feedtocks. The most

common method used is bydirectly store the feedstock at low temperature (Wu et al.,

2010) but it is highly energy consuming. Other methods such as concentration, heat

sterilization and clarification of the juice (Andrzejewski et al., 2013; Billa et al., 1997;

Mamma et al., 1995) are employed as a juice pretreatment prior to storing at low

temperature. On the other hand, Vargas-Ramirez et al. (2013) found that fermentable

sugars in raw thick beet juice was preserved under controlled acidic and alkaline

condition in combination with concentration by mean of evaporation. However, all of

these methods require high cost, high energy consumption and tedious. Abdullah et al.

(2013) proposed on juice recovery and storage processes by using the excess steam

available from palm oil mill processing. Thus, no additional energy is required for OPF
50
juice recovery. Up to date, there are no reports on storage of liquid fermentation

feedstock at mild temperature. Therefore, this study evaluates an alternative technique

of preserving the main sugar for bioethanol production which is glucose by

evaporating the fresh OPF juice prior to storage at mild temperature.

3.2 Materials and methods

The overall experimental outline in this chapter is summarised in Fig. 3.1 whereby the

fresh OPF juice was subjected to evaporation prior to storage at mild temperature. The

samples were analysed at five days interval. The optimum storage condition which

was able to maintain the glucose content and pH of the juice was chosen as the storage

method for OPF juice prior to fermentation.

51
 OPF juice

Evaporation

30%, 50% and 70% water removal

Storage

30°C, 40°C, 50°C and 60°C for 20 days

Analysis of samples at 5 days interval:

 glucose content
 bacterial growth
 pH

Sample at the optimum storage condition

Bioethanol production

Figure 3.1. Diagram of overall experimental outline for the effect of storage

3.2.1 Oil palm frond

Fresh OPF was obtained from palm tree planted at Taman Pertanian Universiti,

Universiti Putra Malaysia, Serdang, Selangor, Malaysia. The petiole part was collected

and squeezed by three roller hydraulic press machine (No.1 Mini mill 7.5kWx 4P x

1/195, 415v x 50 Hz, Matsuo Co.Ltd., Kagoshima, Japan) to obtain the juice. The juice

was centrifuged at 15,000g at 4°C for 15 min to remove the solid materials (Zahari et

al., 2012). The solid free juice was stored in -20°C freezer for further experiments.

52
3.2.2 OPF juice concentration and storage

The solid free juice was concentrated by mean of evaporation using an evaporation

rotary system (Buchi, Switzerland). Evaporation was performed at 55°C under vacuum

(approximately 72 mbar). The juice was subjected to concentration by removing 30%,

50% and 70% (v/v) of water from the fresh juice and named as 30%, 50% and 70%

concentrated juice, respectively. Non-concentrated juice was used as a control.In order

to study the effect of storage temperature, all samples were stored in airtight container

at different storage temperature (30, 40, 50 and 60°C). The glucose content, pH and

bacterial loads were monitored for 20 days to evaluate the storage stability of the juice

at different temperatures. The samples were collected and stored at -20°C freezer for

further analysis.

3.2.3 Oil palm frond juice analyses

Sucrose, glucose and fructose in the OPF juice were determined using HPLC

(Shimadzu LC-20A series, Japan) using the Rezek RCM Monosaccharide Ca2+ (8%)

column (300 x 75 mm) with a refractive index detector operated at 80°C. The mobile

phase was 100% water at a flow rate of 0.6 ml/min. The components were identified

by comparing their retention times with those standards under analytical conditions

and quantified by external standard method. The glucose content was calculated using

eq. 3.1, where Co and C are the glucose concentration before and after storage,

respectively.

Glucose content, % = C⁄C × 100

o

(Equation 3.1)

53
A pH meter was used to determine the pH of OPF juice. The water activity of OPF

juice was determined according to APHA (2001) at 25°C (±0.2°C) using an electronic

dew-point water activity meter (Aqualab Series 3, USA), equipped with a temperature-

controlled system.

3.2.4 Determination of total sugars

The soluble sugar concentration in the OPF juice was measured by the phenol sulfuric

acid method using starch as the standard (Dubois et al., 1956). The diluted OPF juice

(1 ml) was mixed completely with 1 ml of 10% (w/v) phenol solution. The

concentrated sulfuric acid with 95% purity (5 ml) was added to the mixture and left at

room temperature for 30 minutes. The absorbance of the solution was read at 485nm

wavelength using UV-Vis spectrophotometer.

3.2.5 Bacterial counts

The stored OPF juice samples were serial diluted with sterile distilled water (10-2 to

10-4 dilution). Each diluted suspension was pipetted on to nutrient agar plates and were

then incubated at 30, 40, 50 and 60°C for 96 h, respectively based on OPF juice storage

temperature. Agar plates with colony numbers between 25 and 250 were chosen for

colony counting.

54
3.2.6 Bioethanol fermentation

Previous results obtained showed that the glucose content and pH of OPF juice were

stable when stored at 50°C. Therefore, this study was carried out to evaluate the ability

of OPF juice stored at 50°C to produce bioethanol. Fermentation was carried out in

250 ml Erlenmeyer flask containing 100 ml of 50% concentrated OPF juice which was

stored at 50°C for 20 days. For comparison, the freshly prepared 50% concentrated

OPF juice (without storage) was used. Both juices were not sterilized and were not

supplemented. The inoculum preparation, fermentation condition and analysis were

conducted in similar manner as procedure in section 2.2.2 at page 29.

3.3 Results and discussion

3.3.1 Effect of OPF juice concentration on glucose concentration, density and

water activity

Vacuum evaporation is a common technique to concentrate juice at lower temperature.

In this study, part of the water content in OPF juice was evaporated at 55°C leaving

the sugars in the OPF juice. As more water was evaporated, the OPF juice became

more concentrated. A linear relationship was exhibited between OPF juice

concentration (percentage of water removal) and glucose concentration as illustrated

in Fig. 3.2. The removal of 50% and 70% water portion from OPF juice led to the 2.1

and 2.8 folds increment of glucose concentration, respectively from non-concentrated

OPF juice (42.79 g/l).

55
A positive correlation was observed between density and OPF juice concentration

whereby the density of the juicewas slightly increased from989.33 to 1073.13 kg/m3

with the increment of OPF juice concentration from 0% to 70%, respectively.Based

on the results, a small increment of the density value was due to the removal of water

from the OPF juice by evaporation. The obsrved trend is similar to the cashew

(Azoubel et al., 2005) and pink guava (Zainal et al., 2000) juices where the juice

density was increased by enhancing the concentration of soluble solids or sugars. On

the other hand, the density of depectined and clarified peach juice was higher

compared to OPF juice which was reported between 1034.8 - 1292.5 kg/m3 (Ramos

and Ibarz, 1998). These parameters are important to estimate the viscosity and flow

characteristics of the juice as a fermentation medium.

140 1.0 1200

120
1000
0.8
Glucose concentration (g/l)

100
800
Water activity, aw

80
0.6 Density (kg/m3)

600

60
0.4
400
40

0.2
200
20

0 0.0 0
Control 30 50 70

Percentage of water removal (%, v/v)

Water activity (aw)

Density (kg/m3)
Glucose concentration (g/l)

Figure 3.2. Effect of OPF juice concentration (percentage of water removal) on

glucose concentration, juice density and water activity.

56
Water activity (aw) is a measure of water amount available for chemical reactionsand

microbial growth in food (Belitz et al., 2009; Jay, 2000). Low water activity foods with

awlower than 0.85 (Beuchat et al., 2013) have a robust effect on the microorganisms

growth and thus contributes to the long shelf life of a food product. Generally, high

awvalues were obtained in all concentrated OPF juices (30% - 70%) with no

significantdifference of aw values (0.93 - 0.94) as indicated in Fig. 3.2. These values

are comparable to concentrated orange juice which aw values ranged from 0.90 to 0.95

(Grant, 2004) and a slightly higher aw of some non-concentrated fruit juices ranging

from 0.97 - 1.00 (Gabriel, 2008). Maail et al. (2014) have shown that evaporation of

80% water from OPF juice gave similar aw (0.925), whilst lower aw was recorded at

0.836 when 95% water evaporation was performed. Generally, the minimum aw for

most bacteria to grow is 0.90 (Grant, 2004). The results obtained in this study indicated

that there is a high chance of microbial growth in concentrated OPF juice. However,

it depends on another factor which is temperature of storage. The combination of the

high valueof awwith optimum storage temperature of the juice will inducethe growth

of bacteria and fungus. On the other hand, combination of high aw with too low or high

temperature will slow down the microbial spoilage in the OPF juice. Despite the awof

OPF juice was high, it can be seen from the results in Fig. 3.6 where the bacterial

growth were inhibitedat higher temperature storage (50°C and 60°C).Similar

observation were reported byVaquera et al. (2014), Mohamed et al. (2012) and

Romero et al. (2007), where fungal growth rate increased with high aw and mesophilic

temperature but slow at low temperature (6°C and below). However, there is lack of

research on the effect of mild temperature (30°C to 60°C) storage on the high aw juice.

57
Therefore, aw is not a major factor to be considered when the OPF juice is stored at

temperature above 50°C.

3.3.2 Effect of OPF juice concentration and storage temperature on glucose

degradation profile

Fig. 3.3 shows the effect of storage temperature on glucose content in various

concentrated OPF juice. Regardless of juice concentration, the glucose content was

declined from day 1 until 20 days of storage at 30°C. The rate of degradation increased

in the following order: control > 30% > 50% > 70% (Fig. 3.3a). All juices were

completely degraded at 15 days of storage except for 70% concentrated juice with little

glucose remainedat 20 days of storage (C/Cₒ = 20%). The high sugar content in the

concentrated OPF juice allows a limited number of osmotolerant bacteria or yeast to

grow. Moreover, the growth rate of the microorganism was low in this condition

(Membre et al., 1999). A gradual degradation rate of glucose in OPF juice stored at

40°C is illustrated in Fig. 3.3b. The glucose in non-concentrated juice was depleted to

85.9% at 15 days of storage and remained stable thereafter. While the other

concentrated juices (30, 50 and 70%, v/v) have lost about 70% of glucose at the end

of storage period. These findings demonstrated that OPF juice cannot be stored at room

or ambient temperature in tropical country regardless of how concentrated the juice

are.

Interestingly, as presented in Fig. 3.3c and Fig. 3.3d, the glucose content in different

juice concentration were escalated during the first five days of storage and reduced a

little bit and remained stable thereafteruntil 20 days of storage when stored at 50°C

58
and 60°C. The increment of glucose content after storage was not resulted from water

evaporation as the juice was stored in airtight container. Furthermore, the weight lost

recorder after the storage was only 0.64% which is not significantly affected the sugar

content in OPF juice. Initially, the sucrose was hydrolysed to glucose and fructose

resulting the increment of glucose content during the first five days of storage.

30°C
40°C
120 0%
0% 120
Glucose content, C/Co (%)

Glucose content, C/Co (%)

100 30%
30%
100
80 50% 50%
80
70% 70%
60
60
40
40
20
20
0
0
0 5 10 15 20
0 5 10 15 20

Storage time (day) Storage time (day)

(a) (b)

50°C 60°C
160 160
Glucose content, C/Co (%)

Glucose content, C/Co (%)

140 140
120 120
100 100
80 80
60 60
0% 30% 0% 30%
40 40
50% 70% 50% 70%
20 20
0 0
0 5 10 15 20 0 5 10 15 20
Storage time (day) Storage time (day)

(c) (d)

Figure 3.3. Glucose degradation profile in OPF juice stored at different temperatures (a)
30°C, (b) 40°C, (c) 50°C and (d) 60°C

59
This assumption was verified by the HPLC chromatogram of sugars profile during 20

days of storage at 50°C (Fig. 3.4). The sucrose concentration was declined completely

at 20 days of storage in all OPF juices stored at 50°C. Basically, heat exposure on OPF

juice during storage enhanced sucrose inversion. A rising pattern of glucose content

was observed in loquat (Shao et al., 2012) and peach fruits (Lara et al., 2009) after

being exposed to heat. Panpae and his co-workers (2008) verified that the process of

sucrose inversion was strongly influenced by temperature and pH. Those research

works agrees well with our finding where the increment of temperature led to the

enhancement of sucrose inversion in OPF juice.

60
 (a)

(b)

Figure 3.4. HPLC chromatograms showing the change of sugar profile over time at
50°C in (a) sucrose solution and (b) 50% concentrated juice

However, the sucrose decomposition process is depended on two factors. Firstly, the

inversion of sucrose due to the combined effect of temperature and pH, and secondly,

the destruction of sucrose by enzymes (Van Der Pol and Alexander, 1955).

Single factor which is mild heat (50°C) storage exposed to sucrose solution (Fig. 3.4a)

resulted in low inversion rate of sucrose into glucose and fructose. On the contrary,

61
more sucrose in OPF juice was converted into glucose and fructose at the same storage

condition (Fig 3.4b). This finding suggests the presence of invertase enzyme in OPF

juice which enhanced the inversion rate of sucrose into glucose and fructose. The

invertase enzyme is believed originated from the OPF petiole tissue and diffuse into

the pressed juice. Chandra et al. (2012) have reported that there is positive correlation

between soluble acid invertase activity and stem elongation in sugarcane. The

invertases hydrolyse sucrose to glucose and fructose, and play a crucial role in the

control of metabolic fluxes, sucrose partitioning, and ultimately plant development and

crop productivity. The cytoplasmic neutral invertase is higher in older tissues and is

thus mainly involved in controlling sugar flux in mature storage tissues (Sachdeva et

al., 2003). Optimum pH and optimum temperature range of invertases from fresh cane

juice were 4.5–5.0 and 35–45°C, however invertases from juice of stale cane were

having optimum pH of 4.0–4.5 and optimum temperature 40–55°C. Invertases

identified from juice of stale canes were kinetically more efficient in comparison to

the enzymes identified from juice of fresh canes, as they were having higher Vmax /Km

values than invertases from fresh cane juice (Bhatia et al., 2012). In connection with

these findings and similar characteristics between OPF and sugarcane, it is suggested

that the presence of invertases enzyme in OPF juice together with temperature

exposure at 50°C enhanced the sucrose hydrolysis in OPF juice during the storage

period. Moreover, Bassetti et al. (2000) have reported that the free invertase enzyme

at pH 5.0 is stable up to 50°C for a period of 4 h. Further investigation is needed on

the presence of the enzymes in the fresh and stored OPF juices.

The sucrose is commonly degraded at high temperature more than 100°C as being

reported by Junior and Massaguer (2006) and Šimkovic et al. (2003). The rate constant

62
k was obtained by the standard integral method according to the following equation

(Plazl et al., 1995):

𝐶𝑠𝑢𝑐
𝑙𝑛 ( ) = −𝑘𝑡
𝐶0,𝑠𝑢𝑐

Equation 3.2

where Co, is the initial concentration of sucrose and t, is time of storage. The measured

rate constants of control sucrose solution and OPF juice are shown in Fig. 3.5. The

reaction rate was found to be first order kinetics with the rate constant (k) value in

sucrose solution was very low (0.014 day-1) as compared to k value in OPF juice (0.144

day-1) during storage at 50°C for 20 days. This result is in agreement with Van Der Pol

and Alexander (1955) whereby possible sucrose losses due to the temperature and pH

effect are small under normal operating conditions and can be neglected. The results

obtained in this study implies that the sucrose inversion only occur at mild temperature

in the presence of invertase enzyme.

63
 1

ln (Csuc/ Co, suc)

sucrose solution; k = 0.014 day-1
-1

-2

-3 OPF juice; k = 0.144 day-1

-4
0 5 10 15 20

Storage time (d)

Figure 3.5. Hydrolysis of sucrose to glucose and fructose during 20 days of

storage at 50°C.

At the same time, the formation of degradation products as well as Maillard reaction

products during the storage should be considered for further investigation.

Carbohydrates undergo different changes, like the caramelization and hydrolysis

during heat exposure (Matusek et al., 2008).

Overall, there was no glucose degradation in all concentrated OPF juice when stored

at 50°C and 60°C. However, storage at 50°C was chosen as an optimum storage

method due to less heat energy consumption.

64
3.3.3 Bacterial counts and pH profile of OPF juice during storage

The survival of microorganisms is depended on temperature and incubation period, as

well as the number of microorganisms present in the juice. An average of initial

bacteria present in various concentrated OPF juice (0-70%) was 2.20 x 104 CFU/ml, in

agreement with other finding at 2.8 x 104CFU/ml in 60 to 80% concentrated OPF juice

(Maail et al., 2014). The enumeration of bacteria was extensive in all OPF juices stored

at 30°C and 40°C corresponding with the glucose reduction as shown in Fig. 3.6a,b.

Regardless of water removal percentage (up to 70% v/v) from OPF juice, the

microorganismwas still survived when stored at mesophilic temperature. The results

obtained was comparable to microbial count in the 60-80% concentrated OPF juice

stored at 30°C (Maail et al., 2014). Despite the higher concentration of OPF juice (90-

95%), there were still microbial growth by one log increment.

However, the bacteria cell number dropped after 4 days of storage associated with the

reduction of pH values (Table 3.1) and this indicated that organic acids were produced

during the storage . These results were confirmed by the presence of lactic acid and

acetic acid at the end of storage period (data not shown). The results obtained

suggested that growth of bacteria was inhibited by low pH and also the inhibitory

substances generated by lactic acid bacteria such as organic acids, lactic acid, hydrogen

peroxide, diacetyl and bacteriocins (Wang et al., 2002). These results are in agreement

with other researchers (Justé et al., 2008; Hein et al., 2002) as they suggested that the

pH drop during sugar beet juice degradation is mainly due to lactic acid increment. On

the other hand, the total bacterial counts in raw sorghum juice was 1.9 × 107 CFU/ml,

increased by 2-logs over the next 4 h of storage, subsequently stabilised, then slightly

decreased by 144 h storage at room temperature ~25°C. While the pH values of the

65
raw sweet sorghum juice similarly remained stable up to 20 h storage then decreased

to pH 3.75 at 144 h (Eggleston et al., 2014).

30°C 40°C
9 0% 0%
30% 8
8 30%
Bacterial count,log(CFU/ml)

Bacterial count, log(CFU/ml)

50% 7
7 50%
70%
6 70%
6
5
5
4
4
3
3
2 2

1 1

0 0
0 10 20 0 5 10 15 20
Storage time (day) Storage time (day)

(a) (b)

50°C 60°C
6
5
Bacterial count, log(CFU/ml)

0% 30%
Bacterial count, log(CFU/ml)

5
50% 70% 4
4 0% 30%
3
3 50% 70%
2
2
1
1

0 0
0 5 10 15 20 0 5 10 15 20
Storage time (day) Storage time (day)

(c) (d)

Figure 3.6. Bacterial growth in OPF juice stored at different temperatures (a) 30°C,
(b) 40°C, (c) 50°C and (d) 60°C.

Despite dawdling growth of the bacteria, the glucose content in the juice was kept

declined until 15 days of storage at 30°C (Fig. 3.3a) due to consumption of the glucose

by the fungus and yeast survived in the juice. At the end of storage period (20 days),

66
the number of survived bacteria was in the range of 102 to 103CFU/ml. The amount of

bacteria might have been reduced if the storage period was prolonged. Mohaibes and

Heinonen-Tanski (2004) suggested that mesophilic condition requires up to 45 days in

order to destroy pathogens in farm slurry and food waste.

Table 3.1. pH profile of various concentrated OPF juice stored at different

temperatures for 20 days
pH at different storage temperaturesa
OPF juice
concentration 30°C 40°C 50°C 60°C
(%, v/v) b
Initial Final Initial Final Initial Final Initial Final

0 4.82 3.78 4.76 4.16 4.76 4.86 4.76 3.85

30 5.06 3.64 5.04 3.35 4.94 4.58 4.96 4.19

50 4.78 3.91 4.81 3.85 4.70 4.48 4.78 3.85

70 4.78 3.69 4.79 3.86 4.70 4.50 4.71 3.89

a
All values are average of triplicate samples
b
Equivalent to percentage of water removal

Generally, the number of survived microorganism gradually dropped over time when

stored at 50°C (Fig. 3.6c). The result is in agreement with Mohaibes and Heinonen-

Tanski (2004), where all pathogens were destroyed after 3 days of storage at this

temperature. After 6th day of storage, only thermophilic bacteria survived and slowly

proliferated and maintainedat 1x103 CFU/ml until the end of storage period. This

happened due to acidic condition (pH 4.7) of OPF juice which slow down the growth

of thermophilic bacteria. It is believed that the microbial activity was dormant since

the pH of all juices were maintained at pH around 4.70 to 4.50 from day 1 until day 20

indicated that there was no microbial activity during the storage (Table 3.1). There was

67
also no glucose consumption recorded from 6th day until the end of storage period (Fig.

3.3). The glucose is minimally consumed by the bacteria or only undergone minimum

degradation when stored at 50°C even thoug the number of bacteria presents in OPF

juice was 1x103 CFU/ml.

However when the OPF juice was stored at 60°C, all the bacteria were gone starting

from 2nd day onwards. All bacteria which initially present in the juice could not survive

at 60°C for a long time. The common thermo-acidophilic and spore-forming can

survive in fruit juice with low acidity was identified as Alicyclobacillus genus bacteria

which have optimum growth temperature from 45 to 55°C (Chen et al., 2006). The

result obtained confirmed that this potential microbe in OPF juice were inhibited when

exposed to storage temperature at 60°C. The pH of the OPF juice also dropped from

4.8 to 3.9 (average) by 20 days of storage (Table 3.1). Although there were no

microbial survived at this temperature, the pH drop might contributed by the Maillard

reaction products such as brown melanoidins, fulfural, acetic acid and formic acid

(Jönsson et al., 2013). The Maillard reaction is a series of chemical reactions between

amino and carbonyl compounds resulting in complex changes in biological and food

systems. The reaction may take place slowly at low temperatures around 30°C, but

proceeds rapidly at higher temperatures (Azhar, 1996).

Based on the criteria observed from this experiment, storage at 50°C was selected as

the optimum condition of OPF juice storage due to high glucose content preserved,

stable pH and minimum microbial survived during the 20 days of storage. The stored

juice at the predetermined optimum condition (50°C, 20 days) was further fermented

for ethanol production.

68
The findings suggest a new storage method of liquid fermentation feedstock. Hence,

this treatment (storage at 50°C) up to three weeks is proposed as an alternative

preservation method of fermentation feedstock for bioethanol production. The storage

period at this temperature might be prolonged and further research is needed to

evaluate the effect of sugars degradation as well as the important nutritional contents

for fermentation feedstock.

3.3.4 Microbial community in oil palm plantations

Generally oil palm are planted in tropical countries such as Malaysia, Indonesia,

Thailand, Nigeria, Columbia, Papua New Guinea, Honduras, Ecuador, Cote d'Ivoire

and Brazil. The ideal requirements for oil palm are:

a. Annual rainfall of 2000 mm or greater, evenly distributed without a marked

dry season, and preferably at least 100 mm each month.

b. A mean maximum temperature of about 29-30°C and a mean minimum

temperature of about 22-24°C.

c. Sunshine of 5-7 h/day in all months and solar radiation of 15 MJ/m2 per day.

The oil palm thrives under Malaysia’s tropical climate which is marked by all-year-

round temperatures ranging from 25 to 30°C and evenly distributed rainfall of 2000

mm per year. Tropical countries that experience several months of drought season

drastically reduced yield of FFB (Rizal and Tsan, 2008). The tropical climate is

considered as the best growth condition of many microorganisms especially

mesophiles. The microbial communities present in the oil palm plantation are generally

69
dependent on the environmental condition as well as the type of soil. Moreover, the

variety of microbial communities in soil is also influenced by the types of fertilizer

used by the oil palm tree. Application of EFB in oil palm estates is practiced to enrich

soil organic matter and improve water availability. Situmorang et al. (2014) found that

application of EFB has increased the soil bacterial biodiversity especially some

beneficial genera involved in soil fertility. On the other hand, oil palm plantations

which used inorganic fertiliser have often been noted for an increase in soil

acidification and decrease in soil carbon stocks with increasing time under cultivation

(Pauli et al., 2014). Therefore it expected that less bacterial biodiversity be found in

the soil which applied 100% inorganic fertiliser.

It is believed that bacteria present in OPF juice are originated from the oil palm

plantations and enters the interior of the OPF petiole through cut ends and/ or any

wound sites of the petiole and survives at the expense of stored sugars. Moreover, the

rainfall also influence the microbial community around the oil palm especially when

the FFB is harvesting during the rainy day. As a result, the microbes are easily

absorbed onto the petiole cut and this lead to increasing total number of microbes in

the fresh OPF juice. All these hypotheses are a new phenomenon that require scientifc

investigation to be carried out in the future.

3.3.5 Potential of bioethanol production from the stored OPF juice

The ability of fresh and stored OPF juice to produce bioethanol were assessed. The

comparison of results by freshly prepared 50% concentrated OPF juice with the juice

stored at 50°C is presented in Table 3.2. The results obtained show that the stored OPF

70
juice at 50°C for 20 days gives approximately 15% more bioethanol concentration as

compared to the concentration of bioethanol produced by freshly prepared OPF juice.

The higher bioethanol concentration obtained is due to higher sugars (sucrose, glucose

and fructose) content in the stored juice which accounted for 126 g/l prior to

fermentation process as compared to freshly prepared OPF juice which is only 116 g/l.

Higher sugars in the stored OPF juice is believed to be derived from the hydrolysis of

polysaccharides present in the juice as the total sugar content by phenol sulfuric

method showed higher value than the total of sucrose, glucose and fructose. Simple

sugars, oligosaccharides, polysaccharides, and their derivatives, including the methyl

ethers with free or potentially free reducing groups, give an orange-yellow colour

when treated with phenol and concentrated sulfuric acid (Dubois et al., 1956). On the

other hand, the yield of bioethanol per sugar consumed of fresh and stored OPF juices

were 0.35 and 0.37, respectively.The high yield of bioethanol obtained without

sterilization, exclusion of nutrient supplementation and without pH adjustmentshowed

that theglucose content in non-sterilized OPF juice stored at 50°C was preserved and

safe to be used as fermentation medium for bioethanol production.

71
Table 3.2. Comparison of parameters by freshly prepared 50% concentrated OPF juice
with stored juice at 50°C.

Parameters Fresh OPF juicea Stored at 50°Ca

Sucrose (g/l) 19.6±0.62 1.80±0.10

Glucose (g/l) 89.83±4.51 99.73±12.08

Fructose (g/l) 6.93±0.38 16.63±2.02

Total sugars (sucrose, glucose 116.37±5.44 126.35±0.92

and fructose)

Total carbohydrate (g/l)b 146.80±3.12 151±1.81

Bioethanol produced (g/l)c 27.60±2.14 31.75±0.35

Bioethanol yield (g/g) c 0.35±0.02 0.37±0.01

Sugars consumption (%)c 67.57±2.00 67.63±2.01

Productivity (g/l.h) c 1.15 1.32

a
OPF juice was subjected to 50% concentration by evaporation
b
phenol sulfuric method
c
without nutrient supplementation

72
3.4 Conclusion

An alternative storage method is necessary to preserve fermentable sugars in OPF juice

which was qualified as bioethanol feedstock. Despite 70% water removal by

evaporation was not significantly reduced the aw of the OPF juice, storage at 50°C and

60°C managed to preserve the glucose content. However, evaporation of at least 50%

of water removal is possible to minimise the size of storage container. Storage at 50°C

was found to be promising preservation method as the glucose concentration was

stable during 20 days storage. Furthermore, the high yield of bioethanol obtained from

the stored OPF juice has granted the potential of the feedstock for bioethanol

production at bigger scale.

73
 CHAPTER 4:

EFFICIENT FERMENTABLE SUGARS PRODUCTION AND

BIOETHANOL PRODUCTION FROM OIL PALM FROND BY

INTEGRATED TECHNOLOGY APPROACH TO AN EXISTING

PALM OIL MILL

4.1 Introduction

Oil palm biomass are abundantly generated at oil palm plantation and palm oil mill

(POM). Apart from biomass generated at the POM, the largest volume of biomass is

produced at the plantations namely oil palm frond (OPF). It is estimated that the same

ratio of fresh OPF to FFB were produced annually. As big volumes of fresh OPF are

available daily during the harvesting of FFB, it must be processed immediately to

avoid moisture lost. A mechanism of OPF collection and transportation from oil palm

plantation to the POM was proposed using an additional cart to the current truck of

FFB transportation (Roslan, 2014). While a mechanism of fermentable sugars

production from OPF petiole is proposed in this study by using the excess energy

available at the POM.

Most of POMs manage to stand alone without relying on external energy for operation.

Current practice is by deploying a cogeneration approach to cater for steam and

electricity demands for the milling process (Nasrin et al., 2011). Most POM operation

74
utilise readily available biomass as boiler fuel at the mill which comprise of mesocarp

fibre and shell with the ratio from 90:10, 80:20 or 70:30, respectively (Vijaya et al.,

2008). The steam produced at the mill is normally far more sufficient to be used in the

POM and the excess steam produced is released to the atmosphere. As reported by

Chiew et al. (2011), there are 23.8 MJ of electricity and 24.2 MJ of steam which remain

unused in the palm oil process or released to the air after meeting the electricity and

steam demands of the POM. On the other hand, (Fonade, 1976) reported that 190 kg

of steam was exhausted for every tonne of FFB processed. POM management do not

put attention on this issue as they get the water and biomass fuel at no charge. Previous

report by Zahari et al. (2015) have proposed and shown the economic viability of

sugars from OPF as fermentation feedstock for the production of the bioplastic, poly(3-

hydroxybutyrate), P(3HB) within an integrated palm biomass biorefinery. Hence, by

adapting the similar concept, this study was aimed to assess the economic feasibility

of integrating a bioethanol production plant from OPF petioles to an existing POM.

This technology proposes the use of excess steam and electricity generated at the mills

for fermentable sugar production from OPF petiole before being used as fermentation

feedstock for bioethanol production. This approach would be economically efficient if

the excess energy at the POM could be tapped to meet the energy demand of the

proposed concept.

4.2 Materials and methods

In this study, it is assumed that fermentable sugars produced from OPF will be

transported to a centralized biorefinery plant for bioethanol production from at least

six POMs within a 80 km radius that have average capacity to process oil palm FFB

75
at 240,000 t/y/mill. There are 434 of POMs in all over Malaysia (MPOB, 2014).

Additionally, it is assumed that the biorefinery plant will be located at one of the six

mills to utilize the surplus energy from the POM. There are several criteria to be

considered in choosing the ideal POM for biorefinery plant to be attached to. The

location of the biorefinery plant should be easily connected to petroleum refinery

complex since the bioethanol is targeted as petrol additive. The POM has enough

excess energy obtained from palm oil processing lines to run the biorefinery plant.

Finally, there is back up of electricity store from the biogas plant nearby the POM if

there is necessity to have additional electricity supply. In 2011, there were only 12.9%

of the total POMs that have completed biogas plants installed in their mills while 3.8%

under construction and another 35.2% under planning (Chin et al., 2013). Figure 4.1

shows the proposed concept of integrated OPF renewable sugars and biorefinery plant

for the production of bioethanol.

76
Figure 4.1. Schematic diagram of the integrated OPF renewable sugars and
biorefinery plant for the production of bioethanol to existing palm oil mill (POM).
Average distance from each POM to biorefinery plant is 80 km radius.

4.2.1 Process description

A block flow diagram with the major processing steps and products of the biorefinery

is shown in Fig 4.2. The base case scenario considers the design of a biorefinery with

a processing capacity of 113,300 tonnes of sugars produced from 345,600 tonnes of

OPF petioles per year from six neighbouring POMs.

77
 Fermentable sugar production from OPF
OPF Petioles petiole at existing POMs

Cleaning

Treatment and
Pressing OPF fibre
saccharification

Juice

Sugars

Filtration and
concentration

Storage

Bioethanol
Fermentation production at
centralised
biorefinery plant

Distillation and
rectification

Bioethanol

Figure 4.2. Schematic flow diagram of biorefinery concept for the production of
bioethanol and from fresh OPF.

78
4.2.1.1 Fermentable sugars production from OPF

Basically the steps involved in sugar production from OPF is similar to sugarcane as

their physical characteristics are not significantly different (Albarelli et al., 2014). A

brief description of the steps involved at the fermentable sugars production process is

given below.

Cleaning of OPF petiole and extraction of fermentable sugar

Initially, OPF petiole about 1 m length went through a cleaning step to remove the

contaminants brought during harvest. It was considered a dry cleaning system using

air at this step. OPF was extracted through compressing sap system (Zahari et al.,

2015) to obtain the juice which contain sugars from the OPF. Apart from OPF juice,

pressed OPF fibre was also produced as a by-product of the OPF pressing process.

Pressed OPF fibre contains a substantial amount of carbohydrate, which is also useful

as fermentation feedstock (Zahari et al., 2014).

Juice treatment

The extracted OPF juice undergoes a physical treatment consisting of cyclones and

filters for removing solids and insoluble contaminants. The filtered OPF juice was

directed to the evaporation system to remove part of the water content. The

concentrated juice is finally stored in a storage container with temperature maintained

at 50°C prior to use as a fermentation medium for bioethanol production.

79
Fermentable sugar extraction from OPF fibre

The OPF pressed fibre undergoes a physical- mechanical pre-treatment before being

hydrolysed to glucose and xylose by saccharification using 20 FPU of cellulase.

Approximately 0.469 g and 0.298 g of maximum glucose and xylose concentrations,

respectively could be obtained per g of OPF petiole from the saccharification method

with 95% of holocellulose being converted into mixed sugars (Zahari et al., 2014). The

sugars are stored in storage container prior to use as a fermentation medium for

bioethanol production.

4.2.1.2 Bioethanol production from fermentable sugar

The renewable sugars from the storage tank at neighbouring POMs is transported to

the biorefinery plant for bioethanol production. There are only two basic steps

involved in bioethanol production from sugars, fermentation and ethanol recovery. A

brief description of the steps involved at the bioethanol production process is given

below.

Fermentation

The concentrated juice is sterilised prior to fermentation. The sterilisation is carried

out by an HTST-type treatment (high temperature short time). In this treatment, the

pressure of the concentrated juice is increased to 0.6 MPa, the mixture is heated to 403

K and at then cooled to 305 K (Palacios-Bereche et al., 2013). This step is

recommended due to the benefits of preservation of fermentable sugars and thermal

80
inactivation of bacterial contaminants (Junior and Massaguer, 2006). Since the major

sugars presented in the OPF renewable sugars are glucose and xylose, there are two

promising strategies to be performed in order to efficiently convert both sugars to

bioethanol. Taniguchi et al. (1997) have shown that co-culture system composed of

two fermentors and two microfiltration modules for efficient ethanol production from

a mixture of glucose and xylose by co-culture of Pichia stipitis and Saccharomyces

cerevisiae. When P. stipitis and S. cerevisiae were cultivated individually under

different oxygen supply conditions in the new co-culture system, the yield and

productivity of ethanol from a glucose and xylose mixture were higher than in single

culture of P. stipitis alone. However, this approach seems to be high in capital and

operational cost due to the complicated system. While another option would be more

economically feasible by using suitable recombinant strain of S. cerevisiae that can

convert both sugars to bioethanol simultaneously. Matsushika et al. (2009) has found

that the flocculent yeast strain MA-R4 had the highest ethanol production when

fermenting not only a mixture of glucose and xylose, but also mixed sugars in the

detoxified hydrolysate of wood chips. These results collectively suggest that yeast

MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol

production from mixed sugars present in lignocellulosic hydrolysates.

Distillation

After fermentation, the liquor, containing bioethanol, is taken to the distillation system

to remove the water. Fermented liquor is heated to a suitable temperature before

entering the first distillation column. Hydrous ethanol (95% ethanol and 5% water)

81
obtained from stripping and rectification stages can be blended effectively with

gasoline without phase separation (Palacios-Bereche et al., 2014).

4.2.2 Fermentable sugar production cost

As being reported by Zahari et al. (2014b), the total production cost of renewable

sugars from OPF are mainly contributed by raw material (OPF petiole) cost,

transportation, harvesting and collection cost of OPF from the oil palm plantation to

the mill, pre-processing cost and the cost of enzymes used for the saccharification of

OPF fibre. All costs used in this study were determined based on the current situation

in Malaysia and valued in US Dollar ($).

Harvesting and collection cost of oil palm frond

The fresh oil palm fronds are obtained during harvesting of fresh fruit bunch where

only petiole part is cut and collected while the leafy part are left as top soil replacement

and natural fertiliser. Different collection methods could be adopted to collect the

fronds using simple manual collection with a wheelbarrow, to collection with a buffalo

cart or motorised cart, to advanced mechanisation. The choice of collection method

for a specific plantation depends on the terrain (e.g., elevation, spacing of trees), labour

constraints and economies of scale (MIA, 2013). Depending on the collection method,

the estimated collection cost is range from $4.78–20.02 per dry tonne.As a basis for

calculation, the cost for harvesting and collection of OPF was estimated as $ 10/t OPF

(Zahari et al., 2015).

82
Transportation cost of oil palm frond to the palm oil mill

Current cost estimates, based on the density of the product and the distance

transported, range from $ 0.2 to 2.99 per kilometre per tonne (MIA, 2013). However,

these estimates are based on road transport by truck, and the actual transport cost could

be lower in regions where transport by train and/or barge is feasible, and where costs

can be shared with return cargo. The average transport cost in Malaysia is taken to be

$ 10/t for a 100 km distance as quoted by The Malaysian Transport Association (Zahari

et al., 2015). However, the cost might be less at a price of $ 7.14 per tonne of OPF

petiole (FELDA, personal interview, 2014). The attachment of additional cart to the

existing FFB transportation truck would reduce the cost of OPF petiole mobilization

(Roslan, 2014). As a basis for calculation, the transportation cost was estimated at $

10/t OPF processed for less than 100 km distance.

Price of fresh OPF petiole

For the purpose of assessing the economic feasibility of mobilisation, the volume of

oil palm biomass must be mobilised at an average cost of less than $ 47 per tonne at

mill gate (MIA, 2013). Subtracting $ 20 for the costs of harvesting, collection and

transportation of OPF, the farmers will earn about $ 27 per tonne of the OPF collected.

Pre-processing cost

Different biomass types can undergo different forms of pre-processing in order to

reduce the moisture content, reduce the weight or volume to be transported and/or in

83
preparation for a specific end use. Depending on the type of biomass and the extent of

pre-processing required, the estimated cost ranged from $ 5.08-164.35 per tonne for

mesocarp fibres, fronds, trunks and EFBs (MIA, 2013). With drying accounting for a

large proportion of pre-processing cost, it is likely that both plantations and

downstream industries will explore scenarios that do not require biomass to be dried.

Since fresh OPF was used in this case study, there will be no drying process required.

Therefore, the pre- processing cost was estimated at $ 5/t OPF (Zahari et al., 2015).

Cost of enzymes for saccharification

Currently, the estimated cost of enzymes is $ 0.04 to 0.07/kg glucose (Zahari et al.,

2015). As a basis for calculation, the cost of enzymes for saccharification of OPF fibre

to obtain fermentable sugars (glucose and xylose) is estimated at $ 20/t OPF fibre

processed (Lee and Ofori-Boateng, 2013).

Utility and electricity cost

Basically water, steam and electricity are the utilities required in production of sugars

from OPF. However, in this study the cost of the utility can be exempted with the

assumption that the required energy and utility would be obtained from surplus energy

at the existing palm oil mill.

Labour cost

Labour cost was estimated based on four operators at sugar production line and the

current salary rate is set by Suruhanjaya Perkhimatan Awam Malaysia (SPA, 2014).

84
4.2.3 Bioethanol production cost

Once mass and energy balances for the bioethanol production processes have been

estimated, the production cost can be determined. As a basis for calculation, the data

for cost estimation were taken from the first generation bioethanol from sugar cane,

which was reported by (Macrelli et al., 2012). In this case study, a flocculent S.

cerevisiae strains (MA-R4) is used for the production of bioethanol using fermentable

sugars from OPF due to ability of the yeast to maximally consumed glucose and xylose

corresponds to 82.4% of the theoretical yield (Matsushika et al., 2009). The resulting

beer from fermentation is sent to a distillation column followed by a rectification

column. The steam is extracted from excess steam turbines and back pressure receiver

(BPR) at POM processing and electricity generated in the POM (Wang et al., 2014).

In biorefinery concept, the plant is proposed to be constructed next to a POM where

the excess steam and electricity from the mill can be tapped and connected to the

bioethanol production plant. The project was assumed to operate for 10 years

considering fixed capital cost, production cost and revenues. The fixed capital cost

consists of equipment cost for bioethanol production plant including the costs for

installation, piping and instrumentation. The equipment cost of the current OPF

capacity was calculated using Williams method (Eq. 4.1) (Sánchez-Segado et al.,

2012) with sugarcane juice as a reference (Dias et al., 2010).

New capacity 0.6

New cost = Original cost ( )
Original capacity

Equation 4.1

85
Since the largest cost is process equipment, 10% was chosen as an average for

simplicity, i.e. linear full depreciation in ten years (Macrelli et al., 2012). Basically,

the production cost was estimated based on annual operating and maintenance cost

excluding the capital cost per annual bioethanol production capacity. On the other

hand, annual operation and maintenance cost includes salary for plant manager,

engineer and operators; energy and utility; repair and maintenance; fermentable sugars

from OPF; chemicals and yeast. Labour cost was estimated based on nine number of

workers as per current salary rate set by Suruhanjaya Perkhidmatan Awam Malaysia

(SPA, 2014). The repair and maintenance was taken to be 4% of fixed capital

investment. The energy and utility cost includes electricity and water resource which

were calculated based on current industrial tariff set by Tenaga Nasional Berhad and

Syarikat Air Negeri Sembilan.

4.3 Results and discussion

4.3.1 Energy cogeneration and utilisation at palm oil mill and biorefinery plant

The process flow diagram of renewable sugars production from OPF proposed at the

POM is illustrated in Fig. 4.3. The process starts from the oil palm plantation where

the FFB and OPF petioles are collected and transported to the POM. The FFB undergo

palm oil processing line starting from sterilization until CPO production. The steam

required for FFB processing is supplied by the cogeneration system at the POM which

comprise of boiler, turbine and BPR. Biomass fibres and shell obtained after oil

extraction is returned to the boiler as biofuel. Approximately 15% w/w of FFB

86
processed is ended as mesocarp fibre and 8% is palm kernel shell after the oil pressing

was fed into the boiler. It is estimated that for the annual mill capacity of 240,000

tonnes/yr of FFB processing, a total of 55,200 tonnes of mesocarp fibre and shell

(0.66:0.34) are burned in boiler. With the calorific value of 19,068 and 20,108 kJ/kg,

in mesocarp fibre and palm kernel shell, respectively (Chiew et al., 2011), it managed

to produce 299,325 tonnes of high pressure steam (20 bars). This amount of steam

would generate 9.98 GWh of electricity. Considering that 17 kWh is required to

process each tonne of FFB, approximately 4.08 GWh/yr of electricity would be

required. This shows that the excess and available power (5.9 GWh/yr) could be tapped

to run the proposed sugar recovery machines as well as bioethanol production plant.

On the other hand, approximately 510 kg of lower pressure steam from BPR is required

to process each tonne of FFB (Fonade, 1976; Vijaya et al., 2008). Overall, 176,925

tonnes of excess steam would be available each year at every mill. This source of

energy could also be tapped to run the fermentable sugar production from OPF, sugar

storage as well as ethanol recovery.

The power and steam requirement for juice extraction and recovery from OPF was

estimated based on sugarcane juice processing as a reference (Table 4.1 and Fig. 4.3).

The power needed to run the milling and extraction of OPF juice was approximately

1.04 GWh/year, assuming that 18 kWh is needed to process each tonne of OPF petiole.

It shows that no extra power would be required to run the juice extraction machine as

the turbine provide 5.9 GWh/yr of excess electricity. Concentrating process of the juice

by evaporator requires steam. Specific energy consumption for evaporation process is

assumed where 1 kg of steam is required per 1 kg of water evaporated (Ahmad et al.,

2003). Hence, only 26,000 tonnes of steam per year is needed to remove 90% water

87
from OPF juice to produce 2,880 t/y sugar syrup. Less amount of steam is required if

multiple effects evaporator system is applied for the concentration process. The

concentrated OPF juice with sugar concentration of approximately 800 g/l is combined

with the sugars recoverd from OPF pressed fibre (16,788 t/y) in storage tank with

temperature maintained at 50°C before being transported to biorefinery plant for

bioethanol production. Approximately 160 tonnes of steam required annually to

maintain this mild temperature storage, which the energy is also obtained from the

excess steam.

Table 4.1. Estimated energy and utility requirement for bioethanol production from
OPF*

Power demand for cane preparation and juice extraction 16-18 kWh/t cane a, b, c

Hence, electricity demand for OPF juice extraction 1.04 GWh/y

Steam demand for OPF juice evaporation d 26,000 t/ye

Steam demand for distillation of ethanol 4 kg/l ethanolf

Hence, total steam demand for distillation of ethanol 162,000 t/y g

Electricity demand for ethanol process 12 kWh/t cane h

Hence, electricity demand 4.15 GWh/y

Water usage 0.0106 m3/l ethanoli

*Calculation was adopted from sugarcane juice processing

a
Ensinas et al.(2007)
b
Dias et al. (2010)
c
Magalhaes (2010)
d
Single effect falling film evaporator for 90%, w/w of water removal from OPF juice at each POM
e
Specific energy consumption is assumed where 1 kg of steam is required per 1 kg of water evaporated.
f
Bizzo et al. (2014)
g
consider 15% ethanol is produced in fermentation broth, 2 distillation columns required
h
Palacios-Bereche et al.(2013)
I
BBI Biofuels Canada (2010)

88
As a result of this integrated approach, the high cost of energy consumption for sugar

recovery may be avoided and this help to reduce the total cost of bioethanol production

from OPF juice. In addition, the clean water obtained from the vapour condensate after

evaporator can be recycled to a water resource tank (26,000 t/y). It can be used for

other processes without treatment and hence reduce the cost of water supply and water

treatment.

However, the biorefinery plant attached to existing POM may require external energy

in order to complete the bioethanol production process. The estimated excess steam

and electricity available after the sugar recovery from OPF were 151,000 t/y and 4.86

GWh/y, respectively. The steam demand required for the current ethanol distillation

process is estimated at 4 kg for each litre of ethanol (Antonio Bizzo et al., 2014). With

the assumption that 85% of water should be removed from the fermentation broth,

about 160,000 tonnes of steam would be required to distillate the ethanol using two

distillation columns. Roughly, about 9,000 tonnes of additional steam must be supplied

to biorefinery plant in order to complete the bioethanol production. However, it is not

an issue as the additional steam can be obtained by feeding the OPF fibre residue to

the boiler to produce steam. With the calorific value of OPF fibre at 12,552 kJ/kg

(FAZA, 2014), it is estimated 42,100 tonnes of steam could be produced from 12,000

tonnes of pressed OPF fibre yearly. Moreover, the surplus of steam produced can be

converted to electricity and sold to Tenaga Nasional Berhad.

89
 FFB OPF petiole
FFB
240,000 t/y

Processing of Sterilization Oil palm plantations

crude palm oil
palm oil
(CPO)
48,000 t/y
Steam
Steam 64,800 t/y OPF petiole OPF fibre treatment and
57,600 t/y 2.75 bar 57,600 t/y saccharification tank
OPF fibre
BPR 28,800 OPF fibre
3.2 bar residue
12,012
Electricity Excess steam
4.1 GWh/y 176,925 t/y
Milling and juice extraction
Cushion
Turbine tank
260C, OPF Juice
18.5 bar 28,800 t/y
Generator
10 GWh/y Steam Steam;
299,325 t/y 26,000 t/y
300C, 20 bar
Evaporator Renewable
(90% w/w water removal) sugars
16,788 t/y
Shell and fibers Boiler
55,200 t/y
Recycle of clean
water 26,000 t/y
Fermentable
sugars
2,880 t/y

Storage tank,
Water tank 50°C
Electricity Steam;
1.04 GWh/y 160 t/y

proposed waste steam utilisation

Transportation to
proposed electricity utilisation
biorefinery plant
proposed clean water recycling

Crude palm oil processing line Sugars production line

Figure 4.3. Schematic diagram of integrated technology concept of fermentable sugars recovery from OPF at one of the POMs.

90
4.3.2 Fermentable sugar production cost

To evaluate the economic viability of OPF as fermentation feedstock for bioethanol

production, it is important to estimate the production cost of fermentable sugars from

OPF. As the transportation cost of OPF from plantation is one of the main cost for

sugar production, it is therefore proposed in this study to processed the OPF at the

nearest POM before being transported to the main biorefinery for bioethanol

production. In addition, this study also proposed only one biorefinery plant for

bioethanol production, where by the fermentable sugar from OPF is obtained from 6

neighbouring POMs. The total OPF processed in the POMs for fermentable sugars

production is estimated at 345,600 t/y (MPOB, 2014). This capacity is more than

reported by Zahari et al. (2014a) as the data in this study was based on the higher

average weight of OPF petiole (3 kg) obtained from the oil palm trees aged from 8 to

20 years old. Annually, it is estimated that 25 million tonnes of fresh OPF petioles can

be collected from the whole plantations in Malaysia. Data in Table 4.2 summarized

the estimated annual capacity of fermentable sugars produced from 6 POMs. Upon

concentration of the juice to remove 90% of the water content, the sugar obtained was

17,280 tonnes.

With specific gravity of the concentrated juice was 1100 kg/m3, therefore the volume

of 90% concentrated OPF juice obtained was 15,709,091 litres. For each litre of

concentrated OPF juice, it is estimated to contain 800 g of sugars (Zahari et al., 2012).

Hence, an amount of 12,570 tonnes sugars can be obtained from the juicy part only.

91
 Table 4.2. Estimated total OPF processed and sugar produced at 6 palm oil mills
per year. Calculation was made based on the current practice at the plantation for
every 1 FFB processed, 2 OPF will be pruned.
Capacity of the millsa 1.44x109 kg/y of FFB

processed

Average weight of FFBb 25 kg/FFB

Hence, amount of FFB processed 57.6x106 FFB/y

Average weight of OPFpetiolec 3 kg/OPF petiole

Total weight of OPF petiole generated 345,600 t/y

Amount of juice obtained (50% w/w) 172,800 t/y

Upon concentrating (90% water removal) 17,280 t concentrated juice

containing 800 g/l sugars

Amount of OPF petiole fibre obtained (50% w/w) 172,800 t/y

Amount of glucose extracted from OPF fibre 61,593 t

Amount of xylose extracted from OPF fibre 39,136 t

a
MPOB (2014)
b
Shamsudin et al., (2012)
c
Average of OPF petiole weight for the oil palm tree ages from 8 - 20 years

On the other hand, OPF fibre was found to contain 80.58% hollocellulose based on the

fibre weight. The extraction of sugars from OPF fibre was carried out by wet disc

milling followed by enzymatic saccharification to obtain glucose and xylose. Based

on Roslan (2014), maximum glucose and xylose concentrations of 0.469 g and 0.298

g, respectively per g of OPF petiole could be obtained from the saccharification

method with 95% of holocellulose being converted into mixed sugars. Therefore, from

92
172,800 tonnes of OPF fibre, the glucose and xylose obtained were 61,593 t and 39,

136 t, respectively. Hence the total sugars obtained from the whole OPF petiole was

113,299 tonnes at the biorefinery plant annually. These sugars are fermentable

feedstock for bioethanol production. The overall mass balance for the production of

fermentable sugars from OPF is presented in Fig 4.4.

93
 PROCESS
INPUT OUTPUT
PALM OIL MILL (POM) BIOREFINERY

72,071 t/y
OPF FIBRE
72,071 t/y RESIDUE

OPF PRESSED
PHYSICAL
FIBRE FOR
TREATMENT AND
BIOSUGARS
SACCHARIFICATION
PRODUCTION
100,729 t/y sugars
50% 100,729 t/y sugars
pressed based on
OPF fibre 80%holocellulose in
OPF, 95% conversion
OIL PALM to glucose and xylose FERMENTABLE BIOETHANOL 57,000 t/y
FRONDS MILLING SUGARS PRODUCTION BIOETHANOL
PLANT
(345,600 t/y) (113,983 t/y)
(51% yield)
50% OPF juice
Total OPF from 172,800 t/y
six (6) POMs (80g/l sugars) 17,280 t/y sugar syrup
12,570 t/y sugars
(800 g/l sugars)
OPF JUICE
TREATMENT CONCENTRATED
AND SUGAR JUICE
EVAPORATION

155,520 t/y

90% water removal CLEAN WATER

FOR
RECYCLING

Figure 4.4. Overall mass balance for the production of fermentable sugars from oil palm frond (OPF) from 6 palm oil mills and
subsequently bioethanol production at a centralised biorefinery plant (adapting from Zahari et al., (2014b).

94
Detailed calculation for the cost estimation of fermentable sugars production from

345,600 t/y of OPF is presented in Table 4.3. Major cost was contributed by the cost

of OPF (37.4%) followed by cost of enzyme for saccharification process (27.7%). As

the proposed concept should benefit the 3Ps (profit, people and planet), the abundant

OPF will benefit the whole farmers by giving additional income at approximately $

1.47 billion a year. Apart from the job and profit creation from this project, the earth

will be greener by the reduction of oil palm wastage.

Based on the amount of fermentable sugars currently obtained from OPF juice, it was

estimated that approximately 0.040 kg of fermentable sugars per kg OPF could be

generated (Zahari et al., 2012). Additionally, about 0.77 kg of fermentable sugars

could be obtained from 1 kg of OPF fibre by saccharification process based on the

maximum theoretical yield of holocellulose to fermentable sugars (Zahari et al., 2014).

Therefore, it is estimated that 425 kg of dry mass of fermentable sugars could be

produced from one tonne of fresh OPF processed (Zahari et al., 2015). The current

price of raw sugar from sugarcane is around $ 0.58/kg (USDA, 2014). Therefore, the

value of fermentable sugars obtained from OPF is estimated at around $ 247/t OPF.

This shows that the value of fermentable sugars from OPF is 3 times higher than the

production cost of fermentable sugars from OPF i.e., $ 72/t OPF as shown in Table

4.3. The estimated cost in this study was higher than the value estimated by Zahari et

al. (2014b) as they did not include the price of the OPF to farmers.

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Table 4.3. Cost estimation for renewable sugars production from 345,600 t/y of oil
palm frond (OPF) processed.

Item Cost ($) % total cost

Transportation 3,456,000 13.9

@ $ 10/t OPF

Harvesting and collection cost 3,456,000 13.9

@ $ 10/t OPF

9,331,200 37.4
Price of OPF petiole
@ $ 27/t OPF

Pre-processing cost 1,728,000 6.9

@ $ 5/t OPF processed

Enzyme cost for saccharifcation process 6,912,000 27.7

@ $ 20/t OPF processed

Labour cost for one year 36,000 0.1

@ $750/labour

Total cost ($/ year) 24,919,200 100.0

Specific production cost of fermentable sugars 72

($/t OPF processed)

4.3.3 Production cost of bioethanol production from OPF

Approximately 57,000 tonnes or equivalent to 73.7 million litres per year of bioethanol

could be produced from 345,600 tonnes of fresh OPF. The bioethanol production cost

breakdown is consists of two major inputs which are capital cost as well as operation

and maintenance cost. The cost of equipment include bioreactors, centrifugation unit,

absorption column, distillation column, rectification unit and storage tank including

the costs of installation, piping and instrumentation. The estimated value is calculated

96
using data from simulation of ethanol production from sugarcane in Brazil (BBI

Biofuels Canada, 2010) as the OPF petiole’s physical characteristic is similar to

sugarcane. All these equipment and installation cost about $50.4 million. The

estimated production cost of bioethanol at biorefinery plant is summarised in Table

4.4.

The total annual operating and maintenance cost for the biorefiney plant estimated to

be at $38.3 million. The maintenance cost is estimated at 4% of total investment cost

(Sánchez-Segado et al., 2012). Based on the yield of bioethanol obtained at 51% from

the sugars consumed, approximately $ 28.5 million is spent on the fermentable sugars

from OPF supplied by neighbouring mills at a price of $ 0.25/kg. Even though this

mill gate price of OPF sugar is half of the market price, the neighbouring mills

producing fermentable sugars from OPF still receive additional profits of $ 1.51

million per year. On the other hand, the cost of transportation to transport the

fermentable sugars from the neighbouring POMs to the central POM is around $ 1.14

million annually. In comparison, the overall cost of sugar production from sugarcane

is higher as it includes operating expenses and land cost to plant the sugarcane (Crago

et al., 2010). On the other hand, there is no need to grow additional crops in order to

obtain the OPF as the biomass is obtained during harvesting of FFB, the main product

of oil palm industry.

The operation of integrated technology approach requires manpower of 9 workers

comprising of 1 plant manager, 1 engineer, and 7 operators. In addition, 4.15 million

kWh/yr (Table 4.1) at a price of $ 0.12/kWh (TNB, 2014) would be utilised for

operating the bioethanol plant and 781,000 m3 of fresh water (BBI Biofuels Canada,

97
2010) to run the process at a price of $ 0.49 per cubic meter of fresh water

(Kementerian Tenaga, 2014). Interestingly, the cost of utility could be waived as the

excess steam, electricity and water can be obtained from the palm oil processing mill.

This technology managed to cut the cost around $ 4.32 million per year.

Table 4.4. Production cost of bioethanol from OPF at centralized biorefinery plant.

Share of
Description Quantity Unit total cost
(%)
Yield (bioethanol) 51 %

Product volume (bioethanol) 73,700,000 Litres/year

Capital cost 50,400,000 US $

Equipment depreciation 5,037,000 US $/year 14.8

Labour cost 117,400 US $/year 0.3

Raw material cost 28,500,000 US $/year 83.9

Chemicals and yeast 1,474,000 US $/year 4.3

Transportation of renewable 1,140,000 US $/year 3.4

sugars from neighbouring POMs
Energy and utility (4,324,500) US $/year -12.7

Operation and maintenance cost 2,015,000 US $/year 5.9

Product cost 0.46 US $/Litre

1US $ = 3.22 MYR

Value in parentheses is a negative value - energy and utility obtained from palm oil mill

The expected revenue from bioethanol is $58.21 million per year, which is calculated

from the annual expected bioethanol produced multiplied with the bioethanol price of

$ 0.79 per litre (Moncada et al., 2013). For the case of sugarcane bioethanol, the

98
producer can improve the profitability of ethanol production by selling surplus

electricity produced from cogeneration systems of sugarcane bagasse and trash which

is used as fuels in bioethanol production, supplying steam and electricity (Dias et al.,

2013). The reduction on ethanol production costs as low as $ 0.18 per litre can be

obtained by bioethanol production with electricity co-product credit (BBI Biofuels

Canada, 2010). Despite the integrated technology approach obtaining the steam and

electricity from the POM, it is not able to reach as low as production cost of sugarcane

bioethanol as the surplus of electricity is not considered in this study. In addition, cost

of interconnection facilities to the national grid must also be taken into consideration.

However, if the non-hydrolysed OPF fibre was considered as a biofuel to produce heat

and power, the surplus of electricity could be sold, subsequently contribute to the lower

production cost of bioethanol. It is normal that bioethanol cost of a new plant will be

significantly higher than that for an nth plant, which is mainly due to a much higher

total project investment (Chovau et al., 2013).

99
Table 4.5. Production cost of bioethanol from various feedstocks

Types of Production scale (litre Cost of production per

References
feedstock ethanol/year) litre (USD/litre ethanol)

Corn 47,619,048 0.46 BBI Biofuels Canada (2010)

Sugarcane 108,000,000 0.18a BBI Biofuels Canada ( 2010)

180,000,000 0.313b Dias et al. (2010)
Not stated 0.34b van den Wall Bake et al. (2009)

Lignocellulosic 19,047,619 0.60 BBI Biofuels Canada ( 2010)

EFB 36,500,000 0.49a Quintero et al. (2013)

OPF 73,700,000 0.46a This study

a
with electricity co-product credit

b
without electricity co-product credit

100
The bioethanol production cost from OPF was estimated at $ 0.46/litre, similar to

ethanol from corn and lower from SGB. Since the EFB is also abundant oil palm

biomass and can be easily obtained at palm oil mill after oil extraction, the bioethanol

production cost was also compared. Quintero et al. (2013) reported the bioethanol

production cost from EFB at $ 0.49/litre which is a bit higher than OPF. Production

cost for ethanol in Brazil is the world’s lowest with the average production cost

approximately $ 0.165 per litre, according to the Brazilian Sugarcane Industry

Association (UNICA). Factors contributing to Brazil’s competitiveness include

favourable climate conditions, low labour costs, and mature infrastructure built over

at least three decades (Xavier, 2007). Brazilian ethanol with attached co-generation

facilities has been shown to consistently result in a production cost lower than its

corresponding petroleum product, although this depends significantly upon net

production costs due to an electricity co-product credit (BBI Biofuels Canada, 2010).

4.3.4 Future direction of bioethanol generation in Malaysia

4.3.4.1 Potential of oil palm biomass for bioethanol production

As the world second largest palm oil producer, Malaysia has the potential of producing

SGB from the abundant oil palm biomass. Today, a majority of oil palm biomass is

left in the field while the rest is mulched and returned to the field as fertilizer. In

addition, in a business as usual scenario, by 2020, Malaysia’s palm oil industry will

utilise 12 million tonnes of biomass per annum for use in wood products and

bioenergy. Moreover, producing bioethanol and biobased chemicals from oil palm

biomass will offer increased wealth as well as new and better jobs. However, the

technologies to convert lignocellulosic biomass into bioethanol will only be available

101
on a commercial scale between 2013 and 2015 (MIA, 2013). In December 2010, Sime

Darby Plantation had announced its collaboration with Mitsui Engineering and

Shipbuilding Co. of Japan to construct and operate a bioethanol demonstration plant

which will convert EFBs into bioethanol located next to the Tennamaram palm oil mill

at Bestari Jaya (formerly Batang Berjuntai), Selangor. The plant can process 1.25

tonnes of EFBs a day using the hydrothermal pretreatment and enzymatic hydrolysis

technology. It is understood that Sime Darby is still collecting operational data to

confirm the technical feasibility of commercial scale production of bioethanol from

EFBs (Choong, 2012).

However, bioethanol from fresh OPF petiole is looked to have superior potential to

EFB in term of bioethanol production capacity as summarised in Table 4.6. In general,

it was estimated that 11,700 million litres of bioethanol can be obtained from both

juicy and pressed fibre of OPF petioles collected in the whole oil palm plantation in

Malaysia. This figure is far more higher than expected bioethanol productivity from

EFB.

102
Table 4.6. Potential value of sugar and ethanol from OPF and EFB
OPF EFB* Reference
Sugars production 3.49** 0.324a a
Shamsudin et al. (2012)
(t/ha/y) 0.594 b b
Cui et al. (2014)

Ethanol 2300 372c c

Tan et al. (2013)
production 588d d
Tye et al. (2011)
(l/ha/y) 487e e
Quintero et al. (2013)

*Average weight based on dry basis: 1.55 tonnes of EFB collected per hectare (Sulaiman et al., 2011).

** Percentage of sugar yield from OPF was estimated at 33% (w/w)

Total planted area approximately 5.1 million hectares (MPOB, 2014)

4.3.4.2 National Biofuel Policy

The Malaysia National Biofuel Policy (NBP) was launched on 21st March 2006 is

aimed to use environmentally friendly, sustainable and viable sources of energy to

reduce the dependency on depleting fossil fuels; and enhanced prosperity and well-

being of all the stakeholders in the agriculture and commodity based industries through

stable and remunerative prices. Biodiesel is among the list of products or activities that

are encouraged under the Promotion of Investments Act 1986. The biodiesel projects

are eligible to be considered for pioneer status or investment tax allowance (Lunjew,

2007). The Malaysian government hoped to take advantage of the increasing interest

in biodiesel and the country’s leading position in the production of palm oil. Under the

thrusts of biofuel for transport policy, the Malaysia government acknowledges that

diesel use by the transport sector is highly subsidised and therefore this sector will

receive the highest priority under Malaysia's biofuel agenda. The plan was to blend

5% palm biodiesel into regular diesel (producing B5) and make it available country

103
wide for the usage by land and sea transports. However, on December 2012, this has

not fully achieved due to the high CPO prices (Abdul-Manan et al., 2014; Sorda et al.,

2010).

The bioethanol production from abundant oil palm biomass holds great potential as

bioethanol feedstock. However, the bioethanol process is not yet scientifically feasible

nor economically viable (Rittgers and Wahab, 2013). As a steep learning curve is

expected in the first years of commercialisation, second generation ethanol may

initially not be cost-competitive with first generation ethanol from sugarcane. There

are strong indications, however, that second generation bioethanol is likely to be better

accepted in the US and EU than first generation bioethanol (MIA, 2013). Currently,

bioethanol production is not yet commercially significant in Malaysia due to lack of

enforcement and supporting policy from government. The Brazilian's experience with

bioethanol from sugarcane has shown to the world the importance of government

policy and support towards renewable energy. Currently, there are not much policies

being implemented that encourage bioethanol from lignocellulose except some

government subsidies and funds in the United States. As far as Brazil National Alcohol

Programme (NAP) or commonly called PROALCOOL is concerned, policy or strategy

is still inadequate to ensure that SGB would be a major source of renewable energy

(Tan et al., 2008).

104
Goh and Lee (2010); Tan et al. (2008) outlined few important strategies or policies in

order to promote SGB as a substitute to fossil fuel by referring to the example in Brazil:

i. Government and private grants funding in research and development.

ii. Offer incentives such as subsidy to bioethanol producers for each litre

produced.

iii. Low-interest loans to the producers to increase plant capacity.

iv. A systematic infrastructure to collect, transport and store cellulosic

feedstock built by cooperation between government and private sectors.

v. Production of ethanol-fueled vehicles.

vi. Reduction of vehicle tax for flexible fuel vehicles (FFVs).

vii. Mandatory blending of gasoline with bioethanol.

viii. Restriction of selling price of bioethanol to be higher than gasoline.

ix. Implementation of carbon-based fuel tax policy as a guideline for taxation

of energy fuels.

Introducing a mandate for bioethanol blending of 10 percent (E10) in Malaysia would

generate a domestic demand for 1 million tonnes of bioethanol per annum (MIA,

2013). This capacity is possible based on the current capacity of oil palm plantation in

Malaysia.

105
4.4 Conclusion

The analysis of materials flow and energy balance shows that integrated bioethanol

plant to existing POM is feasible to be carried out. The technology resulted in no

additional energy and utility requirements to recover OPF juice as well as for

bioethanol production. The low bioethanol production cost from OPF at $ 0.46/litre is

similar to the production cost of corn bioethanol, giving a good promise of

commercialization in the near future. The government should make a move to bring

this agenda into reality by introducing the policy on second generation bioethanol. As

the world’s most exciting oil palm industry cluster, Malaysia generates a great deal of

lignocellulosic waste which has the potential to be converted to SGB.

106
 CHAPTER 5:

CONCLUDING REMARKS AND SUGGESTIONS FOR FUTURE

RESEARCH

Rising concern over depleting fossil fuel and greenhouse gas emisson has resulted in

a high level of interest in biofuel originating from renewable resources such as sugars,

starches and lignocellulosic materials. Technology of the first generation bioethanol

from sugar and starchy crops are already matured since it began about four decades

ago. However, second generation bioethanol from lignocellulosic still experiencing

high cost of pretreatment and technological uncertainty resulting the delay in

commercialization. Malaysia is blessed to have abundant lignocellulosic biomass from

the main agriculture crop namely palm oil industry.

The novelty of this study is the finding of a new renewable and non-food sugar

feedstock from oil palm biomass namely oil palm frond juice for bioethanol

production. The specialty of this biomass is owed to the rich sugar content as well as

nutrient content of the OPF juice which was suitable as a fermentation feedstock.

Interestingly, the technology of bioethanol production from OPF juice is simple as the

first generation bioethanol from sugarcane. The comparable bioethanol yield obtained

from OPF juice to sugarcane juice demonstrated that the fresh OPF juice is a complete

fermentation medium for bioethanol production without nutrient supplementation and

pH correction. However, supplementation of OPF juice with yeast extract and nitrogen
107
source was able to improve the yield of bioethanol. The unnecessary pretreatment and

enzymatic saccharification of the OPF juice has promoted the juice as an attractive

bioethanol feedstock. These results could be a fundamental reference for future pilot

scale of first generation bioethanol production from OPF juice.

Another significant finding from this study is the alternative storage method that is

necessary to preserve fermentable sugars in OPF petiole juice for bioethanol feedstock.

Evaporation of OPF juice up to 70% of water removal did not substantially reduced

the aw of the OPF juice. The good part was the storage at 50°C and above managed to

preserve the glucose content regardless of OPF juice concentration. However,

evaporation of at least 50% of water removal is possible to minimise the size of storage

container. The storage at 50°C was found to be promising preservation method as the

glucose concentration was stable during 20 days storage. Furthermore, the high yield

of bioethanol obtained from the stored OPF juice has granted the potential of the

feedstock for bioethanol production at larger scale.

Finally, the analysis of materials flow and energy balance shows that integrated

bioethanol plant to existing palm oil mill is feasible to be carried out. This technology

resulted in no additional energy and utility requirements to recover fermentable sugars

from OPF petiole as well as for bioethanol production as the excess steam and

electricity available at the mill were estimated to be 177,000 tonnes and 5.9 GWh per

year, respectively. These excess energy resulting low bioethanol production cost from

OPF at $ 0.46/litre,similar to the production cost of corn bioethanol and cheaper than

SGB, thus giving a good insight of commercialisation in the near future.

108
Looking at the prospect of the new biofuel generation, Malaysia government should

take this challenge as stepping stone to commercialise bioethanol production from

OPF. However, there are few aspects that need further investigation before up scaling

bioethanol production from OPF. Since the major fermentable sugars from OPF are

glucose and xylose, further studies should be carried out to maximize bioethanol yield

and productivity by using a better yeast strain such as the flocculent strain MA-R4

which proven to have the highest ethanol production when fermenting a mixture of

glucose and xylose and also mixed sugars in the detoxified hydrolysate of wood chips.

Secondly, further investigation should be carried out on the effect of plant age and

location of the OPF collected on the chemical composition of the OPF petiole. On the

other hand, the presence of invertase enzyme in fresh OPF should be proven in order

to correlate the increment of glucose content in the juice during storage at 50°C. Since

all sugars from juicy part and pressed fibre of OPF were assumed stored in the same

storage tank, the effect of storage of the mixed sugars comprised of sucrose, glucose,

fructose and xylose also need further investigation. Finally, detail studies should be

done on a simulation solution for a complete proposed biorefinery plant technology

approach for bioethanol production. The process and cost models can be developed

using SuperPro Designer software, a simulation programme that is able to estimate

both process and economic parameters.

109
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